J. Phytopathology 146, 549-556 (1998)® 1998 Blackwell Wissetischafts-Vedag, BerlinISSN 0931-1785

Institui National de la Recherche Agronomique, Station de Recherches sur les Phvloplasmes, Dijon. France

The Role of Hyalesthes obsoletus (Hemiptera: Cixiidae) in the Occurrence ofBois noir of Grapevines in France

R, SFORZA, D . CLAIR, X. D.MRE, J. LARRUE and E. BOUDON-PADIEU

Authors' address: Institut National de la Recherche Agronomique, Station de Reeherches sur les Phytoplasmes, 21034Dijoti Cedex, France (correspondence to E. Boudon-Padieu)

With 5 figures

Received December 29, !997: accepted March 19, 1998

AbstractAn epidemiological study on a grapevine yellows diseasecalled bois noir was carried out for 3 years in the Rhonevalley (France). This yellows is caused by a stolbur typephytoplasma. Vectors and alternative host plants weresearched, and the inoculation period was determined.Detection of stolbur phytoplasma in insects and plantswas obtained using primers STOLl 1 f2,rl. In addition,a nested polymerase chain reaction (PCR) was used withprimers P1/P7 and fU5/rU3 for detection in stolbur-infected plants with low titre. Several thousand insectswere captured and species of Hemiptera were listed.Fourteen wild or reared Hemiptera species were used intransmission trials. Thirty-four wild species were moni-tored for phytoplasma DNA by PCR. A planthopper.Hvalesthes obsoletus Sign., tested positive for stolbur ata level of 28% (98,/343) in 1995 and 38% (205,529) in1996. In 1995, two leafhoppers, Mocydia crocea (1 78)and Euscelis tineolatus (2/309), were infected at a muchlower ratio. Successful experimental transmission tograpevine, periwinkle and thorn apple was only obtainedwith H. obsoletus. Among wild plant species, hoary cress(Cardaria draba L.), bindweed (Convolvulus arvensis L.),sweet cherry {Prunus sp,), plum (Prunus domestica L) ,lilac (Syringa vulgaris L.), fig tree [Ficus carica L.) andelm {Ulmus sp.) were shown to be stolbur infected andhoary cress was identified as a new host plant for H.obsoletus in France. The role of some fallow lands closeto vineyards as sites where the inoculum of stolbur phyto-plasma was maintained in insect vector and reservoirplants, is discussed. The natural inoculation period tograpevine was shown to extend from June to August,corresponding to the adult activity of H. obsoletus.Together with the close relationships of the phyto-plasmas involved in vergilbungskrankheit, a Germangrapevine disease, and bois noir. The results ofthis study suggest that the two yellows are veryclose.

ZusammenfassungBedeutung von Hyalesthes obsoletus (Hemiptera: Cixiidae) furdas Auftreten der Bois-noir-Krankheit der Weinrebe in Frank-reichIm franzosischen Rhonetal wurde eine 3-jahrige epide-miologische Studie iiber die Bois-noir-Vergilbungskrank-heit der Weinrebe durchgefuhrt. Diese Krankheit wirdvon einem Phytoplasma des Stolbur-Typs ausgelost. Eswurde nach Vektoren und alternativen Wirtspflanzengesucht, und die Inokulationsperiode wurde bestimmt.Die Detektion von Stolbur-Phytoplasmen in insektenund Pftanzen erfolgte mit Hilfe der Primer STOL 1! f2/rl.Zudem wurde eine genestete PCR mit den Primem P1 /P7und fU5 rU3 zur Detektion in stolburinfizierten Pflanzenmit niedrigem Titer durchgefuhrt. Mehrere tausendInsekten wurden gefangen, und die Hemiptera-Artenwurden aufgelistet. 14 wilde oder geziichtete Hemiptera-Arten wurden in Ubertragungsversuchen verwendet, und34 Wildarten wurden mittels PCR auf Phytoplasma-DNA untersucht. Der Fulgoride Hvalesthes obsoletusSign, war 1995 mit 28% (98/343) und 1996 nut 38%(205/529) der getesteten Tiere stolburpositiv. 1995 warendie beiden Zwergzikaden Mocydia crocea (Ijl^) und Eus-cetis lineolatus (2/309) ebenfalls infiziert, jedach in vielgeringerem AusmaB. Eine experimentelle Ubertragungauf Weinrebe, lmmergrun und Stechapfel war nur mitH. obsoletus erfolgreich. Unter den Wildpflanzenartenwaren Pfeilkresse (Cardaria draba L.), Ackerwinde (Con-volvulus arvensis L.), Suflkirsche (Prunus sp.), Pflaume(Prunus domestica L.), Flieder (Syringa vulgaris L.),Feigenbaum (Ficus carica L.) und Ulme (Ulmus sp.) stol-burinfiziert. Die Pfeilkresse erwies sich in unseren Unter-suchungen erstmals als Wirtspflanze von H. obsoletus inFrankreich. Die Bedeutung neben Weinanlagen liegenderBrachflachen als Reservoir des Stolburphytoplasmas inlnsektenvektoren und befallenen Pflanzen wird disku-tiert. Die natilrliche Inokulationsperiode der Weinrebereicht von Juni bis August, was dem Aktivitatszeitraumder Imagines von H. obsoletus entspricht. Ebenso wie die

