Bio-ethological observations on Reptalus quinquecostatusand its relationship with Stolbur phytoplasma

in Tuscany vineyardsBruno Bagnoli - Elisabetta Gargani

Agrobiology and Pedology Research CentreFlorence - Italy

Current status and perspectives of phytoplasma disease research and management Sitges, Spain, February 1th and 2nd, 2010

To date in Tuscany Flavescence dorée is poorly present and luckily no vine growing area is affected by serious phenomena of disease epidemic spreading.

On the contrary Bois noir is widespread not only in old and traditional vineyards of autochthonous varieties but also in young vine crops of ‘Chardonnay’, ‘Merlot’ and ‘Sangiovese’ varieties.

T

During the investigations carried out in Central Italy in the last five years on the system “vectors – yellows – grapevine”, various species of leaf-and planthoppers are found to be positive for the Stolbur phytoplasma reaching the first step to be considered potential vectors of BN.

Thamnotettix zelleri (Kirschbaum)

Anoplotettix putoni Ribaut Hyalesthes luteipes Fieber

Hyalesthes scotti Ferrari

Bagnoli et al., 2005, 2008

Trivellone et al., 2005, 2007

Pasquini et.al., 2008

Reptalus quinquecostatus (Dufour)

Among these species, R. quinquecostatus has appeared a leading candidate to explain, togheter or as an alternative to Hyalestes obsoletus Signoret (still the only ascertained vector), BN diffusion in the vineyard.

Hyalesthes obsoletus Reptalus quinquecostatus

Alma et al., 2002

Maixner, 1994, 2006

Sforza et al., 1998

2006

0/5

6/12

2/21/1

3/10

4/10

0/1

0/5 1/3

0/2

0

5

10

15

20

30 June 14 July 28 July 10 August

No. a

dults

cap

ture

d

Interrow VineElm

2005

5/12

7/7

0/2 1/1

5/17

1/30/1

2/10 8/10

3/51/3

0

5

10

15

20

29 June 14 July 28 July 9 August

No.

adu

lts c

aptu

red Interrow

VineElm

Since 2005, a vineyard of ‘Syrah’ and ‘Sangiovese’ varieties located at “Colle Verde”estate (Lucca) has been configured as a specific and interesting case study. In this vine growing area R. quinquecostatus shows, from late June to late July, a considerable mobility in the agro-ecosystem, it is the most demographically significant Auchenorrinchspecies in the vineyard and it can feed on the leaf midribs and young shoots of the vine.

Trivellone et al., 2005, 2007

The cixiid adult population sampled both on elm and on vineyard (interrow and grapevine) has always shown very high infection rates of the order of 40-50%.

2005habitat

Positive/ tested % tuf type

vineyard 20/43 46.5 I and IIelm 15/28 53.5 I and II

Total 35/71 49.3

*specimen with mixed infection

2006habitat

Positive/ tested % tuf type

vineyard 34/64 53.1 I and II *elm 5/13 38.5 I

Total 39/77 50.6

Trivellone et al. 2005, 2007

In 2007, laboratory tests performed on wild adults with the method of artificial membrane of Tanne et al. (2001), allowed us to verify that males and females of R. quinquecostatus, despite the short life span in the Eppendorf modified as rearing chambers, can inoculate the Stolbur phytoplasma (only tuf type I) in an artificial feeding medium with an efficiency of 40% (Pinzauti et al., 2008).

Eppendorf modifiedas insect chamber

Cotonec

Feeding medium: 200 μl of 5% sucrose in TE (10 mM Tris and 1 mM EDTA at pH 8)

The specimens were placed individually in Eppendorf modified and maintained in climatic chamber (25 ± 1 ° C, 75% RH, 16:8 L: D) until the death of individuals

cap

At the end of each test, the specific determination was confirmed (on dead specimens), then either insects and sucrose solutions were stored at -30 ° C until DNA extraction

Figure 1 MspI restriction fragment length polymorphism profiles, in 2.5% agarose gel, of nestedpolymerase chain reaction products amplified with primer pair TufAYf2/r2. In (A) and (B), VK patterns of 25 wild infected Reptalus quinquecostatus. M, GeneRuler 100 bp DNA Ladder Plus Fermentas); VK-I, positive control from Bois noir-infected grapevine sample (VK-I); VK-II, positive control from Bois noir-infected grapevine sample (VK-II); lanes (1–10, 13–17, 19, 21–23 and 25), VK-I infected specimens; lanes (11–12, 18 and 20), VK-II infected specimens; lane (24), VK-I/VK-II infected specimens.

