Full text of DOI:10.1111/j.1744-7348.2003.tb00252.x

Ann. appl. Biol. (2003), 142:285-290
285
Printed in UK
Epidemiology of apple proliferation (AP) in northwestern Italy: evaluation of
the frequency of AP-positive psyllids in naturally infected populations of
Cacopsylla melanoneura (Homoptera: Psyllidae)
By ROSEMARIE TEDESCHI, CRISTINA VISENTIN, ALBERTO ALMA and DOMENICO BOSCO*
Di.Va.P.R.A., Entomologia e Zoologia applicate all’Ambiente “Carlo Vidano”, Università degli Studi di
Torino, via L. Da Vinci 44, I-10095 Grugliasco (TO), Italy
(Accepted 28 November 2002; Received 19 August 2002)
Summary
Adults of Cacopsylla melanoneura, vector of the apple proliferation (AP) phytoplasma, were collected
every 2 weeks from January until May in 2000 and 2001 by the beating tray method in eight apple
orchards of the Aosta Valley (northwestern Italy). Total DNA was extracted from batches of five
insects and amplified with the universal phytoplasma primers P1/P7 in direct PCR. A nested PCR
assay was then performed on P1/P7 amplicons using the primers fO1/rO1, specific for the AP-
phytoplasma group. The digestion of fO1/rO1 amplicons with SspI restriction endonuclease confirmed
that C. melanoneura adults harboured the AP phytoplasma. The data obtained with PCR were used to
estimate the proportion of AP-positive insects in overwintered and offspring adults. Percentages of
AP-positive insects of 3.6% and 0.8% were estimated in 2000 among overwintered and offspring
psyllids respectively. In 2001 only the overwintered insects were found infected, with an estimated
proportion of 2.8%. The seasonal abundance of the vector was measured using yellow sticky traps. C.
melanoneura was always present at a low population level, and the highest density was recorded from
mid-February until mid-March in both years. The results show that the overwintered population is
higher and spends a longer period in apple orchards, suggesting the crucial role of the overwintered
adults in vectoring AP.
Key words: AP-phytoplasma, Cacopsylla melanoneura, apple, epidemiology, PCR detection
Introduction
(Tedeschi et al., 2002) have been shown to vector
apple proliferation (AP).
Phytoplama-associated diseases are among the
most important threats of woody plants in Europe,
especially of grapevine, pome and stone fruits. The
role of insect vectors in spreading the disease is of
major importance since phytoplasmas, unlike many
plant viruses, are not mechanically transmitted.
Phytoplasma inoculum can be introduced by both
infected propagation material and by infective
vectors, then the further spread of the disease is due
to the feeding activity of phloem-sucking insects.
Psyllids have a crucial role in the transmission of
phytoplasmas belonging to the phylogenetic apple
proliferation cluster which comprises the most
important fruit tree phytoplasmas widespread in
temperate European regions. Cacopsylla pyricola
(Förster) in England (Jensen et al., 1964) and C. pyri
(L.) in France (Lemoine, 1991) and Italy (Carraro
et al., 1998) have been reported as vectors of the
Pear Decline (PD) agent; C. pruni (Scopoli) is able
to transmit the European stone fruit yellows (ESFY)
(Carraro et al., 2001a); while C. costalis in
northeastern Italy (Frisinghelli et al., 2000) and C.
melanoneura (Förster) in northwestern Italy
Molecular tools now enable the sensitive detection
of phytoplasmal DNA in insects (Bosco et al., 2002b)
and they can be usefully applied also for the
screening of insect field populations. Phytoplasma
detection in field-collected insects has been used to:
i) search for new, unknown vectors (Hill & Sinclair,
2000); ii) study seasonal or geographic distribution
of infective vectors (Rahardja et al., 1992; Carraro
et al., 2001b); iii) predict phytoplasma disease
outbreaks (Goodwin et al., 1999); iv) monitor for
vector pressure (Weber & Maixner, 1998).
The aim of our research was to assess the seasonal
distribution of the vector C. melanoneura, and the
identification of a crucial period in which AP-
positive psyllids are present in apple orchards, thus
potentially transmitting the disease.
