This is an author version of the contribution published on:
Questa è la versione dell’autore dell’opera:
Journal of Pest Science, 87: 671-679. ISSN: 1612-4758. DOI
10.1007/s10340-014-0593-3]
Galetto L., Miliordos D., Roggia C., Rashidi M., Sacco D., Marzachì C., Bosco D., 2014.
Acquisition capability of the grapevine Flavescence dorée by the leafhopper vector
Scaphoideus titanus Ball correlates with phytoplasma titre in the source plant.
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Luciana GALETTO1#, Dimitrios MILIORDOS2, Chiara ROGGIA2, Mahnaz RASHIDI1,2, Dario SACCO2, Cristina
MARZACHI’1, Domenico BOSCO2
Acquisition capability of the grapevine Flavescence dorée by the leafhopper vector Scaphoideus titanus
Ball correlates with phytoplasma titre in the source plant
1 Istituto di Virologia Vegetale, CNR, Strada delle Cacce 73, 10135 Torino, Italy.
2 Dipartimento di Scienze Agrarie, Forestali e Agroalimentari (DISAFA), Università degli Studi di Torino, Largo Paolo Braccini 2, 10095 Grugliasco (TO), Italy.
Abstract
Flavescence dorée (FD) is one of the most economically important grapevine diseases in Southern Europe, and it is associated with phytoplasmas, phloem-limited wall-less bacteria. Recovery from disease naturally occurs in infected grapevines during the following seasons after infection. The capability of the leafhopper vector Scaphoideus titanus to acquire FD phytoplasma (FDP) from recovered and infected grapevines of Barbera and Nebbiolo varieties was investigated in North-western Italy vineyards monitored from 2007 to 2011. Pathogen concentration was quantified by real-time PCR in FDP infected grapevines and broad beans, also used as source plants under controlled conditions, to correlate acquisition capabilities and phytoplasma titre in source plants. S. titanus acquired FDP from infected, but not from recovered, grapevines. FDP titre was higher in Barbera than in Nebbiolo and higher in summer than in spring, and acquisition efficiency and pathogen titre in source plants were positively correlated, both in field and laboratory conditions. Recovered plants do not represent a source of inoculum for the vector and therefore do not contribute to FDP spread. The inability of recovered plants to serve as FDP acquisition sources for the vector, as well as the effect of the season and of the two grapevine varieties on the FDP acquisition efficiency are relevant results to re-design disease management practices, especially since insecticide treatments against the vector are not fully effective and newly designed successful control strategies are required.
Keywords: Vitis vinifera, FD, Recovery, Barbera, Nebbiolo
Introduction
Flavescence dorée (FD) is a severe grapevine disease and represents a threat for wine production in many European viticulture areas (Belli et al. 2010). Main symptoms are leaf yellows or reddening, growth reduction, downward rolling of leaves, lack of new shoot lignifications and bunch withering. FD is associated
with the Flavescence dorée phytoplasma (FDP). Phytoplasmas are mollicutes that colonize both phloem of host plants and several organs of insect vectors and actively multiply in both hosts (Hogenhout et al. 2008). Leafhoppers, planthoppers and psyllids transmit these pathogens in a persistent propagative manner and phytoplasma diseases have been observed on hundreds of plant species all over the world (Foissac and Wilson 2010). In nature, FDP is specifically transmitted by Scaphoideus titanus Ball (Schvester et al. 1963), a monovoltine nearctic leafhopper, monophagous on grapevine. FDP is also routinely maintained under laboratory conditions in broad bean by Euscelidius variegatus Kirschbaum transmission (Caudwell et al. 1972). This latter system is used to investigate details of FDP cycle (Salar et al. 2013). Infected grapevines usually show symptoms the year after inoculation, but according to the variety and possibly the age of the plant, longer or shorter latencies have been reported (Caudwell et al. 1987; authors’ unpublished results). Phytoplasmas are classified using restriction fragment length polymorphism (RFLP) and sequence analysis of PCR-amplicons of 16S ribosomal and few other genes; FD is associated with phytoplasmas belonging to the 16SrV-C and -D subgroups (Martini et al. 2002; Angelini et al. 2001).