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550 SFORZA et al.

engen Beziehungen zwischen den an der in Deutschlandbeobachteten Vergilbungskrankheit der Weinrebe betei-ligten Phytoplasmen und den Erregern der Bois-noir-Krankheit legen unsere Ergebnisse nahe, daO die beidenKrankheiten eng miteinander verwandt sind.

IntroductionGrapevine yellows (GY) are severe diseases of grapevine(Vitis vinifera L.). and are associated with phytoplasmaswhich are specifically transmitted by phloem feedinginsects. Leafhopper and pianthopper species are mainphytoplasma vectors, but other Hemiptera vectors, stichas psyllids, have been reported (D'Arcy and Nault, 1982).In France and Germany, two GY have been known andstudied for a long time, bois noir (BN) (Caudwell, 1961;Caudwell etal., 1972) and vergilbungskrankheit (VK)(GfSrtel, 1965). Recently, the phytoplasmas associatedto one and the other GY have been characterized andboth shown to belong to the stolbur group (Daire etal.,1993; Maixner etal., 1995). Stolbur type phytoplasmas(SP) have also been detected in other GY occurring inseveral countries of Western Europe and Israel (Daireetal,, 1993; Bertaccini etal., 1995; Lavina etal., 1995;Tanne etal., 1995). Stolbur was the name of an epidemicdisease on solanaceous crop which was described in East-ern Europe in the 1940s and progressed to other countries(Suchov and Vovk, 1946; Valenta etal., 1961; Marchouxetal., 1970b). The SP is very ubiquitous and it can bedetected in a wide range of plants (Garnier etal., 1990;Fosetal., 1992; Maixner etal., 1995).

SP seem to be very homogenous, although a few recentreports have shown a genomic variability in tomato iso-lates (Minucci and Boccardo, 1997; Vibio etal., 1997).However, a monoclonal antibody was reported (Gamieretal., 1990) which reacts with a number of referencestrains and wild isolates (Fos etal., 1992). In addition,Daire etal. (1997a) could not demonstrate a genomicvariability among stolbur phytoplasmas, includinggrapevine isolates from different countries. The SP associ-ated to BN and VK and other related GY have not beendifferentiated up to now, although they occur under arange of different environmental conditions and indifferent cultivars (Daire etal., 1993; Bertaccini etal.,1995; Lavina etal., 1995; Maixner etal., 1995; Daire etal.,1997b).

In France, BN occurs in all viticultural areas (Daireetal., 1997b). Untii now, the vector of BN was unknownin France. In the case of VK, Maixner (1994) showedthat the pianthopper Hyalesthes obsoletus Signoret 1865(Hemiptera; Cixiidae) was an efficient vector of SP inGermany. In the present study, investigations were car-ried out to identify one or more vector species of SP ingrapevine in south-eastern France. Particular attentionwas devoted to H. obsoletus and its potential vector rolein BN disease. Moreover, when the months in whichtransmission occurs in the fields was determined and thebiology of putative vectors according to their relation-ships with their host plants and grapevine were inves-tigated.

