Volume 63, Issue 1 p. 31-41
Original Article
Free Access

Flavescence dorée phytoplasma titre in field-infected Barbera and Nebbiolo grapevines

C. Roggia

C. Roggia

Istituto di Virologia Vegetale, CNR, Strada delle Cacce 73, Torino, 10135 Italy

University of Torino, DISAFA, Via Leonardo da Vinci 44, Grugliasco (TO), Italy

Search for more papers by this author
P. Caciagli

P. Caciagli

Istituto di Virologia Vegetale, CNR, Strada delle Cacce 73, Torino, 10135 Italy

Search for more papers by this author
L. Galetto

L. Galetto

Istituto di Virologia Vegetale, CNR, Strada delle Cacce 73, Torino, 10135 Italy

Search for more papers by this author
D. Pacifico

D. Pacifico

Istituto di Virologia Vegetale, CNR, Strada delle Cacce 73, Torino, 10135 Italy

Search for more papers by this author
F. Veratti

F. Veratti

Istituto di Virologia Vegetale, CNR, Strada delle Cacce 73, Torino, 10135 Italy

Search for more papers by this author
D. Bosco

D. Bosco

University of Torino, DISAFA, Via Leonardo da Vinci 44, Grugliasco (TO), Italy

Search for more papers by this author
C. Marzachì

Corresponding Author

C. Marzachì

Istituto di Virologia Vegetale, CNR, Strada delle Cacce 73, Torino, 10135 Italy

E-mail: [email protected]Search for more papers by this author
First published: 02 May 2013
Citations: 41

Abstract

Flavescence dorée phytoplasma (FDP) titre in two red grapevine cultivars, Barbera and Nebbiolo, was measured over the vegetative seasons of two consecutive years in two vineyards of the Piemonte Region (northwestern Italy), with a double absolute quantification of FDP cells and grapevine DNA in real-time PCR. The relationships of pathogen concentration to cultivar susceptibility and symptom severity were investigated. FDP titre was always higher in cv. Barbera than in cv. Nebbiolo infected vines, and this difference was significant at early and late summer samplings of 2008 and at early summer sampling of 2009. A seasonal trend in FDP concentration (low in spring, high in early summer and intermediate in late summer) was conserved for cvs Barbera and Nebbiolo in both years and vineyards. Considering both cultivars and years from both vineyards, a significant positive correlation between FDP concentration and symptom severity was found in the spring samples. Regarding the FDP strains (-C or -D), no differences in pathogen titres were detected for either cultivar. Similarly, the presence of another grapevine yellows phytoplasma, bois noir, a subgroup 16SrXII-A phytoplasma, in mixed infection with FDP strains had no effect on FDP concentration. These results demonstrate for the first time that grapevine cultivars with different susceptibility to FDP support different pathogen titres.

Introduction

Phytoplasmas are wall-less plant pathogenic bacteria of the class Mollicutes associated with diseases of numerous plant species (Bertaccini, 2007). They are phloem-limited and transmitted by phloem-sucking leafhoppers, plant-hoppers and psyllids (Marzachi et al., 2004; Weintraub & Beanland, 2006). Phytoplasmas are classified on the basis of the highly conserved 16S rRNA sequence into more than 30 groups within the ‘Candidatus Phytoplasma’ genus (Firrao et al., 2004; Zhao et al., 2010).

Flavescence dorée (FD) is an epidemic, economically important, quarantine disease of grapevine in France, Italy and Spain (Boudon-Padieu, 2003). The disease is associated with a phytoplasma belonging to the 16SrV (Elm yellows) taxonomic group (Lee et al., 2000). FD epidemics are caused by phytoplasma strains belonging to three phylogenetic strain clusters, according to multilocus sequence analysis of map, uvrB-degV and secY loci (Arnaud et al., 2007). In Italy the disease is caused by phytoplasma isolates belonging to clusters 2 and 3, and in Piemonte (northwestern Italy) FD strains within the highly variable cluster 3 (FD-C) are the most prevalent (Martini et al., 2002), although isolates of the less variable cluster 2 (FD-D) are also present. The leafhopper Scaphoideus titanus is the specific vector of the different FDP strains to grapevine under natural conditions (Schvester et al., 1963; Mori et al., 2002; Papura et al., 2009). Recently, a role as potential vector of FDP from alternative hosts to grapevine has been suggested for the planthopper Dictyophara europea (Filippin et al., 2009).

FD-infected grapevines usually show symptoms the year after inoculation (Morone et al., 2007), although longer latencies have been reported (Osler et al., 2002). Yellowing, downward curling of leaves, fruit abortion, stunting, and lack of lignification of new shoots are among the most important symptoms (Caudwell, 1983, 1990). Infected vines show dramatically reduced grape production, but following the first year of symptom expression, a spontaneous and cultivar-dependent remission of symptoms may occur, and symptomless plants usually do not contain detectable FD phytoplasma (Morone et al., 2007).

Control of the disease relies mainly on compulsory insecticide treatments to reduce vector population and rogueing of infected plants. No resistance is available to FD, but cultivars with different susceptibility to the disease have been reported (Kuzmanovic et al., 2008). Barbera and Nebbiolo are two traditional and economically important grapevine cultivars of Piemonte for the production of red wines. Cultivar Barbera is highly susceptible to FD and shows severe symptoms starting with growth reduction at the beginning of the vegetative season, whilst symptoms on cv. Nebbiolo vines are milder, especially at the beginning of the vegetative season (Morone et al., 2001).