In Italy, FDP is especially prevalent in Northern viticultural areas, where, since 2000, compulsory insecticide treatments were enforced by the Plant Protection Services to suppress vector population and control FD epidemics. In the Piemonte region, all originally infected areas were declared “FD-outbreak areas” and removal of infected plants, together with the application of insecticides against the vector, were the main strategy for disease management. Nevertheless, in 2007 the disease was so widespread that some sites were declared “FD-settlement areas” and a new management strategy, exploiting the possible spontaneous remission of symptoms (recovery) instead of eradication of infected plants was proposed.
Following pruning of infected branches, a high recovery efficiency was observed for several cultivars, such as Barbera (Morone et al. 2007) and Prosecco (Musetti et al. 2007). Thus, following the first year of symptom expression, the development of the disease may vary according to the grapevine cultivar. The recovered plant remains symptomless if not exposed to new infective vectors and the phytoplasma cannot be detected, but whether these plants represent a source of infection for the vector it is still unknown.
Recovery from phytoplasma diseases has been reported also for grapevines infected by Bois Noir (Bellomo et al. 2007; Romanazzi et al. 2009), another important grapevine yellows (GY) (Mori et al. 2012), for apple trees infected by apple proliferation (Musetti et al. 2004), for apricots infected by European stone fruit yellows (Musetti et al. 2005), and for pears infected by pear decline (Seemüller et al. 1984). Recovery may be induced by pruning infected branches, but also by stressful agronomical practices, like uprooting and plant pulling (Romanazzi and Murolo 2008). Treatments with resistance elicitors have also been reported to induce recovery (Romanazzi et al. 2013). Different susceptibility to GY and different recovery efficiencies have been observed among different grapevine cultivars (Osler et al. 2003; Morone et al. 2007; Garau et al. 2004; Speich et al. 2006; Bellomo et al. 2007). Nebbiolo is more tolerant to FDP infection, with milder symptoms and lower FDP titre compared to Barbera (Roggia et al. 2013). In another study, FDP-infected Pinot blanc grapevines, a very susceptible cultivar, resulted a better source of inoculum for the insect vector compared to Merlot, considered a tolerant variety (Bressan et al. 2005).
The main aim of this work was to investigate acquisition efficiency of S. titanus from infected and recovered grapevines, cultivar Barbera and Nebbiolo, in two different periods of the vegetative season, under field conditions. Moreover, the correlation among FDP acquisition efficiency by S. titanus and phytoplasma titre in source plants was investigated under field and laboratory conditions using grapevines and broad beans, respectively. The results of this work are relevant to re-design the disease management strategy in areas where FD has become endemic, because insecticide control of vector population is not fully effective in preventing the spread of the disease.
Materials and Methods
Plant and insect material
Vineyards of different viticultural areas of North-western Italy were monitored for the presence of FD in 2007: Montegioco (Alessandria province), Cocconato (Asti province), Castagnito and Monteu Roero (Cuneo province). Grapevines (Vitis vinifera, L.) of the two most important cultivars of Piemonte region, Barbera
and Nebbiolo, were used in the study. Basal, median and apical leaves of symptomatic grapevine shoots were sampled according to Roggia et al. (2013), and analysed for the presence of FD phytoplasma by nested PCR and PCR-RFLP. According to the results, infected plants were labeled. Cocconato vineyard was selected for further analysis and in the following year, phytoplasma detection was carried out on symptomatic plants, newly infected, and on all the plants resulted positive in the previous year, regardless of symptom presence, in order to identify recovered plants. Therefore, every year since 2008, plants were classified as “FDP-infected”, if symptomatic and PCR positive in the current season, or as “recovered”, if symptomless and PCR-negative in the current season but symptomatic and FDP-positive in the previous year. Recovered plants were monitored for FDP presence three times a year by nested PCR: end of May-beginning of June, July and August. Flavescence dorée infection was monitored in Cocconato vineyard until 2011.
FDP-infected broad bean seedlings (Vicia faba L.) were used as host plants to check S. titanus transmission capability and for further acquisition experiments under laboratory conditions.
To obtain phytoplasma-free S. titanus for the acquisition experiments, two year-old grapevine branches hosting leafhopper eggs, were collected during winter in heavily infested vineyards. Branches were kept inside net cages for egg hatching in the greenhouse; potted grapevines from in vitro propagation and broad bean seedlings were provided for nymph rearing.
