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PROGETTO TERMOVIT

FITOPLASMI INDIVIDUATI IN
VITI TERMOTRATTATE
Paola Casati
Fabio Quaglino
Riccagioia, 31-01-2012
Influenza della T sulla sopravvivenza delle
talee termotrattate
Piante ottenute dalle talee
termotrattate
VARIETA’

NT

A

B

C

TOTALE

barbera

119

103

70

23

315

chardonnay

113

93

46

44

296

croatina

78

77

71

37

263

pinot nero

100

41

22

23

186

riesling italico

122

67

46

38

273

riesling renano

69

70

22

22

183

uva rara

86

48

78

21

687

499

355

208

233
1749
Non
trattato

Tesi A
48°C
45 min

Tesi B
50°C
45 min

Tesi C
52°C
30 min

•Assenza di sintomi su piante non trattate e trattate
•Assenza di sintomi non significa assenza di fitoplasmi
•Numerosi casi di infezioni latenti
IDENTIFICAZIONE dei
FITOPLASMI
Per valutare l’efficacia della termoterapia,
tutte le viti sopravvissute al trattamento
devono essere analizzate

SCELTA DELLA TECNICA DA
IMPIEGARE
COSA SONO I FITOPLASMI?
parassiti endocellulari obbligati
localizzati nelle cellule floematiche della pianta
non coltivabili in vitro

Nessuna possibilità di isolamento
Metodi di Biologia Molecolare: basati sul
riconoscimento e l’amplificazione di porzioni
specifiche del DNA del fitoplasma
Estrazione DNA da
nervature/piccioli

ACCTGGCTGATGTGTGTAATGTGCCTGTATGCC
TGGACCGACTACACACATTACACGGACATACGG
PCR
ACCTGGCTGATGTGTGTAATGTGCCTGTATGCC
TGGACCGAC
Primer
Primer
CTGTATGCC
TGGACCGACTACACACATTACACGGACATACGG

ACCTGGCTGATGTGTGTAATGTGCCTGTATGCC
TGGACCGACTACAGAC…………
T
C

A C

G

G

T G A

GTGTAATGTGC CTGTATGCC
TGGACCGACTACACACATTACACGGACATACGG
Real-time PCR

ACCTGGCTGATGTGTGTAATGTGCCTGTATGCC
TGGACCGAC
primer
primer
sonda

CTGTATGCC
TGTGTAATG
TGGACCGACTACACACATTACACGGACATACGG
IDENTIFICAZIONE dei
FITOPLASMI
Preparazione dei campioni per l’estrazione del DNA

Campioni: piccioli raccolti da 5-7 piante della stessa varietà,
sottoposte alle stesse condizioni di termoterepia
IDENTIFICAZIONE dei
FITOPLASMI
VARIETA’

NT

A

B

C

TOTALE

barbera

119

103

70

23

315

chardonnay

113

93

46

44

296

croatina

78

77

71

37

263

pinot nero

100

41

22

23

186

riesling italico

122

67

46

38

273

riesling renano

69

70

22

22

183

uva rara

86

48

78

21

687

499

355

208

233
1749

265 campioni (circa 1300 piante di vite)
Estrazione del DNA
IDENTIFICAZIONE dei
FITOPLASMI
Real-time PCR condotta impiegando primers e
sonde, specifiche per l’identificazione dei
fitoplasmi di FD e LN

Primers, sonde e condizioni di reazione
ottimali sono state sviluppati nel corso del
progetto DIACERVIT, finanziato dalla
Regione Lombardia (responsabile scientifico
Dott.ssa Casati)
Analisi real-time PCR
Amplificazione
associata a curva di
emissione di
fluorescenza
IDENTIFICAZIONE dei
FITOPLASMI
Analisi ripetute negli anni 2009-10-11
% infezione calcolate su campioni
infetti almeno una volta nel triennio
(possibili infezioni latenti; morte di
piante infette)
IDENTIFICAZIONE dei
FITOPLASMI
Varietà

Tesi

% infezione

Barbera

NT
A (48°C x 45 min)
B (50°C x 45 min)
C (52°C x 30 min)
NT
A (48°C x 45 min)
B (50°C x 45 min)
C (52°C x 30 min)

0
0
9
0
47
0
0
0

Chardonnay

FD
LN
IDENTIFICAZIONE dei
FITOPLASMI
Varietà

Tesi

% infezione

Croatina

NT
A (48°C x 45 min)
B (50°C x 45 min)
C (52°C x 30 min)
NT
A (48°C x 45 min)
B (50°C x 45 min)
C (52°C x 30 min)

75
13
8
14
54
17
0
0

Pinot nero

FD e LN
LN
LN
LN
FD e LN
FD
IDENTIFICAZIONE dei
FITOPLASMI
Varietà
Riesling Italico

Tesi

NT
A (48°C x 45 min)
B (50°C x 45 min)
C (52°C x 30 min)
Riesling Renano
NT
A (48°C x 45 min)
B (50°C x 45 min)
C (52°C x 30 min)
Uva rara
NT
A (48°C x 45 min)
B (50°C x 45 min)
C (52°C x 30 min)