In this study, a new approach to real-time quantification of FD phytoplasma in grapevine was used to determine: (i) relationships between cultivar susceptibility and pathogen concentration in the plant, (ii) relationships between pathogen presence/concentration and symptom severity, and (iii) FD phytoplasma titre over the vegetative season in order to identify the best timing for sampling and detection.

Materials and methods

Vineyard description

Two vineyards located in Piemonte (northwestern Italy) were chosen: Cocconato (Asti province) and Monteu Roero (Cuneo province). Barbera and Nebbiolo cultivars were planted at the Cocconato vineyard in 1999 on SO4 rootstock. Nebbiolo was planted at Monteu Roero vineyard in 1998 also on SO4 rootstock. Both vineyards were subjected to conventional agronomic and phytosanitary practices. An electronic meteorological station was present in Cocconato between the neighbouring Barbera and Nebbiolo vineyards. For the Monteu Roero vineyard, climatic data were collected at a station located in a neighbouring municipality (Canale) in the same exposure and altitude conditions. Mean monthly temperature (°C) and number of days in each month with minimum temperature below 0°C (freezing days) were calculated from data collected at both stations.

Establishment of a correct sampling procedure

To establish a correct sampling procedure, five FD-infected cv. Barbera and Nebbiolo plants were selected in the Cocconato vineyard at growth stage 17–19 (12–16 leaves separated; Coombe, 1995). Shoots with and without symptoms were separately labelled on each plant and samples consisting of three basal, three median and three apical leaves (a total of nine leaves) were separately collected from each shoot. Total DNA was extracted from each sample, and the presence of FD phytoplasma was assessed by nested PCR as detailed below. FD phytoplasma titre was then measured in each sample as described below.

Grapevine sample collection

Samples were collected in 2008 and 2009 only from grapevines with symptoms. Each sample consisted of three basal, three median and three apical leaves from one shoot with symptoms, for a total of nine leaves. In spring samplings, shoots with symptoms were severely stunted and dwarfed, and therefore the nine leaves were collected from two or three stunted shoots. Cultivar Barbera samples were collected at Cocconato, while cv. Nebbiolo samples were collected at both Cocconato and Monteu Roero, as not enough FD-infected plants were present at Cocconato. For sample collection, vineyards were surveyed three times a year: late spring (May–early June), early summer (July–early August) and late summer (September). During each survey, grapevines with symptoms were labelled (Margaria et al., 2009) and scored for symptom severity. The same vines were sampled in both years provided that they had symptoms. New vines with symptoms were sampled to replace recovered vines sampled in the previous year. In spring, symptom severity of each infected plant was rated on a scale of 1 to 3 according to the percentage of shoots with symptoms on the fruit-bearing branch (1: <30%; 2: 30–65%; 3: >65%). During early and late summer surveys, symptom severity of each infected grapevine was rated as A, B, C or D, according to the proportion of the plant canopy showing phytoplasma-specific symptoms (leaf downward rolling, sectorial leaf and vein reddening, bunch drying, premature leaf dropping, presence of brown pustules on canes, and, for late summer surveys, lack of cane lignification): A: plants lacking FD-specific symptoms, but not looking healthy; B: <30% FD-specific symptoms; C: 30–60%; D: >60%.

Total DNA extraction and phytoplasma diagnosis

Total DNA was extracted from 1·5 g of leaf veins following a phytoplasma enrichment protocol (Marzachi et al., 1999) and dissolved in 100 μL sterile double distilled water (SDW). DNA concentration of extracts was quantified with a NanoDrop (ND-1000 Spectrophotometer; NanoDrop Technologies, Inc.). PCR was employed for phytoplasma detection. Universal primer pair P1/P7 was used in direct PCRs as previously described (Deng & Hiruki, 1991; Schneider et al., 1995). Reaction products were diluted 1:40 in SDW and used as templates in nested reactions driven by primers R16(I)F1/R1 or R16(V)F1/R1 for the specific detection of 16S groups I and V, respectively (Lee et al., 1994). Amplicons were separated by electrophoresis in 1% agarose gels buffered in 0·5 × TBE (90 mm Tris-borate, 2 mm EDTA) and visualized under UV light after staining with ethidium bromide. To identify bois noir, a group 16SrXII (stolbur group) phytoplasma infection, 2 μL aliquots of fragments amplified with R16(I)F1/R1 primers were digested for 2 h with 1 U MseI (Invitrogen) at 37°C, according to the manufacturer's recommendations. Digestion products were separated in 5% polyacrylamide gels buffered in 1 × TBE along with a 1 kb plus DNA size marker (Gibco BRL) and visualized by UV-light after staining with ethidium bromide. A Piemonte strain of FDP (FD-C), acquired from grapevine by Scaphoideus titanus and transmitted to Vicia faba, a French strain of FDP (FD-D), kindly provided by Dr E. Boudon-Padieu and graft-maintained in periwinkle in the collection of the Istituto di Virologia Vegetale, CNR, and a Sardinian strain of stolbur phytoplasma from tomato (T2_92), also maintained in periwinkle (Minucci & Boccardo, 1997), were used as reference isolates and positive controls in PCR and RFLP experiments. A healthy cv. Barbera grapevine from in vitro propagation, maintained in an insect-proof greenhouse, was employed as negative control in PCR experiments.