Field experiments
Acquisition experiments were performed in Cocconato vineyard in early June, (spring acquisition access period, AAP) and mid August (summer AAP) of 2008. These periods were chosen in order to avoid the effect of insecticides applied in field at mid-June and mid-July against nymphs and adults of S. titanus, respectively. For AAP on Barbera, vector nymphs were fed on seven recovered and six FDP-infected grapevines in spring, whereas summer AAPs were performed on four different recovered and six FDP-infected grapevines. Spring AAPs on Nebbiolo were carried out on five recovered and nine FDP-FDP-infected
grapevines, whereas six different FDP-infected and two recovered Nebbiolo plants were used for summer AAP.
Fourth and 5th instar nymphs of laboratory reared S. titanus were isolated inside nylon net cages on grapevine branches for seven days. About 50 insects were caged on a single branch of recovered or FDP-infected grapevines. At the end of AAP, caged branches were cut from the plant, insects were collected, transferred to grapevines and allowed to complete the latent period for three weeks in the climatic chamber at 24 °C, L:D 16:8h. Each group of insects collected from the same branch was isolated together on the same plant. After latency, a sub-set of the insects, were caged onto broad beans (four insects per plant per one week) to check their FDP transmission ability. All insects were singly tested by PCR for the presence of FDP; broad bean plants exposed to vectors were observed for symptom appearance and assayed by diagnostic PCR one month after inoculation. The proportion of transmission by single insects was calculated using the Swallow estimator (Swallow 1985). DNAs extracted from i) FDP source grapevines, ii) representative S. titanus fed on FDP source plants, and iii) inoculated broad beans were analysed by PCR-RFLP to confirm the identity of the phytoplasma strain.
To perform the correlation analysis between phytoplasma concentration in source plants and acquisition efficiency, FDP titre was quantified in all Barbera and Nebbiolo symptomatic plants resulted PCR-positive, following the first (early June) and the second (late July) samplings of the season in Cocconato vineyard in 2008.
Laboratory experiments
Broad bean was used as host plant to characterize FDP acquisition efficiency by S. titanus under laboratory controlled conditions (24 °C, L:D 16:8h). About 200 S. titanus nymphs were isolated for one week AAP on 12 FDP-infected broad beans, and then maintained on healthy grapevines to complete the latent period. At the end of AAP, leaves of source broad beans (40 days post inoculation) were sampled for DNA extraction,
phytoplasma detection and quantification by qPCR. To characterize FDP acquisition efficiency, S. titanus adults were sampled at 30 days post acquisition for DNA extraction and phytoplasma detection by PCR.
DNA extraction, phytoplasma detection by PCR and FDP strain characterization by PCR-RFLP
Total DNA was extracted from grapevines (1.5 g), broad beans (0.1 g) and single insects according to Galetto et al. (2005).
For FDP diagnosis, two µl of DNA was used in direct PCR with universal primers P1/P7 (Schneider et al. 1995). Reaction products were used as templates in nested PCRs driven by primers R16(V)F1/R1 (Lee et al. 1994). Reaction and cycling conditions were as detailed in the original papers. PCR products were separated in a 1% agarose gel, buffered in TBE (90 mM Tris borate and 2 mM EDTA, pH 8.3), stained with ethidium bromide, and visualised under UV light.
For FDP characterization, Restriction Fragment Length Polymorphism (RFLP) was performed on PCR products of ribosomal 16S and secY genes. For the former, P1/P7 amplicons were used as templates in nested PCRs with primers M1/B6, while for the latter, two µl of DNA was used in direct PCR with universal primers FD9f2/r followed by nested assays with FD9f3/r2, according to Angelini et al. (2001) and Martini et al. (2002). Amplicons from both nested PCRs were digested with TaqI endonuclease (Invitrogen, Paisley, UK) for 1 h at 37°C and the restriction profiles visualized after electrophoresis on ethidium bromide-stained acrylamide gels.