Materials and Methods
Insect samplings and population dynamics
To set up a protocol for the detection of AP
phytoplasmas in C. melanoneura samples,
preliminary assays were carried out using overwintered
*Corresponding Author E-mail: domenico.bosco@unito.it
© 2003 Association of Applied Biologists

286
R TEDESCHI ET AL.
adults collected in an old apple orchard located in
the province of Torino, northwestern Italy, where
all plants have been infected byAP for several years.
A total number of 40 insects (28 females and 12
males) were collected and singly tested.
P7 (Schneider et al., 1995), then the amplicons were
diluted 1:40 and subjected to nested PCR assays
using the AP-group specific primer pair fO1/rO1
(Lorenz et al., 1995). Reaction and cycling
conditions were as in the original papers for both
primer pairs. The PCR products were analysed by
electrophoresis through 1% agarose gel, stained with
ethidium bromide and visualised on an UV
transilluminator. The specific amplification of the
apple-infecting AP-phytoplasma was attained by
RFLP analysis as described in Lorenz et al. (1995).
The proportion of infected insects in overwintered
and offspring groups was estimated by its
The investigations were conducted in the Aosta
Valley, a region where disorders due to AP
phytoplasmas have been repeatedly observed in the
last few years. Eight apple orchards with different
ages and different degrees of infection were chosen
(Tedeschi et al., 2002). Living psyllids were
collected every 2 wk from the end of January until
the end of May in 2000 and 2001 by the beating tray
method. A white plastic tray (250 mm × 350 mm)
was held under tree branches and the branches were
struck several times with a stick to make the insects
fall and be collected. Adults were then identified in
the laboratory under the stereomicroscope.
ˆ
maximum-likelihood estimator, p, calculated
1/k
ˆ
according with Swallow (1985): p = 1 – H , where
H is the observed fraction of healthy groups and k is
the number of insects per group, five in this case.
During the same periods, the population dynamics
of C. melanoneura was monitored by yellow sticky
traps (145 mm × 205 mm) (Forschungsanstalt
Wadenswil, Switzerland). Three yellow sticky traps
per orchard were hung 1.5 m above the ground on a
diagonal line in the eight apple orchards. The traps
were changed weekly and insects were identified
and counted under a stereomicroscope.
Results
Insect population dynamics and plant symptoms
The average (± SEM) number of psyllids sampled
by yellow sticky traps in the eight apple orchards in
2000 and 2001 was calculated. The results (Fig. 1)
revealed that the overwintering specimens started
to colonise apple orchards at the end of January in
100
Symptom surveys
2000
Each year the plants were checked for the presence
of AP symptoms in all the eight apple orchards. The
number of symptomatic plants was recorded in
autumn because witches’-brooms and rosettes at the
apex of the shoots were more evident after the leaves
had fallen.
80
60
40
20
Phytoplasma detection
In order to check for the presence of AP in the
insects collected with the beating tray, in the different
orchards, batches of five adult psyllids were ground
in Eppendorf tubes with 0.5 ml of pre-warmed
(60°C) CTAB buffer (2% w/v CTAB, 1.4 M NaCL,
20 mM EDTA, 100 mM Tris-HCl pH 8, 0.2% β-
mercaptoethanol), following a phytoplasma DNA
extraction procedure described previously (Marzachì
et al., 1998). The suspension was incubated for 30
min and, after extraction with chloroform-isoamyl
alcohol (24:1, v/v), DNA was precipitated from the
aqueous phase with 0.7 volume of cold isopropanol
and microcentrifuged. The pellet was washed twice
with 70% ethanol, dried and re-suspended in 20 µl
sterile distilled water.
100
2001
80
60
40
20
0
1 2 3 4 5 6 7 8 9 10111213141516171819
Further trials were carried out, following the
procedure described above, to extract total DNA
from single overwintered adults (20 females and 20
males) collected in the two most infected orchards,
2 and 4, at the beginning of March.
Sampling date
Fig. 1. Population dynamics of Cacopsylla
melanoneura adults caught by yellow sticky traps.
Traps were changed weekly from 21 January to 25
May in 2000 and from 19 January to 25 May in 2001.