Figure 2 MspI restriction fragment length polymorphism profiles, in 2.5% agarose gel, of nestedpolymerase chain reaction products amplified with primer pair TufAYf2/r2. Characterisation of phytoplasma in the 10 sucrose-feeding media that tested positive. M1, GeneRuler 100 bp DNA LadderPlus (Fermentas); M2, X174 DNA/BsuRI (Marker 9; Fermentas); STOL nr, positive control not restrictedfrom Bois noir-infected grapevine sample; STOL, positive control (VK-I).

Pinzauti et al., 2008

77tested

specimens

25positive

for Stolbur(32.5%)

10inoculated

in the feeding medium (40%)

4tuf type II

1tuf type Ituf type II

20tuf type I

9inoculated(tuf type I)

1inoculated

(only tuf type I)

X did not inoculate

Bagnoli et al., 2008Pinzauti et al., 2008

Further laboratory tests on Sangiovese grafted vines allowed us to verify that the insect can inoculate Stolbur phytoplasma on vine plants as well (Bagnoli et al., 2008).

These results lead to the conclusion that R. quinquecostatus is a competent species in Stolbur phytoplasma transmission and it can have a more or less importance (to be defined) in BN spreading in the vineyard.

The lack of correspondence between the biological characteristics of the cixiid (mobility in the agro-ecosystem, population density, feeding on vine, high rate of infection and ability to transmit Stolbur phytoplasma) and the present very low spreading of disease symptoms in the “Colle Verde” vineyard, leads to consider that in the complex flow chart, that from phytoplasma acquisition gets to the expression of the disease symptoms, could be included phenomena of disease tolerance on vines, expressed as phytoplasma recovery.

REFERENCESAlma A., Soldi G., Tedeschi R., Marzachı` C., 2002 - Ruolo di Hyalesthes

obsoletus Signoret (Homoptera Cixiidae) nella trasmissione del LegnoNero della vite in Italia. - Petria, 12: 411-412.

Bagnoli B., Pinzauti F., Trivellone V., 2005 - Indagine preliminare sugli auchenorrinchi potenziali vettori di stolbur in un'area viticola del Lazio. -Atti III Incontro Nazionale sulle Malattie da Fitoplasmi, Milano, 22-24/06/2005, Petria, 15 (1/2): 55-58.

Bagnoli B., Pinzauti F., Trivellone V., 2008 - Reptalus quinquecostatus(Dufour): dati bio-etologici e rapporti con il fitoplasma dello stolbur in aree viticole toscane. - Petria, 18 (2): 225-228.

Maixner M., 1994 - Transmission of German grapevine yellows (Vergilbungskrankheit) by the planthopper Hyalesthes obsoletus(Auchenorrhyncha: Cixiidae). - Vitis, 33: 103-104.

Maixner M., 2006 - Grapevine yellows - current developments and unsolved questions. - In 15th Meeting of the International Council for the Study of Virus and Virus-Like Diseases of the Grapevine (ICVG) (ed. J.T. Burger), 3-7 April 2006, Stellenbosch, South Africa.

Pasquini G., Ferretti L., Bagnoli B., Gentili A., Albanese G., Rapisarda C., Cavalieri V., Barba M., 2008 - Il Legno nero della vite nel centro-sud Italia. - Atti del "Workshop" 4° Incontro nazionale sulle malattie da fitoplasmi, Roma 28-30 maggio 2008, Petria, 18 (2): 218-22.

Pinzauti F., Trivellone V., Bagnoli B., 2008 - Ability of Reptalus quinquecostatus (Hemiptera: Cixiidae) to inoculate stolbur phytoplasma to artificial feeding medium. - Annals of Applied Biology, 153: 299-305.

Sforza R., Clair D., Daire X., Larrue J., Boudon-Padieu E., 1998 - The role of Hyalesthes obsoletus (Hemiptera: Cixiidae) in the occurrence of bois noir of grapevines in France. - Journal of Phytopathology, 146: 549–556.

Tanne E., Boudon-Padieu E., Clair D., Davidovich M., Melamed S., Klein M., 2001 - Detection of phytoplasma by polymerase chain reaction of insect feeding medium and its use in determining vectoring ability. -Phytopathology, 91: 741–746.

Trivellone V., Pinzauti F., Bagnoli B., 2005 - Reptalus quinquecostatus(Dufour) (Auchenorrhyncha Cixiidae) as a possibile vector of Stolbur-phytoplasma in a vineyard in Tuscany. - Redia, 88: 103-108.

Trivellone V., Pinzauti F., Bagnoli B., 2007 - Reptalus quinquecostatus(Dufour) (Cixiidae): potenziale vettore di stolbur in un ambiente viticolo toscano. - XXI Congresso Nazionale Italiano di Entomologia, Campobasso 11-16 Giugno 2007, Proceedings, p. 174.