FDP quantification by qPCR
FDP titre in infected grapevines and broad beans was measured as number of FDP cells per ng of plant DNA, as previously detailed (Marzachì and Bosco 2005; Roggia et al. 2013). To quantify FDP and grapevine DNAs, FdSecyFw/Rv and Vitis18SF1/R1 (Roggia et al. 2013) were used, respectively. To determine amount of broad bean DNA, a new primer pair was designed on V. faba ribosomal gene (X17535) using Primer3 software
(Untergrasser et al., 2012). To confirm the exclusive presence of the expected specific amplicon, 5 μL of PCR products were analyzed by electrophoresis in 1.5% agarose gel buffered in TBE. Standard curves for the absolute quantification of FDP and host DNA were obtained by dilution of i) plasmid p26SecYFD, containing the appropriate secY gene target sequence from a local isolate of FDP, and ii) total DNA extracted from healthy grapevine and broad bean, respectively. Three quantities of p26SecYFD (0.5 ng, 5 pg, and 5 fg), were used, corresponding to 1.17·108, 1.17·106, 1.17·103 cells of FDP. For the absolute quantification of host DNA, four quantities of total DNA extracted from healthy plants (50 ng, 5 ng, 0.5 and 0.05 ng) were used. Plant sample DNAs were diluted in ddH20 to a final concentration of 1 ng/μl, and used (5 μl) as template in real time assays together with iQTM SYBR® Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) and specific primer pairs at a final concentration of 300 nM, in a volume of 25 μL. The PCR was performed in 96-well plates in a Chromo4 Real-Time PCR (Bio-Rad Laboratories, Hercules, CA, USA) thermal cycler, following cycling conditions described by Roggia et al., 2013. For each sample, plant and FDP-DNA were amplified in the same plate with their corresponding reaction mixture, together with the standard dilutions of healthy plant DNA and p26SecYFD. Each sample was run in triplicate in the same plate. For each PCR, water instead of DNA was used as negative control. Threshold levels, threshold cycles, and standard curves were automatically calculated by the Bio-Rad Opticon MonitorSoftware, version 3.1. Per-well baseline cycles were determined automatically. Specificity of the reaction was tested by running a melting curve analyses of the amplicons following each quantification reaction.
Statistical analysis
The proportions of PCR positive insects, following feeding on different cultivars (Barbera and Nebbiolo) and during spring or summer season, and the FDP titres measured in source grapevines of both cultivars (Barbera and Nebbiolo) during spring or summer season were compared using a two ways analysis of variance including simple effects and interaction. As data did not respect Anova assumptions, they were transformed. Data about proportions of PCR positive insects were transformed using the arc sine of the
square root, as it is usual for ratios (Camussi et al., 1995), while data about FDP titres were rank transformed, according to Conover & Iman (1981), as no other transformation led to respect ANOVA assumptions. The proportions of PCR positive insects, following feeding on grapevines or broad beans, were compared with the z-test. The FDP titres measured in source grapevines or broad beans were compared with Mann-Whitney Runk Sum Test.
The acquisition efficiency of S. titanus calculated from field experiments was correlated with the mean FDP amount measured in grapevine plants. To perform correlation analysis five categories were considered: i) FDP titre measured in spring-sampled Barbera plants (N=18) and the proportion of positive insects fed on six FDP-infected Barbera in spring, ii) FDP titre measured in summer-sampled Barbera plants (N=24) and the proportion of positive insects fed on six FDP-infected Barbera in summer, iii) FDP titre measured in spring-sampled Nebbiolo plants (N=8) and the proportion of positive insects fed on nine FDP-infected Nebbiolo in spring, and iv) FDP titre measured in summer-sampled Nebbiolo plants (N=28) and the proportion of positive insects fed on six FDP-infected Nebbiolo in summer. A similar experiment was also performed under laboratory conditions using broad beans as source plants, category v): FDP titre measured in broad bean plants (N=12) and the proportion of positive insects fed on these plants. Rank Order Correlation was used to correlate FDP titre in source plants and vector acquisition efficiencies.
All statistical tests were performed using SPSS ver. 20.0.0 (2011).
Results
Acquisition efficiency from infected and recovered grapevines
Field acquisition experiments were performed on 23 Barbera (11 recovered and 12 FDP-infected) and 22 Nebbiolo grapevines (7 recovered and 15 FDP-infected). Recovered plants never showed FDP symptoms and were always PCR negative until 2011 survey. TaqI RFLP profiles for ribosomal 16S and secY genes are shown
in Fig. 1. Eleven and 13 isolates from FDP-infected Barbera and Nebbiolo, respectively, were identified as 16S-C/secY-C; one isolate from Barbera was 16S-C/secY-D; two isolates from Nebbiolo were 16Sr-D/secY-D. Nevertheless, the results for different phytoplasma strains were pooled together as acquisition and transmission efficiencies were similar.