Mean number of insects per apple orchard per sampling
date.
Purified DNAs were amplified using the
phytoplasma universal ribosomal primer pairs P1/

Frequency of AP-positive Cacopsylla melanoneura
287
both years and reached, in 2000, two peaks, one at
mid-February with 56.6 ± 23.3 (± SEM) and the
second at the beginning of March with 56.9 ± 8.8 (±
SEM) psyllids. In 2001 the two peaks were reached
in the second half of February with 45.0 ± 10.8
insects and at mid-March with 45.1 ± 17.1 psyllids.
In both years eggs of C. melanoneura were observed
at the base of the shoots and on the stalks from the
end of March until mid-April, while nymphs were
present on young leaves from mid-April until the
first half of May. During the whole month of May,
adults of the offspring generation were sampled and
peaks of 13.6 ± 4.2 in 2000 and 5.7 ± 2.8 adults in
2001 were recorded.
The molecular analysis performed on individual
insects collected in orchards 2 and 4 showed that
two out of 40 specimens were positive (so that a
proportion of 0.05 could be calculated). Of the 40
insects collected in the 100% infected orchard and
individually analysed, 18 gave positive amplification
for AP phytoplasmas, so that almost half of the
psyllids were AP-positive.
The PCR assays performed on single insects
provided evidence that AP-positive insects were
randomly distributed between males, seven positive
out of 32, and females, 13 positive out of 48 (χ2 =
0.069, P = 0.792).
Numbers and percentages of plants with AP
symptoms in the eight orchards in the 2 years are
summarised in Table 1. The disease spread in all the
orchards and the increases ranged from 2.8% to
35.9%.
Discussion
Recent studies demonstrated that phytoplasmas
associated with the AP disease are vectored by
psyllids. Molecular analysis also revealed presence
of the phytoplasmas in the body of C. costalis and
C. melanoneura, while transmission trials confirmed
the ability of these insects to transmit AP
(Frisinghelli et al., 2000; Tomasi et al., 2000; Poggi
Pollini et al., 2002; Tedeschi et al., 2002).
PCR detection of AP phytoplasmas in C.
melanoneura
DNA from batches of both overwintered and
offspring adults primed with fO1/rO1 pair gave AP-
group specific amplicons of the expected size (1050
bp) (Fig. 2) .
The present work now provides evidence of the
role of overwintered and offspring populations of
C. melanoneura in carrying AP in the Aosta Valley,
northwestern Italy. It is worth noting that the
percentages of AP-positive psyllids never exceeded
3.6%. A possible explanation for this is that the
phytoplasma titre in the trees is low in the period
the vector is feeding. Phytoplasmas reach the highest
titre only later in the season, in late summer and
autumn (Schaper & Seemüller, 1982). The results
obtained with C. melanoneura adults collected in
the most heavily infected orchard, located in Torino
province, showed that 45% of the insects were
positive for AP. We suggest that a very high number
of infected trees in the orchard can increase the
proportion of infected insects, even if a direct
correlation could not be found, mainly because of
The results of phytoplasma PCR detection in the
insects sampled in 2000 and 2001 are shown in Table
2. In 2000 AP-positive psyllids of the overwintered
generation were collected in five out of eight
orchards, while AP-phytoplasma was found only in
one batch of offspring adult, from one orchard.
In 2001 AP-positive insects were found in seven
out of eight orchards. No offspring adults from four
orchards showed the presence of apple proliferation
phytoplasmas.
An estimation of 3.5% of AP-positive insects
among overwintered psyllids and of 0.8% among
offspring specimens was obtained in 2000. In 2001
the estimated proportion of infected insects was
0.029 for overwintered adults (Table 3).