% infezione
69
30
66
0
89
15
50
0
64
0
53
0

LN
FD e LN
FD e LN
FD e LN
FD e LN
LN
LN
FD e LN
IDENTIFICAZIONE dei
FITOPLASMI
Tesi
% infezione Fitoplasmi
NT
60
FD e LN
A (48°C x 45 min)
9
FD e LN
B (50°C x 45 min)
26
FD e LN
C (52°C x 30 min)
3
LN
Percentuale di FD e LN in vite
% infezione
FD

% infezione
LN
DISCUSSIONE e CONCLUSIONI

•Sopravvivenza piante dipende dalla
temperatura utilizzata
•% infezione piante NT superiore a
% infezione delle piante trattate
tesi
NT
A
B
C

% sopravvissute
65
47
33
19

% infezione
60
9
26
3
DISCUSSIONE e CONCLUSIONI
•Soltanto il trattamento a 52°C x 30
min elimina FD
•Più difficile eliminare LN
•Scarsa presenza di FD in materiale
di partenza non trattato
DISCUSSIONE e CONCLUSIONI

•Termoterapia sempre efficace per
Chardonnay
•Diversa efficacia delle condizioni di
trattamento verso le varietà
esaminate
Grazie per l’attenzione
Varietà

Tesi

2009
N. campioni N. piante
totalia
infetti

Croatina

A
B
C
Pinot nero
A
Riesling Italico A
B
Riesling Renano
A
B
Uva rara
B

Varietà
Barbera
Chardonnay
Croatina
Pinot nero
Riesling I
Riesling R
Uva rara
TOTALE

2
1
1
1
3
6
2
2
7

A
80
70
73
30
47
63
44
407

infette
0
0
7
5
9
7
28
56

10
4
5
5
14
34
11
12
35

%
0
0
9
17
19
11
64
14

2010 b
N. campioni
infetti
1
1
1
1
1
1
0
1
2

B
53
49
57
22
44
22
67
314

2011 c
N. piante

N. piante
totali
8
2
4
3
9
19
6
10
21

infette
0
0
3
0
26
10
0
39

morte
2
2
1
2
5
15
5
2
14

%
0
0
5
0
59
45
0
12

infette
4
1
1
2
2
8
1
7
14

C
22
38
36
19
30
25
18
188

totali
7
2
4
2
7
16
5
9
21

infette
0
0
2
0
0
0
0
2

morte
1
0
0
1
2
3
1
1
0

%
0
0
5
0
0
0
0
1
IDENTIFICAZIONE dei FITOPLASMI

0.6 um

M.E. TEM

Microscopia elettronica: sezione del floema di vite infetta.
Tempi molto lunghi, scarsa sensibilità e specificità, costi
elevati.
IDENTIFICAZIONE dei FITOPLASMI

DNA con sequenze
specifiche
Inserzione di sonde marcate che
riconoscono il DNA del fitoplasma

Microscopia confocale: sezione del floema di vite infetta.
Tempi molto lunghi, ottima specificità, bassa sensibilità e
costi elevati.
IDENTIFICAZIONE dei FITOPLASMI
Varietà
Barbera

Chardonnay

Croatina

Pinot nero

a
Tesi N. Campioni

NT
A
B
C
NT
A
B
C
NT
A
B
C
NT
A
B
C

8
17
11
4
13
15
11
8
8
16
13
7
13
6
4
4

Positivi a real-time PCR % infezione
FD
LN
FD + LN
0
0
0
0
0
0
0
0
1
0
0
9
0
0
0
0
0
8
0
47
0
0
0
0
0
0
0
0
0
0
0
0
0
5
1
75
0
2
0
13
0
1
0
8
0
1
0
14
0
6
1
54
1
0
0
17
0
0
0
0
0
0
0
0
IDENTIFICAZIONE dei FITOPLASMI
Varietà
Riesling Italico

a
Tesi N. Campioni

NT
A
B
C
Riesling Renano NT
A
B
C
Uva rara
NT
A
B
C

13
10
9
6
9
13
4
5
11
10
13
4

Positivi a real-time PCR % infezione
FD
LN
FD + LN
0
9
0
69
2
1
0
30
4
1
1
66
0
0
0
0
1
7
0
89
1
1
0
15
0
2
0
50
0
0
0
0
0
7
0
64
0
0
0
0
1
6
0
53
0
0
0
0
Varietà

Tesi

Croatina

A
B
C
A
A
B
A
B
B

Pinot nero
Riesling Italico
Riesling Renano
Uva rara

2009
2010 b
N. campioni N. piante N. campioni
N. piante
totalia
infetti
infetti
totali
morte
2
10
1
8
2
1
4
1
2
2
1
5
1
4
1
1
5
1
3
2
3
14
1
9
5
6
34
1
19
15
2
11
0
6
5
2
12
1
10
2
7
35
2
21
14

Variazione del numero dei campioni
infetti nel tempo

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Quaglino, casati fitoplasmi individuati in viti termotrattate