Materials and MethodsTrapping of adults and nymphsTrapping of leaOioppers and planthoppers were moni-tored in three vineyard blocks and adjacent fallow landsin 1995 and 1996; Fl , F2 and F3 were fallow lands onthe border of VI, V2 and V3, respectively. In 1996, afourth fallow land, F3", situated 100 metres from V3 andF3 blocks, was included. Adults were captured by yellowtraps and D-vac. In 1995, three yellow sticky traps, 60 cmby 30 cm (Biosystemes, Cergy-Pontoise, France), andthree yellow water-bowls were placed in each block. In1996, only three yellow sticky traps were used in eachblock. The yellow traps were deposited on the earth, in astar-shaped array at 10 metres from each other. Theywere replaced weekly from 1 April to 1 October in 1995and 1996. Insects were captured by D-vac (Ventura, CA,USA) suction every 15 days in the same period. Inaddition, the trapping of adults of H. obsokius wasextended to other viticultural areas in 1995 and 1996, inthe south-eastern region (Languedoc) and north-easternregion (Burgundy). Nymphs of H. obsoletus were hand-picked from roots of different host plants in winter in1995 and 1996.

Adults were identified (Ribaut, 1936; Ribaut, 1952;Delia Gitistina, 1989) and counted using a stereo-binocular dissecting microscope (Nikon SMZ-U).

Insect rearingRearing of insects was carried out under controlled con-ditions (23 C, photoperiod I6h;8h, humidity 80%).Pathogen-free individuals of two leafhopper species, Sca-phoideus titanus Ball, the vector of flavescence doree, andEuscelidius variegatus Kbm, an experimental phyto-piasma vector, were reared, respectively, on healthygrapevine and maize as previously described (Caudwelland Larrue, 1970). Rearing of the pianthopper H. obso-letus was undertaken during this study. The adults wereobtained in controlled conditions from eggs of wildfemales; a few adults were fed on stolbur-infected lav-ender (unpublished data).

Insect transmissionTransmission trials were performed with presumably nat-urally infected specimens of Hemiptera species, i.e. Mocy-dia crocea, Euscelis lineolatus. Aphrodes sp.,Psammotetlix sp., Jassargus obtusivalvis, Fieheriellaflori,Neoaliturus fenestratus, Asiraca clavicornis, Hyalesthesscoiti and H. obsoletus. Groups of 10-15 adults, capturedby D-vac, were identified, fed on healthy plants until theydied, then stored at — 20 C and analysed by polymerasechain reaction (PCR). The healthy plants used in trans-mission were cuttings of grapevine (cv Chardonnay),seedlings of periwinkle (Catharanthus roseus L.) and ofthorn apple {Datura stramonium L.). Healthy cuttings ofgrapevine from hot-water treated material were providedby Etablissement National Technique pour FAme-horation de la Viticulture (E.N.T.A.V.). Healthy lab-oratory-reared insects served as control. The duration ofsurvival of wild H. obsoletus was checked on healthybindweed (Convolvulus arvensis L.) and on grapevineunder controlled conditions.

Roie of Hyatesthes obsoleius in Bois noir Occurrence of Grapevines in France 551

Diseased cuttings were collected from canes on BN-infected grapevines in the field. They were grown in thegreenhouse and checked for SP by PCR. The ability ofacquisition/transmission of SP was carried out with S.titanus. Reared healthy specimens were fed for 3 weeks ondiseased cuttings, then transferred to healthy grapevinecuttings until they died. Reared specimens of E. var-iegatus were fed for 2 weeks on SP-infected periwinklereferred to as P3-I, then transferred to healthy periwinkleseedlings until they died. After exposure to insects, theplants were sprayed with Dichlorvos (Bayer, Puteaux,France) and transferred to a greenhouse. Plants withsymptoms of yellows that were confirmed by PCR {seebelow) were considered to have been infected.