Table 1. Incidence of diseased apple trees based on
symptom expression in eight orchards of the Aosta
Valley during 2000 and 2001
1 2 3 4 5 6 7 8 9 1011
2000
2001
Diseased
Orchard plants/total
Diseased
%
plants/total
%
700 bp
350 bp
1
2
3
4
5
6
7
8
28/1848
45/177
3/117
33/42
9/146
0/248
11/206
4/97
1.5
25.4
2.6
78.6
6.2
0
626/1848
87/150
45/117
35/41
15/139
7/248
33.9
58.0
38.5
85.4
10.8
2.8
1050 bp
Fig. 2. PCR amplification with the specific primer pair
fO1/rO1 of total DNA from field collected Cacopsylla
melanoneura adults. 1-3, single adult; 4-6, batches of
5 adults; 7, water control; 8, 1kb DNA ladder (Gibco
BRL, Rockville, MD); 9 and 10, RFLP with SspI
restriction enzyme of fO1/rO1 amplicons; 11, 100 bp
DNA ladder (Gibco BRL, Rockville, MD).
5.3
4.1
19/202
-*
9.4
-
* data not available

288
R TEDESCHI ET AL.
Table 2. Number of AP-positive batches of five Cacopsylla melanoneura adults (positive/tested) collected by beat
tray samples in 2000 (a) and 2001 (b) in the eight apple orchards
a
Apple orchards
Date
1
2
3
4
5
6
7
8
21 January
4 February
18 February
3 March
16 March
30 March
13 April
-
-
0/1
0/1
-
-
-
-
-
-
0/1
-
0/1
0/4
0/2
0/1
-
-
-
-
-
-
-
-
-
0/2
0/2
-
3/4
0/2
-
0/1
0/1
0/1
0/2
0/3
0/2
0/1
-
0/1
0/2
1/3
1/4
3/5
-
0/1
0/6
1/11
2/4
-
0/1
0/2
1/11
1/5
0/2
-
0/1
1/1
0/1
2/2
0/1
-
27 April
-
-
11 May
25 May
-
-
0/4
-
-
0/1
0/5
0/1
1/5
0/1
0/5
-
-
-
0/5
-
b
Apple orchards
Date
1
2
3
4
5
6
7
8
19 January
2 February
16 February
2 March
16 March
30 March
13 April
-
-
-
0/2
0/8
-
1/1
-
-
-
-
-
-
-
-
-
-
-
-
1/1
1/3
3/13
1/9
-
0/1
0/1
0/2
0/12
0/7
0/1
0/1
-
1/3
6/31
1/12
0/2
-
0/2
1/16
2/17
1/2
0/1
-
3/10
6/27
0/20
1/2
1/2
-
0/2
2/11
0/8
0/1
1/2
-
0/2
0/4
2/7
0/1
0/2
-
-
-
27 April
1/16
11 May
25 May
-
-
0/5
0/1
0/4
-
-
0/2
-
-
-
0/2
-
-
0/1
-
the migratory habit of the vector. In spite of the low
number of AP-positive psyllids, the disease
incidence is increasing in the Aosta Valley. A low
percentage of AP-positive C. melanoneura has also
been reported in Bolzano province of northeastern
Italy, where, on average, 4% of the overwintered
adults were AP carriers (calculated from Poggi
Pollini et al., 2002).
replication processes), and, shortly after acquisition,
phytoplasmas are present at a low titre (Liu et al.,
1994; Bosco et al., 2002a). For example, C. pruni
needs to feed on an infected plant for 2-4 days to
acquire the European stone fruit yellows
phytoplasma, then a latent period of 2-3 wk is
required for completing the latency period (Carraro
et al., 2001a). We can then hypothesise that the C.
melanoneura offspring adults collected in the
orchards were in the early stage of infection (they
leave apple trees as soon as they emerge) and thus
the phytoplasma titre was below the detection
threshold. The hypothesis that the psyllid adults, after
leaving apple orchards, feed on other plant species
infected by AP, cannot be excluded. Phytoplasma
DNA was amplified with “AP-specific” ribosomal
primers from different plants showing yellowing
and/or decline symptoms: rosaceous fruit trees such
as pear, Nashi pear, Japanese plum and sweet cherry
and also other woody plants including hazel,
hawthorn, oak and hornbeam (Seemüller, 2002).