S. titanus never acquired FDP from recovered Barbera plants, and all insects but one (0.8%) fed on recovered Nebbiolo grapevines resulted FDP-negative (Table 1). Overall, following feeding on infected Barbera and Nebbiolo plants, 48/150 and 23/200 S. titanus resulted PCR positive for the presence of FDP, respectively. In spring, AAP on Barbera and Nebbiolo resulted in 18/99 and seven out of 120 FDP-positive insects, respectively. In summer, 30/51 and 16/81 S. titanus acquired FDP following feeding on Barbera and Nebbiolo plants, respectively.
The mean proportion of infected insects recorded following feeding on different vines was not influenced by the source plant variety or by the season, considered as factors in a two way analysis of variance (Table 2).
Phytoplasma transmission to broad beans
To check FDP transmission ability by S. titanus fed on infected source grapevines, single broad beans were inoculated by 4 insects, following both spring and summer field acquisitions (Table 3). None of the insects fed on recovered grapevines was able to transmit FDP to broad beans. Following field acquisition from infected Barbera and Nebbiolo grapevines, 34 and 14% S. titanus
were infected, respectively. Estimated proportions of 7 (Barbera) and 3% (Nebbiolo) of the insects successfully transmitted FDP to broad beans.
The same FDP strain (Fig. 1) was found in the each original source grapevine, in S. titanus fed on each original source plant, and in the corresponding broad bean test plant.
Quantification of phytoplasma titre in host plants
Mean phytoplasma titre measured in grapevines is summarized in Table 4. Two way ANOVA for cultivar and season showed that both factors had a significant effect on the amount of pathogen measured in plants, as FDP titre was higher in Barbera than in Nebbiolo and higher in summer than in spring. No significant interaction was found between these two factors (Table 4).
The newly designed primers FabaFw AATTGCAGAATCCCGTGAAC-3’) and FabaRev2 (5’-TCAACCAACCATGAGACGAA-3’) amplified a specific amplicon of the expected size (175 bp) and gel electrophoresis confirmed the absence of nonspecific products (not shown). Melting curve analysis of the amplicon showed a unique melting peak at 85.0°C. The linear correlation coefficient (r) and the efficiency of the broad bean DNA standard curve were 0.998 and 111%, respectively. The mean FDP titre measured in broad beans (Table 4) was higher than the amount of pathogen cells overall recorded in grapevines (Rank Sum Test, U=277, 254 T=737, P=0.024). Consistently, acquisition efficiency was significantly higher following feeding on FDP-infected broad beans than on infected grapevines (z-test, z=7.6, P<0.001).
Acquisition efficiency and pathogen titre in plants
For the correlation analysis, five categories of FDP-infected plants and corresponding acquisition efficiencies were considered (Fig. 2). FDP titres in the source plants and proportion of positive insects were positively correlated (Spearman Rank Order Correlation Coefficient=0.900, P=0.037). The relationship between phytoplasma acquisition capability and mean FDP titre in different categories of infected plants is depicted in Figure 2.
Discussion
This work provides the first evidence that FDP-recovered grapevines are not a suitable source of pathogen for the vector S. titanus. Moreover, FDP titre was higher in Barbera than in Nebbiolo and higher in summer
than in spring, and phytoplasma acquisition efficiency by the vector resulted strongly positive correlated with the FDP titre in source plants.
Different grapevine cultivars show different susceptibility to phytoplasma disease in terms of symptom severity, recovery rate and pathogen titre (Roggia et al. 2013; Morone et al. 2007). Our results confirmed that FDP titre was higher in Barbera than in Nebbiolo infected grapevines, with a seasonal trend in FDP concentration (low in spring and high in summer) for both cultivars (Roggia et al. 2013). S. titanus acquired FDP more efficiently when pathogen titre was higher in source grapevine (e.g. more in Barbera than in Nebbiolo and more in summer than in late spring). In a previous work (Bressan et al. 2005), S. titanus was shown to acquire FDP with significantly higher efficiency from FDP-infected Pinot blanc grapevines, a very susceptible cultivar, than from Merlot, a FDP-tolerant variety. These findings are relevant to the understanding of FD epidemiology. In the Piemonte region, a higher incidence of the disease is observed in Barbera vineyards compared to Nebbiolo ones. The higher pathogen titre measured in Barbera compared to Nebbiolo may be one of the reasons to explain this phenomenon, therefore, secondary spread of the disease (from vine to vine) is more rapid in Barbera vineyards. Primary spread due to infected S. titanus coming from outside the vineyards (fed on wild rootstocks or abandoned grapevines) is likely to occur (Pavan et al. 2012) and therefore the different spread of FDP may also depend, besides the cultivar, upon the surrounding environment of the fields.