Characterization of flavescence dorée phytoplasma isolates

Characterization of flavescence dorée isolates was done by restriction analysis of the partial 16S–23S rDNA operon and secY gene. In the first case, fragments amplified with the P1/P7 primer pair (Deng & Hiruki, 1991; Schneider et al., 1995) were diluted 1:40 with SDW, and reamplified with primers M1/B6 as previously described (Martini et al., 1999). For the partial amplification of the secY gene, total grapevine DNAs were amplified in direct PCR with primers FD9f2/FD9r (Angelini et al., 2001) and then, following 1:40 dilution with SDW, amplified with primers FD9f3/FD9r2 (Martini et al., 2002). Amplicons were digested for 2 h with 1 U TaqI (Invitrogen) at 65°C. Digestion products were separated in 5% polyacrylamide gels in TBE along with a 1 kb plus DNA size marker and visualized as detailed above.

Quantification of flavescence dorée phytoplasma DNA

Flavescence dorée phytoplasma titre in infected grapevines was measured as number of FD phytoplasma cells per ng of grapevine DNA. To determine the number of FD phytoplasma cells, specific primers targeting a portion of the secY gene of the FD phytoplasma were designed using primer3 (Rozen & Skaletsky, 2000) and used in an absolute quantification real-time PCR assay. For the quantification of grapevine DNA, specific primers targeting the 18S rDNA gene of grapevine were designed. The chosen primers were subjected to blastn analysis on GenBank through the NCBI database to test their specificity. To confirm the exclusive presence of the expected specific amplicon, 5 μL of real-time PCR products were analysed by electrophoresis in 1·5% agarose gels buffered in TBE. Standard curves for the absolute quantification of FD phytoplasma and grapevine DNA were obtained by dilution of plasmid p26SecYFD, containing the appropriate secY gene target sequence from FD-C phytoplasma, and of total DNA extracted from healthy grapevine grown from seed, respectively. Three quantities of p26SecYFD (0·5 ng, 5 pg and 5 fg) were used, corresponding to 1·17 × 108, 1·17 × 106 and 1·17 × 103 cells of FDP. For the absolute quantification of grapevine DNA, three quantities of total healthy grapevine DNA (5, 0·05 and 0·005 ng) were used. Grapevine sample DNAs were diluted in SDW to a final concentration of 1 ng μL−1, and used (5 μL) as template in real-time assays together with Platinum SYBR Green qPCR Supermix UDG (Life Technologies) 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 an iCycler (Bio-Rad). Cycling conditions were as follows: one cycle at 95°C for 5 min, then 40 cycles at 95°C for 15 s, and 61°C for 1 min. For each sample, plant and FDP DNAs were amplified in the same plate with their corresponding reaction mixture, together with the three standard dilutions of healthy host 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 iCycler software, v. 3.06070. Per-well baseline cycles were determined automatically. Specificity of the reaction was tested by running a melting curve analysis of the amplicons following each quantification reaction.

Data analysis

For the analysis of phytoplasma concentration, raw data (expressed as log values) were used and the concentration of FDP in each plant was expressed as the difference between the logarithm of the number of FDP cells and the logarithm of ng grapevine DNA (Dlog). FDP titres in cv. Barbera and Nebbiolo grapevines were analysed by GenStat v. 11.1 (VSN International Ltd).

anova and t-test were used to compare titre of FDP in samples, while the Mann–Whitney U-test was used to compare symptom severity. Spearman's rank correlation analysis was used to study relationships between symptom severity and FDP titre in spring, early and late summer samples of the two years.

Results

Symptom severity of infected grapevines and temperature parameters of the vineyards

The number of samples collected and symptom severity ratings of grapevine cultivars Barbera and Nebbiolo surveyed in vineyards during the spring, early summer and late summer of 2008 and 2009 are summarized in Table 1.

Table 1. Number of samples from cv. Barbera and Nebbiolo grapevines showing phytoplasma-specific symptoms of different categories collected during spring, early and late summer surveys of 2008 and 2009
Year Cultivar Springa Early summerb Late summerb
1 2 3 A B C D A B C D
2008 Barbera 1 9 5 2 9 5 0 2 4 4 0
Total plants 15 16 10
2009 Barbera 2 1 0 0 1 2 5 0 1 1 5
Total plants 3 8 7
2008 Nebbiolo 2 1 2 4 6 5 0 3 6 5 0
Total plants 5 15 14
2009 Nebbiolo 2 1 0 0 2 4 7 0 2 3 5
Total plants 3 13 10
  • a In spring, symptom severity of infected grapes was rated on a scale of 1–3 according to the percentage of shoots with symptoms on the fruit-bearing branch (1, <30%; 2, 30–65%; 3, >65%).
  • b In early and late summer surveys, symptom severity of infected grapes was rated as A, B, C or D, according to the proportion of the plant canopy showing phytoplasma-specific symptoms (A, plants lacking FD-specific symptoms, but not looking healthy; B, <30% FD-specific symptoms; C, 30–60%; D, >60%).

Overall, 59 cv. Barbera samples were collected in the two surveys of 2008 (n = 41) and 2009 (n = 18). Details on the number of plants showing symptoms in the different categories in the two years are presented in Table 1. For early summer samplings, in 2008 most plants showed class B and C symptoms, and in 2009 most of them showed class D symptoms. For late summer samplings in 2008, most of the plants showed class B and C symptoms, and in 2009 most of them showed class D symptoms (Table 1).