One of the problems in understanding AP
In both years, the overwintered adults showed a
higher proportion of AP-positive insects when
compared with the offspring population. We
demonstrated that psyllids are AP-infected in
February-March, thus suggesting that the
phytoplasma possibly overwinters in the body of the
vector. Similar results were provided by Davies et
al. (1998) and Carraro et al. (2001b) for C. pyricola
and C. pyri respectively. The very low proportion
of AP-positive insects among the offspring
population can be explained by a lower phytoplasma
titre in their body at the time of sampling and
analysis. Actually, phytoplasma titre in the insect
body increases with time (possibly following

Frequency of AP-positive Cacopsylla melanoneura
289
Table 3. Estimated proportion (pˆ) of AP-infected Cacopsylla melanoneura adults collected by beat tray samplings
in 2000 (a) and 2001 (b) in the eight apple orchards
a
Overwintered
AP
Offspring
AP
Orchard
No. of insects tested
p
ˆ
No. of insects tested
p
ˆ
1
2
3
4
5
6
7
8
10
75
45
110
105
30
-
+
-
+
+
+
+
-
0
20
5
30
30
25
0
-
-
-
+
-
0.036
0.008
50
55
25
-
b
Overwintered
AP
Offspring
AP
ˆ
p
ˆ
p
Orchard
No. of insects tested
No. of insects tested
1
2
3
4
5
6
7
8
55
+
+
+
+
+
+
+
-
0
30
20
10
0
10
0
5
320
190
305
120
80
-
-
-
-
-
0.029
0
130
125
-
epidemiology in northwestern Italy is the lack of
knowledge on summer and winter natural hosts of
C. melanoneura (Tedeschi et al., 2002). Further
studies are then needed to describe the whole cycle
of the insect, and to identify alternative hosts of the
psyllid that could serve as inoculum source.
the Aosta Valley, insecticide treatments should
provide protection during February and March, since
C. melanoneura adults feed on buds, also before
onset of vegetational growth. Later treatments or
very persistent products should be avoided to prevent
poisoning of pollinators during the incoming
flowering period (first half of April in the Aosta
Valley).
We failed to provide evidence of a correlation
between the infectivity of the insects and the
spreading of the disease, probably because the
vectors migrate, the investigated area was restricted
and the period of observations was limited to 2 years.
Our experiments demonstrate that the molecular
detection performed on individual insects is feasible,
but group testing for estimating infection rates is
advisable and cheaper when very large numbers of
insects should be tested. Molecular analysis of
insects is very useful in practice because the presence
of C. melanoneura in itself does not necessarily
imply its role in vectoring AP.
Acknowledgements
This research was supported by a European
Community grant: Interreg II (Italy-France
cooperation) no. 397 “Epidemiologie des maladies
à phytoplasmes des cultures perennes en zone de
montagne (Vallée d’Aoste et Savoie-Haute Savoie)”.
References
Bosco D, Leoncini P, Marzachì C. 2002a. Aspects of
phytoplasma-vector interactions: replications of CY in two
leafhopper species and effects of the mollicute on leafhopper
fitness. Proceedings of the 11th International
Auchenorrhyncha Congress, 5-9 August 2002, Potsdam,
Berlin, Germany, pp. 47-48.
The identification of a critical period for infection
is fundamental for the development of a management
programme to control the disease. The higher
percentage of AP-positive specimens, the longer
period spent in apple orchards and the higher density
of population observed for the overwintered adults
(Tedeschi et al., 2002) suggest the crucial role of
this generation in vectoringAP. If these assumptions
could be demonstrated, then treatments should be
concentrated against overwintered adults as soon as
they colonise apple orchards. In the conditions of
Bosco D, Palermo S, Mason G, Tedeschi R, Marzachì C,
Boccardo G. 2002b. DNA-based methods for the detection
and the identification of phytoplasmas in insect vector
extracts. Molecular Biotechnology 22:9-18.
Carraro L, Loi N, Emarcora P. 2001a. Transmission
characteristics of the European stone fruit yellows
phytoplasma and its vector Cacopsylla pruni. European
Journal of Plant Pathology 107:695-700.

290
R TEDESCHI ET AL.
Carraro L, Loi N, Emarcora P. 2001b. The ‘life cycle’ of
pear decline phytoplasma in the vector Cacopsylla pyri.
Journal of Plant Pathology 83:87-90.
Poggi Pollini C, Zelger R, Wolf M, Bissani R, Giunchedi L.