This is the first evidence that FDP acquisition by the vector is significantly correlated with pathogen titre in grapevine. Most of the S. titanus nymphs develop during late May and June (Bosio and Rossi 2001), when the acquisition efficiency is lower, due to a lower phytoplasma concentration in the plant. However, due to the wide period of egg-hatching of the leafhopper, nymphs developing late in the season are more likely to acquire the pathogen and become infectious. The leafhopper adults are present mainly from mid-July onward and therefore are likely to feed on vines that are good sources of infection. On the other hand, acquisition by nymphs results in infected insects that can spread the disease for a long period, while acquisition by adults, due to the long latent period in the vector (about one month), results in infected insects that will spread the disease for a brief period due to their shorter life span. The control of adult
leafhoppers is important, not only to suppress insects that acquired FDP as nymphs and are indeed infectious, but also to suppress adults that can acquire the pathogen late in the season and eventually transmit it well after the last insecticide treatment and even after grape harvest. Irrespective of cultivars, the higher pathogen titre recorded in summer than in late spring should be considered when setting a more fine-tuned insecticide treatment strategy.
This is also the first evidence that the vector of FDP is unable to acquire the phytoplasma from recovered grapevines. This finding, in line with the absence of phytoplasmas in the recovered grapevines (Morone et al. 2007), is very important for the disease management. Several studies investigated recovery from phytoplasma diseases in arboreous plants, but little is known about the molecular mechanism regulating this phenomenon, that is influenced by host genotype and environmental conditions (Morone et al. 2007). Accumulation of callose in the phloem, hydrogen peroxide and calcium intracellularly, and a low level of antioxidant enzymes have been observed in grapevines recovered from FDP (Musetti et al. 2007; Gambino et al. 2013; Margaria and Palmano 2011; Margaria et al. 2013). The existence of epigenetic changes in European stone fruit yellows-recovered apricots has been suggested (Osler et al. 2014). Moreover, gas exchange performances analogous as those before infection have been observed in grapevines recovered from FDP (Vitali et al. 2013). The very rapid phytoplasma translocation to roots following the inoculum by vectors in the aerial parts, at least recorded herbaceous hosts (Lherminier et al. 1994; Saracco et al. 2006), should exclude the possibility that recovery is related with the time of infection during the season, such as the case of Xylella fastidiosa, another well-studied vector borne grapevine bacterial pathogen (Feil et al., 2003).
For the varieties showing a good recovery efficiency and stability, roguing of infected plants can be avoided, since they do not represent a source of inoculum and they are likely to recover. Recovered grapevines produce less than healthy ones, with a reduction of about 20%, but significantly more than infected ones (Morone et al. 2007; Garau et al. 2007) and yield loss in recovered plants decreases along the following years. Under these circumstances, the maintenance of infected plants to allow natural recovery seems to be a promising strategy in disease management to save money and time of the growers. In the experiments,
one single leafhopper acquired FDP from a recovered Nebbiolo plant; the most likely explanation for this is an incomplete recovery of the source plant that, in spite of the absence of symptoms and negative PCR results, was on the way of recovery but still locally infected at low titre (likely below detection threshold). Actually, it is known that phytoplasma detection in grapevine is difficult, phytoplasmas may be restricted to one or few shoots (Roggia et al. 2013) and false negative results cannot be ruled out. Moreover, Nebbiolo generally shows mild symptoms and low FDP titre.