Overall, 60 cv. Nebbiolo samples were collected in 2008 (= 34) and 2009 (= 26). As for the cv. Barbera plants, details on the number of cv. Nebbiolo vines showing symptoms in the different categories in the two years are presented in Table 1. For early summer surveys, in 2008 none of the infected plants showed symptoms of class D, and in 2009 none of the plants showed symptoms of class A. For late summer surveys, in 2008 most of the plants showed symptoms of classes B and C, and in 2009 about 50% of the infected grapevines showed symptoms of class D (Table 1).

In the Cocconato vineyard, spring symptoms were more severe in cv. Barbera than in cv. Nebbiolo plants in both years (P = 0·991; Table 2). FD symptoms in cv. Barbera and Nebbiolo infected plants at Cocconato were more severe in 2009 than in 2008 (Barbera, early summer: = 0·999, late summer: = 0·996; Nebbiolo, early summer: = 0·975, late summer: = 0·990). In 2008, early summer symptoms at Cocconato were more severe on cv. Barbera than on Nebbiolo (= 0·999). In the same year, early summer symptoms on cv. Nebbiolo plants were more severe in the Monteu Roero vineyard compared to Cocconato (P = 0·999). The presence of BN phytoplasma in mixed infection with FDP in a few plants had no effect on symptom expression on either cv. Barbera or Nebbiolo (Table S1). Also FDP infection of the plant with -C or -D isolates did not influence symptom expression on either cv. Barbera or Nebbiolo (Table S1).

Table 2. Summary of significant to highly significant comparisons between flavescence dorée (FD) symptom severities in cv. Barbera (B) and Nebbiolo (N) grapevines in 2008 and 2009 at Cocconato (C) and Monteu Roero (MR)
Season Vineyard Cultivar Year(s) Comparison n U P
Spring C 2008 + 2009 B>N 17, 3 3·0 0·991
Early summer C B 2009>2008 15, 8 14·5 0·999
N 2009>2008 4, 4 0·0 0·986
2008 B>N 15, 4 2·0 0·999
MR N 2009>2008 11, 9 23·5 0·975
N 2008 MR>C 4, 11 0·0 0·999
Late summer C B 2009>2008 9, 7 7·5 0·996
MR N 2009>2008 8, 7 9·0 0·990
C N 2008 Early summer<Late summer 4, 4 0·0 0·986
  • n, sample number; U, value of U in the Mann–Whitney U-test; P, the exact probability (adjusted for ties) that one group is equal to, greater or smaller than the other group (according to the sign =, >, < specified in the table).

In the Monteu Roero vineyard, mean winter (November–March) and spring (April and May) temperatures in 2008 were 4·8 and 16·1°C, and in 2009 they were 4·1°C (winter) and 14·7°C (spring), with 63 (2008) and 79 (2009) freezing days during the winter. In the Cocconato vineyard, mean winter and spring temperatures in 2008 were 6·7 and 17·1°C, and in 2009 they were 5·6°C (winter) and 15·5°C (spring), with 41 (2008) and 49 (2009) freezing days recorded.

Phytoplasma detection and flavescence dorée strain characterization

Table 3 reports the number of cv. Barbera and Nebbiolo grapevines positive for the presence of FD and BN phytoplasmas alone or as mixed infection among the total samples collected in the surveys of 2008 and 2009. In spring surveys of 2008, almost all Barbera samples with symptoms were singly infected with FDP, and most Nebbiolo plants were doubly infected with FDP and BNP. In spring 2009, all plants with symptoms were singly infected with FDP, irrespective of cultivar identity and vineyard. In early summer surveys of 2008 and 2009, most cv. Barbera and Nebbiolo samples with symptoms were infected with FDP only, although BNP and FDP-doubly infected grapevines were found in both cultivars and vineyards. In 2008 and 2009 late summer surveys, most cv. Barbera and Nebbiolo vines with symptoms were infected with FDP in a single infection; few FDP and BNP mixed infections were also found in plants with symptoms sampled in late summer surveys of both years. BN phytoplasma was never detected as a single infection in grapevines analysed in any survey during either year.

Table 3. Number of cv. Barbera (B) and Nebbiolo (N) grapevines PCR positive for the presence of flavescence dorée phytoplasma (FDP) as either single or mixed infection with bois noir phytoplasma (BNP + FDP) among the total samples collected from vineyards in Cocconato and Monteu Roero during spring (Sp), early (Es) and late summer (Ls) growing seasons of 2008 and 2009
Year Vineyard Cultivar FDP (positive/total sampled) BNP+FDP (positive/total sampled)
Sp Es Ls Sp Es Ls
2008 Cocconato B 14/15 15/16 8/10 1/15 1/16 2/10
Cocconato N 0/1 3/4 2/4 1/1 1/4 2/4
Monteu Roero N 1/4 7/11 9/10 3/4 4/11 1/10
2009 Cocconato B 3/3 8/8 6/7 0/3 0/8 1/7
Cocconato N 2/2 4/4 3/3 0/2 0/4 0/3
Monteu Roero N 1/1 8/9 6/7 0/1 1/9 1/7

Overall, in the Cocconato vineyard, only FD-C type phytoplasmas (16S/secY;= 17) were found in infected cv. Nebbiolo plants sampled in the two years. FD-C phytoplasmas were detected in most cv. Barbera samples collected in all surveys (= 56), and three FD-D isolates were detected in 2008 surveys. FDP isolates from one cv. Nebbiolo and six cv. Barbera vines from the Cocconato vineyard could not be assigned to a type isolate. In the Monteu Roero vineyard, FD-C (= 25) and -D (= 11) phytoplasmas were both present in the surveys of 2008 and 2009. FDP isolates from six grapevines from this vineyard could not be properly assessed.