Plant materialIn 1994, 1995 and 1996, groups of bait plants of differentspecies known to be sensitive to SP were exposed for a 2-week period, beginning in April or May, and ending inOctober. In 1994, two sets of 10 groups of 20 plants (10periwinkles and iO tomato plants) were exposed in Viand V2 (11 May-27 October). In 1995, three sets of 11groups of 15 plants belonging to 14 species (two grape-vines, thorn apple, periwinkle, lavender, clover, tomato,tobacco, courgette, celery, green pepper, thyme, petunia,sugar beet, broadbean), were exposed in VI, V2 and V3(3 April-2 October), and in addition one group of 10periwinkles were exposed in V2 (15 June~ll July). In1996, three sets of nine groups of 15 plants (five grape-vines, five thorn apples and five periwinkles) were exposedin VI. V2 and V3, and nine groups of six plants (threegrapevines and three periwinkles) were exposed in F3' (2May-10 October).

Samples were also collected from plants showing yel-lows or decline symptoms, i.e. grapevines, bindweed andhoary cress (Cardaria draba L.) collected in V and Fblocks, shrubs and trees collected in F blocks such assweet cherry (Prunus sp.), plum (Prunus domeslica L.),lilac (Svringa vulgaris L.), fig tree {Ficus carica L.) andelm (Ulmus sp.).

PCR detection of phytopla»nasProcedures for plants

All the procedures for plant DNA extraction were carriedout as previously described (Daire etai., 1992). PCRamplification was carried out with primers STOL11 f2/r 1,specific for stolbur subgroup strains, according to Daireetal. (1997a). In addition, nested PCR was applied toplants with yellows symptoms which had tested negativewith primers STOL 11 f2/rl. Nested PCR amplificationwas performed with phytoplasma universal ribosomalprimers, that is PI (Deng and Hiruki, 1991) and P7(Smart etal., 1996) for the first amplification andnj5/rU3 (Lorenz etal., 1995) as internal primers for thesecond amplification. The reaction mixture of 40^1 con-tained 50-100 ng of template DNA, Q.lSptu of eachprimer, 250/XM each dNTP, 2 units of Taq DNA poly-merase (Appligene), Taq buffer (Appligene), and wereoverlaid with 40/il of mineral oil. Amplification withP1/P7 was carried out for 30 cycles at 92 C for 45 s, 58T

for 45 s, and 72 C for 1 min 45 s. The PCR products werediluted 1/100 in water, and 1 jxX was added to the samereaction mixture using primer nJ5/rU3. Amplificationwith fU5/rU3 was carried out for 35 cycles at 92 C for30 s, 57 C for 30 s, and 72 C for 45 s. The final PCRproduct was digested with Tru91 and submitted to RFLPanalysis (Daire etal., 1997a). Agarose electrophoresis andpolyacrylamide gel electrophoresis were carried outaccording to standard procedures.

Procedures for insects

The procedures for insect DNA extraction were asdescribed by Maixner etal. (1995). Insect DNA extractswere obtained either from fresh insects or from insectsdried, glued (stored 4 months on sticky traps) or stored4 months in 70% ethanol. Wild insects were crushed bygroups of tw o, five, 10, 15 or 20 of the same species.Specimens of H. obsoietus were analysed individually.Amplification was done with primers STOLll f2/rl for40 cycles. Strain STOL C, isolated from tomato andmaintained in periwinkle, kindly provided by M. T.Cousin, served as positive control and rearing of i/. obso-ietus provided controls.

ResultsSurvey for Hemiptera species

In 1995 and 1996 in the experimental blocks, 40 000 speci-mens of Hemiptera were captured, identified at genus orspecies level and listed in Table 1.

PCR results from wild insectsOut of the hundred species captured in the experimentalblocks, one-third of taxa were tested by PCR (Table 1).But only specimens belonging to three species tested posi-tive. The planthopper H. obsoietus was by far the specieswith the highest ratio of natural infection; 28.5% (98 343)of individuals in 1995 and 38.7% (205 529) in 1996. Inaddition, several specimens of H. obsoietus from differentviticultural areas in France were also infected by SP,in Burgundy in 1995 (1/4) and in 1996 (8/58), and inLanguedocin 1996(3/23).