2002. Indagine sulla presenza di psillidi infetti dal fitoplasma
degli scopazzi del melo (AP = apple proliferation) in provincia
di Bolzano. ATTI Giornate Fitopatologiche 2:607-612.
Rahardja U, Whalon M E, Garcia-Salazar C, TangYT. 1992.
Field detection of X-disease mycoplasma like organism in
Paraphlepsius irroratus (Say) (Homoptera: Cicadellidae)
using a DNA probe. Environmental Entomology 21:81-88.
Schaper U, Seemüller E. 1982. Condition of the phloem and
the persistence of mycoplasma-like organisms associated with
apple proliferation and pear decline. Phytopathology 72:736-
742.
Carraro L, Loi N, Emarcora P, Gregoris A, Osler R. 1998.
Transmission of pear decline by using naturally infected
Cacopsylla pyri. Acta Horticulturae 472:665-668.
Davies D L, Clarck M F, AdamsAN. 1998. The epidemiology
of pear decline in the UK. Acta Horticulturae 472:669-672.
Frisinghelli C, Delaiti L, Grando M S, Forti D, Vindimian
M E. 2000. Cacopsylla costalis (Flor, 1861), as a vector of
Apple Proliferation in Trentino. Journal of Phytopathology
148:425-431.
Goodwin PH, Mahuku GS, Liu H, Xue B G. 1999. Monitoring
phytoplasma in populations of aster leafhoppers from lettuce
fields using the polymerase chain reaction. Crop Protection
18:91-99.
Schneider E, Seemüller E, Smart C D, Kirkpatrick B C.
1995. Phylogenetic classification of plant pathogenic
mycoplasma-like organism or phytoplasmas. In Molecular
and Diagnostic Procedures in Mycoplasmology, Vol I, pp.
369-380. Eds S Razin, J G Tully. San Diego:Academic Press.
Seemüller E. 2002.Apple Proliferation: etiology, epidemiology
and detection. ATTI Giornate Fitopatologiche 1:3-6.
Swallow W H. 1985. Group testing for estimating infection
rates and probabilities of disease transmission.
Phytopathology 75:882-889.
Hill G T, Sinclair W A. 2000. Taxa of leafhoppers carrying
phytoplasmas at site of aster yellows occurence in New York
state. Plant Disease 84:134-138.
Jensen D D, Griggs W H, Gonzales C Q, Schneider H. 1964.
Pear decline virus transmission by pear psylla.
Phytopathology 54:1346-1351.
Lemoine J. 1991. Deperissement du poirier: role de Psylla pyri
dans sa dissemination. Arboriculture Fruitière 442:28-32.
Liu H W, Goodwin P H, Kuske C R. 1994. Quantification of
DNA from the Aster Yellows mycoplasmalike organism in
aster leafhoppers (Macrosteles fascifrons Stål) by a
competitive Polymerase Chain Reaction. Systematic and
Applied Microbiology 17:274-280.
Tedeschi R, Bosco D, Alma A. 2002. Population dynamics of
Cacopsylla melanoneura (Homoptera: Psyllidae), a vector
of Apple Proliferation in northwestern Italy. Journal of
Economic Entomology 95:544-551.
Tomasi F, BranzA, Grando M S, Forno F, Forti D, Vindimian
M E. 2000. Individuazione di fitoplasmi del gruppo AP nelle
psille presenti nei frutteti. Informatore fitopatologico 38:51-
54.
Lorenz K H, Schneider B, Ahrens U, Seemüller E. 1995.
Detection of the Apple Proliferation and Pear Decline
Phytoplasmas by PCR amplification of ribosomal and non-
ribosomal DNA. Phytopathology 85:771-776.
Weber A, Maixner M. 1998. Survey of populations of the
planthopperHyalestesobsoletusSign. (Auchenorrhyncha,Cixiidae)
for infection with the phytoplasma causing grapevine yellows
in Germany. Journal of Applied Entomology 122:375-381.
Marzachì C, Veratti F, Bosco D. 1998. Direct PCR detection
of phytoplasmas in experimentally infected insects. Annals
of Applied Biology 133:45-54.