Quantification of flavescence dorée phytoplasma

The following primers were designed for the absolute quantification of FDP and grapevine DNA: FdSecyFw/FdSecyRv (5′-TGCCTTATGTTACTGCTTCT-3′/5′-TAATAATGATGGGGATTCAA-3′), and Vitis18SF1/Vitis18SR1 (5′-ATGATTAACAGGGACAGTCG-3′/5′-GGTATCTGATCGTCTTCGAG-3′). blast analysis of primers FdSecyFw/FdSecyRv, flanking a 185 bp region of the FD-70 isolate secY gene (accession number: AM238512), showed their specificity for the target gene of several ‘Ca. Phytoplasma vitis’ isolates and phytoplasmas belonging to other 16SrV subgroups. These primers did not match any stolbur or ‘Ca. Phytoplasma asteris’-related sequences, therefore preventing non-specific amplification from two phytoplasmas commonly infecting grapevine. The FdSecyFw/FdSecyRv amplified a phytoplasma-specific amplicon of the expected size only from FD-C and FD-D infected periwinkle DNA, and gel electrophoresis confirmed the absence of non-specific products (not shown). Melting curve analysis of the amplicon obtained with FdSecyFw/FdSecyRv showed a unique melting peak at 76·5°C (Fig. 1c). The Vitis18SF1/Vitis18SR1 primers amplified a grapevine-specific amplicon of the expected size (152 bp) and gel electrophoresis confirmed the absence of non-specific products (not shown). Melting curve analysis of the amplicon showed a unique melting peak at 85·5°C (Fig. 1d). Serial dilutions of FDP plasmid DNA ranging from 1·17 × 108 to 1·17 × 103 copies of plasmid were tested in triplicate and the Ct values were plotted against the copy number. Serial dilution of healthy grapevine DNA ranging from 50 to 0·05 ng were also tested in triplicate and the Ct values were plotted against the DNA concentration. The linear correlation coefficient (r) between the Ct and the logarithm of the DNA copy number was repeatedly greater than 0·995 for the amplification of both targets (Fig. 1a,c). PCR efficiencies of both curves were constantly around 108 and 103% (plant and phytoplasma, respectively).

Details are in the caption following the image
(a) Standard curve of flavescence dorée phytoplasma DNA obtained by plotting threshold cycle (Ct) values vs log of 2 × 107, 2 × 105, 2 × 103 and 2 × 10 copy number of p26SecYFD plasmid; (b) standard curve of plant DNA (Vitis vinifera) obtained by plotting threshold cycle (Ct) values vs log of 50, 5, 0·5 and 0·05 ng of healthy grapevine DNA; (c) melting curve analysis of the amplicon obtained with primers FdSecyFw/FdSecyRv; (d) melting curve analysis of the amplicon obtained with primers Vitis18SF1/Vitis18SR1.

Establishment of a correct sampling procedure

Results of this preliminary experiment are presented in Table 4. FD phytoplasma was detected by nested PCR in all samples from cv. Barbera and Nebbiolo shoots with symptoms, confirming the infected status of the analysed plants. FD phytoplasma was also detected in three samples from symptomless shoots of two cv. Barbera plants, while it was never detected in symptomless shoots of infected cv. Nebbiolo plants. FD phytoplasma titre was measured in all samples from cv. Barbera shoots with symptoms and in two nested PCR positive samples from the two shoots of one cv. Nebbiolo plant with symptoms. FD phytoplasma titre in shoots of the five cv. Barbera plants with symptoms ranged from about 39–1180 cells ng−1 grapevine DNA, and it was above the quantification threshold in one of the three nested PCR positive samples from symptomless cv. Barbera shoots. In cv. Barbera, the variance among FDP titre within shoots of each plant was lower than that among plants (Table 5), therefore collection of leaves from one shoot with symptoms per plant was set as sampling protocol for successive quantification experiments.

Table 4. Number of shoots with and without symptoms from the five cv. Barbera and five cv. Nebbiolo flavescence dorée (FD)-infected grapevines from which samples were collected for successive diagnostic direct (P1/P7 primers) and nested (R16(V)F1/R1 primers) PCRs and FDP quantification. Only samples with a threshold cycle value within that of the lowest standard concentration were considered for FDP quantification
Cultivar Symptoms Shoots (n) Direct PCR (positive/tested) Nested PCR (positive/tested) qPCR (positive/tested) FDP titre (mean)a SE
Barbera Yes 11 9/11 11/11 11/11 345·1 103·94
No 9 1/9 3/9 1/9 9·6
Nebbiolo Yes 7 1/7 7/7 2/7 45·3 5·39
No 13 0/13 0/13 0/13
  • a Titre of FDP is expressed as phytoplasma cells ng−1 plant DNA.
  • SE: standard error.
Table 5. Mean flavescence dorée phytoplasma (FDP) titre (expressed as the difference between the logarithm of the number of FDP cells and the logarithm of ng grapevine DNA, DLog) in shoots with symptoms of five cv. Barbera plants, variance among FDP titres within shoots of each plant and among plants (general, among plants)
Plant Mean FDP titre in shoots (DLog) Variance
1 2·178167 0·07131606
2 2·1135 0·0772245
3 2·850333 0·09827222
4 2·134833 0·0981245
5 2·7865 0·00098272
General, among plants 2·412667 0·16141599