Figure 1 shows the detection of SP by PCR on indi-viduals from different species. Detection on H. obsoietuswas possible independently of storage conditions (lane1-4). SP could aiso be detected on nymphs of the fifthinstar captured on roots of symptomatic bindweed (seebelow) (lane 5). Apart from H. obsoietus, only M. crocea(1/78; 1.3%) (lane 10) and E. lineolatus (2/309: 0.7%)(not shown) were found positive in 1995. All specimenstested in the other species were negative, including thetwo Hyalesthes species namely H. scotti and Hyalesthesluteipes (69 individuals of the two species were tested).

Trapping of HyaJesthes obsoietus and inoculation period to baitplantsFigure 2 shows the results of trapping of H. obsoietus in1995 and 1996 by sticky traps and D-vac suction in vine-yard blocks (VI, V2 and V3) and fallow land blocks (FI,F2, F3 and F3"). Altogether, the first adults were trappedin the middle of Jurte (week 25 in 1995 and week 24 in

552 SFORZA et al.

Table 1List of Hemiptera captured in experimental blocks in Rhone valley (France) in 1995 and 1996 by D-vac suction and yellow traps. Suborderclassification follows Campbell et al. {!995). Bold figures give the ratio of the number of adults which tested positive for stolbur phytoplasma byPCR to the number of adults tested

Suborder(common name)

FamilySubfamily Genus and species

NEOHEMIPTERA FULGOROMORPHA(Planthoppers) CIXIIDAE

DELPHACIDAE

DICTYOPHARIDAE

ISSIDAE

TETTIGOMETRIDAETROPIDtJCHIDAE

EUHEMIPTERA(froghoppers)

(treehoppers)

(leafhoppers)

CICADOMORPHACERCOPIDAE

MEMBRACIDAE

CICADELLIDAETyphlocybinae

IdiocerinaeCicadeliinaeIassinaeMacropsinaePenthimiinaeDorycephalinaeAgallinae

Aphrodinae

EvacanthinaeMegophthalminae

Deltocephalinae

Cixius nen-osus {h. 1758)Hyakslhes ohsolelus Signoret 1865 (303/872)Hyuleslhes luleipes Fieber 1876 (0/69)"Asiraca ilavicimis (F. 1774] (0/2)Javesella pellucida (F. 1774)Bursinia sp. A. costa 1862Dktvophara europea (L. 1758) (0/5)Cafercfa ftone//; {Latreille 1807) (0/2)Hysleropterum sp. Amyot & Serville 1843 (0/1)Tetligonteira griseola Fieber 1865Trypetimorpha occidentalis (Huang &Bourgcin 1993)

Aphrophora sp.Germar 1821 (O/I)CercopLK intermedia {Kbm. !868) (0/3)Cercopis sangwnolenia (Scopoli 1769)Cenrrotus cormilus (L. !758)Gargara genislae (F. 1775)

Arhoridiapanula (Boheman 1845)Emelyanoviana mollicula (Boheman 1845)Empoasia decipiens Paoli 1930Empomca vilis (Gothe 1875) (0/40)Eryihria aureola {(FaUm 1806)Euptervx atrnpunetala (Goeze 1778)Eupiery.x auraia (L. 1758)Baiianocerus pruni (Ribaut. 1952)Ckadelia iiridis (L, 1758)Iassus lanio (L. 1761)

Macropsis fuscula (Zetterstedt 1828) (0/43)Penrhimia nigra iGoeze 1778) (0/4)Eupeli.x ciispidata (F. 1775) (0/4)Agallia eomnhrina Cunh 1833

Anaceralagalliu laevis (Ribaut 1935)Anaceralagallia rihauti (Ossiannilsson 1838)AnaceratagalUa. venosa (Fourcroy 1785) (0/34)Anoscnpus limicola (Edwards I9()8)