Our results seem to suggest that phytoplasma concentration in the plant is a very important feature affecting acquisition, even if other possible factors, such as feeding behaviour on different grapevine varieties or plant species, cannot be ruled out. Roggia et al. (2013) showed a positive correlation between presence of symptoms and FDP detection in grapevine leaf tissue, as the phytoplasma was very rarely found in non symptomatic shoots. It can be concluded that, under field conditions, S. titanus will successfully acquire the phytoplasma only from symptomatic shoots of infected vines. The elimination of symptomatic shoots (when the plant is only partially infected) can be suggested instead of roguing the whole infected grapevine. However, the best strategy to control FDP spread requires a case by case analysis because recovery maybe efficient in a given variety but not in others. As a general rule, phytoplasma titre may be an indicator of the role of the plant as source of acquisition and therefore control strategies should also consider this aspect. The correlation between phytoplasma titre and acquisition efficiency found in the FDP-grapevine-S. titanus association may not be the rule for other phytoplasma-plant-vector associations. This is especially interesting for the FDP-wild rootstock grapevines-S. titanus association that supports the epidemiological cycle of the disease outside the vineyard and represents a serious threat to the viticulture. The results of this work are relevant to re-design disease management practices, re-evaluating the opportunity of roguing FDP-infected plants, that can eventually recover and do not represent a source of inoculum for the vector. This is especially true for organic viticulture, since the allowed insecticides are not totally effective against the vector populations.
Acknowledgements
This research is funded by the Piemonte Region with the projects: “Adoption of a multidisciplinary approach to study the grapevine agroecosystem: analysis of biotic and abiotic factors able to influence yield and quality”, “Studi su fitoplasmi della vite e loro vettori: sensibilità varietale e efficienza di acquisizione di Flavescenza dorata, caratterizzazione, diffusione e vettori di Legno nero, tecniche di riduzione del danno”. The authors declare that they have no conflict of interest.
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Tables
Table 1. Acquisition from recovered Barbera and Nebbiolo grapevines expressed as Flavescence dorée phytoplasma-PCR positive Scaphoideus titanus out of total tested insects.
Cultivar Spring AAPa Summer AAPa Total
Barbera 0/176 0/36 0/212
Nebbiolo 0/96 1/25 1/121
aAcquisition access period (AAP) performed in early June (Spring AAP) or mid August (Summer AAP).
Table 2. Acquisition from Barbera and Nebbiolo Flavescence dorée phytoplasma (FDP)-infected grapevines, expressed as mean proportion of FDP-PCR positive Scaphoideus titanus recorded on different source plants and in different seasons.
Source plant Season N
Proportion of PCR positive insects SEM Transformed data Barbera Spring 6 0.186 0.091 0.360 Summer 6 0.284 0.144 0.433 Nebbiolo Spring 9 0.079 0.058 0.141 Summer 6 0.291 0.131 0.501 Barbera Overall 12 0.235 0.082 0.397 Nebbiolo Overall 15 0.164 0.066 0.285 Overall Spring 15 0.122 0.050 0.229 Summer 12 0.287 0.093 0.467 Overall Overall 27 0.196 0.051 0.334 Analysis of variance Source plant P(F) n.s. Season P(F) n.s.
Source plant x Season P(F) n.s.
Table 3. Flavescence dorée phytoplasma (FDP) transmission to broad bean by Scaphoideus titanus, following field acquisition from Barbera and Nebbiolo grapevines.
Source grapevine cultivar Sanitary status PCR positive/total
inoculating S.titanus
PCR positive/total exposed broad beans
FDP infected 33/96 6/24
Nebbiolo
Recovered 0/40 0/10
Table 4. Mean Flavescence dorée phytoplasma (FDP) titre, expressed as FDP cells/ng plant DNA, measured in different source plants and in different seasons.
Source plant Season N FDP titre SEM Average rank
Barbera Spring 18 1155 509 23.4 Summer 24 14325 2825 62.3 Nebbiolo Spring 8 132 56 13.9 Summer 28 2186 518 37.6 Barbera Overall 42 8681 1908 45.6 Nebbiolo Overall 36 1730 427 32.3 Overall Spring 26 840 362 20.5 Summer 52 7789 1567 49.0 Overall Overall 78 5473 1113 39.5
Broad bean* Overall 12 18969 6964
Analysis of variance
Source plant P(F) 0.000
Season P(F) 0.000
Source plant x Season P(F) n.s.
Figure captions
Fig. 1 TaqI restriction profiles obtained following digestion of M1/B6 (panels a, patterns “16SC” and “16SD”)
and FD9f3/r2 (panels b, patterns “SecYC” and “SecYD”) nested PCR amplicons. M: 1-kb plus DNA size markers (Invitrogen).
Fig. 2 Mean acquisition efficiency of Flavescence dorée phytoplasma (FDP) by the vector Scaphoideus titanus and mean phytoplasma titre (FDP cells/ng plant DNA) measured in grapevines and broad beans source plants.