Flavescence dorée phytoplasma concentration in Barbera and Nebbiolo grapevines

Despite the low number of FD-D phytoplasma infected plants over the two years, tests indicated that no significant differences were recorded in the concentration of FD-C and -D phytoplasmas within each sampling of the different cultivars (Table 6). Further tests also showed that the presence of BNP had no effect on FDP concentration (Table 7). FDP concentration in cv. Barbera grapevines sampled in spring, early and late summer of 2008 and 2009 are illustrated in Figure 2. Mean FDP concentration in Barbera samples was 242 cells ng−1 grapevine DNA in spring 2008, and 2 cells ng−1 grapevine DNA in spring 2009. In early summer samplings, FDP titre was 8856 and 8735 cells ng−1 grapevine DNA in 2008 and 2009, respectively; in late summer, FDP concentration was 6420 cells ng−1 grapevine DNA in 2008 and 671 in 2009. Because of significant differences in the variance, FDP titres in spring and early summer samplings were analysed separately from those of late summer. Significant differences were recorded in FDP titre between sampling seasons and years. FDP concentration was consistently higher in 2008 than in 2009 (3·299 vs 2·456 Dlog; n1 = 31; n2 = 10; SED = 0·2799; least significant difference, LSD, at 0·05 level = 0·5672), and the difference was mainly due to phytoplasma titre in spring, because differences between FDP titre in 2008 and 2009 early summers were not significant (LSD at 0·05 level between two years: spring = 0·0618; early summer = 0·6108). FDP concentration was also significantly higher in 2008 late summer sampling compared to 2009 (P = 0·072). A seasonal trend in FDP concentration (low in spring, high in early summer and intermediate in late summer) was conserved in cv. Barbera vines sampled in both years (Fig. 2).

Table 6. Analysis of variance (anova) for grouping FD-C and FD-D phytoplasma titres of cv. Barbera (B) and Nebbiolo (N) plants sampled at different times in the two vineyards
Cultivar Vineyard Year Season Log[FD-C] (mean) n C Log[FD-D] (mean) n D F pr LSD Comment
B C 2008 Early summer 4·0242 14 3·640 1 0·436 1·034 NS
Late summer 3·8911 7 3·785 1 0·714 0·6746 NS
N MR 2008 Early summer 3·2713 4 2·7967 3 0·206 0·8409 NS
2009 Early summer 2·7845 6 3·5642 2 0·315 1·740 NS
Late summer 3·0948 5 2·551 1 0·397 1·594 NS
  • C, MR: Cocconato and Monteu Roero vineyards, respectively.
  • Log[FD-C], Log[FD-D]: Log of FD-C or -D phytoplasma concentration, respectively.
  • nC, nD: number of samples infected by FD-C or FD-D phytoplasmas, respectively.
  • F pr: F probability; LSD: least significant difference; NS: not significant.
Table 7. Analysis of variance (anova) for grouping flavescence dorée phytoplasma (FDP) titres in FD singly infected and FD and bois noir (BN) doubly infected cv. Barbera (B) and Nebbiolo (N) plants sampled at different times in the two vineyards
Cultivar Vineyard Year Season Log[FD]FD (mean) n FD Log[FD]FD+BN (mean) n FD+BN F pr LSD Comment
B C 2008 Spring 2·384 14 2·370 1 0·989 2·204 NS
Early summer 4·0242 14 3·1773 1 0·100 1·034 NS
2009 Late summer 2·935 6 2·179 1 0·603 3·498 NS
N C+MRx 2008 + 2009 Spring 0·820 4 2·366 2 0·165 2·531 NS
C+MRy 2008 Early summer 3·2644 7 3·3878 4 0·651 0·5955 NS
  • C, MR: Cocconato and Monteu Roero vineyards, respectively.
  • Log[FD]FD, Log[FD]FD+BN: log of FD phytoplasma concentration.
  • nFD, nFD+BN: number of samples infected only by FD or by FD and BN in mixed infection, respectively.
  • F pr: F probability; LSD: least significant difference; NS: not significant.
  • x, y: groups correspond to same symbols in Table 8.
Details are in the caption following the image
Mean flavescence dorée phytoplasma concentration (cells/ng grapevine DNA) and standard error in cv. Barbera and Nebbiolo plants sampled in spring, early and late summer surveys of 2008 and 2009 in Cocconato (C) and Monteu Roero (MR) vineyards.

Flavescence dorée phytoplasma titre in cv. Nebbiolo vines (Fig. 2) was significantly different according to year (2008 and 2009) and sampling season (spring, early and late summer), while it was similar in both vineyards (Table 8). Mean FDP titre in cv. Nebbiolo vines was 134, 1417 and 489 cells ng−1 grapevine DNA in spring, early summer and late summer samples of 2008, respectively. In 2009, mean FDP titre was 2, 545 and 334 cells ng−1 grapevine DNA in spring, early summer and late summer samples, respectively. All data were then pooled to analyse differences between years and sampling seasons. Overall, in 2008, FDP in cv. Nebbiolo was more abundant than in 2009 (2·837 vs 2·310 Dlog; n1 = 32; n2 = 26; SED = 0·1819; LSD at 0·05 level = 0·3651), and the difference was mainly due to the concentration of the spring samples, because differences between the remaining seasons were not significant (LSD at 0·05 level between single seasonal samplings of the two years: spring = 1·0078; early summer = 0·5229; late summer = 0·5909). There was no difference in the FDP titre of early and late summer samples of the two years (LSD at 0·05 level between early and late summer 2008 = 0·5345 and between early and late summer 2009 = 0·5805), but a seasonal trend in FDP concentration (low in spring, high in early summer and intermediate in late summer) was conserved for cv. Nebbiolo in both years and vineyards (Fig. 2).

Table 8. Analysis of variance (anova) for grouping flavescence dorée phytoplasma (FDP) titres from infected cv. Nebbiolo plants collected in two vineyards
Infectiona Year Season Log[FD]C(mean) n C Log[FD]MR(mean) n MR F pr LSD Comment
FD-C 2008 + 2009x Spring 0·349 2 1·291 2 0·549 5·681 NS
2008y Early summer 3·255 3 3·271 4 0·962 0·826 NS
FD-C + BN 2008y Early summer 2·942 1 3·536 3 0·386 2·324 NS
  • a Plants infected with FDP (FD-C) or FDP and BNP (FD-C + BN) in double infection were analysed separately.
  • Log[FD]C, Log[FD]MR: log of FDP titre in plants collected at Cocconato or Monteu Roero vineyards, respectively.
  • nC, nMR: number of infected samples collected at Cocconato or Monteu Roero vineyards, respectively.
  • F pr, F: probability; LSD: least significant difference; NS: not significant.
  • x, y: groups correspond to same symbols in Table 7.

Flavescence dorée phytoplasma concentration was always higher in cv. Barbera than in cv. Nebbiolo infected vines, and this difference was significant at early and late summer samplings of 2008 (early summer: = 0·008; late summer: = 0·002) and at early summer sampling of 2009 (P < 0·001; Fig. 2).

Flavescence dorée phytoplasma concentration and symptom severity

Considering both cvs Barbera and Nebbiolo and both years from both vineyards, the spring samples showed a significant, positive correlation between FDP concentration and symptom severity (Spearman's correlation coefficient r = 0·412; adjusted for ties = 0·367; = 25; d.f. = 23; t = 1·89; P = 0·071), and so did the cv. Barbera samples of 2008 (Spearman's correlation coefficient r = 0·389; = 25; d.f. = 23; t approximation 1·72; P = 0·099), but not 2009, and the cv. Nebbiolo samples of 2009 (Spearman's correlation coefficient r = 0·430; adjusted for ties r = 0·373; n = 22; d.f. = 20; t = 1·98; P = 0·087), but not 2008. Early and late summer samples were analysed together for correlations between FD concentration (as log(FD)) and symptom severity as the two samplings were evaluated on the same severity scale. No other significant correlation was detected. Overall, there was no highly significant correlation between FDP concentration and symptom severity.

Discussion

Flavescence dorée infection in 2008 and 2009 in the two vineyards of Cocconato and Monteu Roero was caused mainly by flavescence dorée type C phytoplasma (FD-C). The presence of this isolate has been stable in the area since the early outbreak of the epidemics in 1998, and it is the most represented one in FD-infected vines of the Piemonte region (Marzachi et al., 2001). FD-D type phytoplasmas were detected sporadically in infected cv. Barbera and Nebbiolo grapevines collected in spring, early and late summer of both years. Grapevines showing early symptoms were mostly infected with FD-C phytoplasma, as reported in other grapevine-growing areas (Angelini et al., 2006), and this isolate was detected throughout the vegetative season. Bois noir disease is also present in Piemonte vineyards (Marzachi et al., 2001), and BNP was found only in mixed infections with FDP in samples from the two vineyards. As only grapevines with symptoms were sampled in the two highly FD-infected locations, the presence of BNP in symptomless plants cannot be ruled out.

For two consecutive years, infected cv. Barbera vines showed more severe spring symptoms than cv. Neb-biolo. This observation coincides with the fact that cv. Barbera is known as a highly FD-sensitive cultivar. Both cultivars showed more severe symptoms in 2009 than in 2008, and in 2009 lower winter and spring mean temperatures and more freezing days were recorded at both localities than in 2008. Low spring temperatures have an immediate effect on grapevine in delaying bud burst (Tomasi et al., 2007), but they also have a long term effect in reducing fruit set (%) and berry number per bunch in a cultivar-specific manner (Ebadi et al., 1995). Temperature stresses also affect Ca2+ homeostasis in grapevine (Wang et al., 2004). Also, low temperatures reduce respiration rates in grapevine (Franck et al., 2011), thus reducing the production of H2O2 (Foyer et al., 2009), a molecule probably involved in defence response pathways to phytoplasmas (Musetti et al., 2007). One can speculate that more severe FD symptoms in both cultivars in early and late summer assessment of 2009 may be an effect of a long-lasting stress of the plant caused by the occurrence of low winter/spring temperatures in that year. Moreover, in 2008, temperature probes recorded lower winter/spring mean temperatures and more freezing days in the Monteu Roero vineyard compared to Cocconato, and significantly more severe early summer symptoms were recorded in cv. Nebbiolo infected vines in Monteu Roero than in Cocconato.

Quantification of FDP DNA was obtained as the ratio of phytoplasma DNA per ng of grapevine DNA as previously described for ‘Ca. Phytoplasma asteris’ (Marzachi & Bosco, 2005). Real-time based PCR assays for diagnosis and characterization of several phytoplasmas, including FDP, have been proposed (Bianco et al., 2004; Galetto et al., 2005; Angelini et al., 2007; Hren et al., 2007; Pelletier et al., 2009; Mehle et al., 2013), but this is one of the first attempts, together with a study by Prezelj et al. (2012), to quantify FDP in field-infected vines of different cultivars. A double absolute quantification assay was developed and only samples with a threshold cycle value within that of the lowest standard concentration were considered. The quantification of less than three phytoplasma cells per ng plant DNA in three spring samples can still be considered reliable according to the MIQE guidelines (Bustin et al., 2009) as it exceeds by three times the most sensitive limit of detection.

It is known that phytoplasmas have uneven distribution in the grapevine canopy, but a direct correlation between the presence of phytoplasma-specific symptoms and FDP detection was clearly shown as all samples collected from shoots with symptoms of both cultivars were positive for the presence of FDP, while all samples from symptomless cv. Nebbiolo and most from symptomless cv. Barbera were nested PCR negative. Similar results are reported for other grapevine varieties (Prezelj et al., 2012) where FDP was mainly associated with tissues with symptoms, although it was also detected in some symptomless tissues of highly infected (with symptoms) plants. Also, the differences between FDP titre in different shoots with symptoms from the same cv. Barbera plant were lower than the differences among plants, therefore confirming that sampling from one shoot with symptoms can provide a reliable and representative evaluation of FDP titre in the infected parts of the plant.

In both cultivars and in both years, the lowest FDP titre was observed in spring, the highest in early summer and an intermediate one at the end of the vegetative season. A continuous increase of FDP titre up to the end of the vegetative season was reported for other grapevine cultivars in Slovenia (Prezelj et al., 2012). Seasonal fluctuations of phytoplasma populations in apple trees infected with apple proliferation phytoplasma have been measured with highest levels occurring from December to May (Baric et al., 2011). Despite the occurrence of more severe symptoms throughout the vegetative season of 2009 compared to 2008, at each sampling date FDP titre was significantly higher in 2008 than in 2009, irrespective of the cultivar. This difference was most evident in the spring samplings, when as many as hundreds of FDP cells per nanogram plant DNA were recorded in 2008 in contrast to only as few as two in 2009. Because the lowest FDP titre was consistently measured in both cultivars, both years and at both sites in spring, the winter temperature together with plant dormancy and pruning, that eliminates most of the infected plant parts, have a cooperative effect in reducing phytoplasma titre in the plant. Multiplication of pathogen resumed with the vegetative season and the differences in FDP titres between the two years were reduced, although FDP-specific symptom expression was more severe in 2009 than in 2008. One can speculate that colder winter/early spring climatic conditions have a long-lasting stress effect on the grapevines, increasing both their sensitivity to FD and symptom severity even in the presence of a low pathogen load. Also, the hypothesis that the pathogen may multiply less efficiently in the stressed plant, therefore reaching a low titre despite the severe symptoms, cannot be ruled out.

Flavescence dorée phytoplasma titre in cv. Barbera grapevines was always higher than in cv. Nebbiolo. This correlates with more severe symptoms expressed by FD-infected cv. Barbera vines compared to cv. Nebbiolo, and provides the most likely explanation for the milder symptoms shown by FDP-infected Nebbiolo. Actually this latter cultivar sustains a low multiplication of the pathogen. Proteomic and transcriptomic analyses of FDP-infected cv. Nebbiolo plants show that most proteins modulated during infection belong to the ‘cell rescue, defence and virulence’ class (Margaria & Palmano, 2011a). On the other hand, preliminary studies on cv. Barbera show a lower presence of proteins of the ‘defence’ category compared to the total identified proteins (Margaria & Palmano, 2011b). Perhaps different responses of the two cultivars to FDP may account for the different multiplication level of the pathogen, but this hypothesis remains untested.

The results indicate that the best time for sampling and detection of FDP infection is early summer, when phytoplasma titre is highest. However, this work, together with that of Prezelj et al. (2012), provides evidence that FDP can be detected as early as at flowering season, or even before in the case of cv. Barbera in the present study. Quantitative real-time PCR is a useful tool for the evaluation of phytoplasma multiplication and can be used when screening for resistance/tolerance. This study has demonstrated that cv. Barbera supports a higher FDP multiplication compared to cv. Nebbiolo, and further studies are required to understand whether this has an effect on vector acquisition and transmission efficiency, as already suggested for Pinot blanc and Merlot cultivars (Bressan et al., 2005).

Acknowledgements

The authors thank the wine growers L. Negro and P. Bava for allowing the collection of samples in their vineyards, F. Spanna (Piemonte Region, Settore Fitosanitario) for providing climatic values at both locations, and C. Lovisolo (University of Torino) for his critical reading of the manuscript. This work was supported by the Italian Regional Grant (Piemonte Region) ‘Studi sui fattori che favoriscono le epidemie di Flavescenza dorata in Piemonte e loro superamento’, and by the Piemonte Region CIPE ‘Adoption of a multidisciplinary approach to study the grapevine agroecosystem: analysis of biotic and abiotic factors able to influence yield and quality’.