Anoscopus sp. Kbm. 1858Aphrodes hieincia (Schrank 1776)Aphrodes makarovi Zachvatkin 1948Elacanlhus inlerruplus (L. 1758)Megophthalmus scahripennis Edwards 1915(0/29)Adarrm muUinoIalus (Boheman 1847) (0/30)Adarru.i tauru.s Ribaut (1952)Alhgidius ahhreiiatus (Lethierry 1878) (0/2)Allygidiiis awmarius (F. 1794)Alhgidius fur calm (Ferrari 1882)Araldus propinquus (Fieber 1869)Arocephalus longiceps (Kbm. 1868) (0/1)Arocephalus .Sagittarius Ribaut (1952)Arthaldeus striifrom (Kbm. 1868)Artianus intersticiaiis (Germar 1821)Cechenotettix quadrinotalus (M. & Rey 1855)Cicaduiaplaiida var. inornata (Horvath 1897)(0/3)Circulifer opacipennis (Lethierry 1876)Conosanus ohsolelus (K.hm. 1858)Eohardya fraudulentus (Horvath 1903)Euscelidius variegatus (Kbm. 1858) (0/31)Euscelis incisus (Kbm. 1858) (0/8)Euscelis lineolatus Brulle 1832 (2/309)Fieheriellaflori (Stal 1864) (0/13)Goldeus harpago (Ribaut 1925)Goniagnathus hrevis (H-S. 1836) (0/8)Graphocraerus ventralis (Fallen 1806)Grypotes staurus Ivanoff 1885Jassargus oblusivahis (Kbm. 186«) (0/278)Lahurrus quadratus (Forel 1864) (0/1)Macrosteles quadripunctulatus (Kbm. 1868)Macrosteles sexnotatus (Fallen 1806)

Hvalesthes scotti Ferrari 1882Oliarus sp. Stal 1862 (0/2)Tachycixius piiosus (Olivier 1791)Laodelphax striatellus (Fallen 1826)Non-determined species

y.s.tu.v coleoptraius (F. 1781) (0/8)

Ceriopis lulnerata Rossi 1807Philtienus spumarius (L. 1758) (0/5)

Sticlocephala hisonia K. & Y. 1977

Eupieryx urticac {F. 1803)Haupiidia proiiiuiali.s (Ribaut 1931)Liguropia funiperi (Lethierry 1876)Ribautiana cruciata (Ribaut 1931)Rihautiana lenerrima (H-S 1834)Zygiiiidiascutellaiis (H. S. 1838)Zygina rhamiii FtTTiri 1882liridkerus u.stulalu.s (M. & Rey i855)

Macropsis sp. Lewis 1834

Ha sinuaia (M. & Rey 1855)(0/17)Drvodurgades antonniaf (Mebchar 1907)Drrodurgadcs resiculatus H-S (1834)

.Aphrodes sp. Curtis 1929 (0/17)"Stroggyloci'phaius agresiis (Fallen 3 806)Stroggylocephalus livens (Zetterstedt 1840)

Mocydia crocea (H. S. 1836) (1/78)Mocydiopsis sp. Ribaut 1839Neoaliturus fenestratus (H-S 1834) (0/t77)Ophiola decumana (Kontkanen 1868)Phlepsius imricalus {H-S 1838) (0/10)Phiepsius .spinulosus Wagner 1963Phlogoletlix Cyclops (M. & Rey 1869)Placoiettix taeniatifrons (Kbm. 1868)Platymetopius filigranus (Scott 1876)Platymetopius major (Kbm. 1868)Platvmetopius rostratus (H-S 1834)Platymeiopius undaius (de Geer 1773)

Psammotettix alienus (Dalhbom 1850)Psammoleltix putoni (Then 1898)Psammotettix striatu.t (Linne 1758)Psammotettix sp. Haupt 1929 (0/S28)'Recilla schmidtgeni (Wagner 1939)Rhoananus hypochlorus (Fieber 1869)Rhopaiopyx elongatus (Wagner 1952) (0/2)Rhytistylusproceps {Kbm. 1868)Scaphoideus titanus Ball 1932 (0/30)Selenocephalus oh.soletus (Germar 1817)Speudotettix mbfusculus (Fallen 1806)Synophropsis lauri (Horvath 1897)fhamnotettix dilutior (Kbm. 1868) (0/11)JTiamnotettix zelleri (Kbm. 1868)

: Total ratio for H. scotti and H. luleipes;': Total ratio for all Aphrodes species;': Total ratio for all Psammotettix species.

Role of Hyaiesihes absokHLs in Bois noir Occurrence of Grapevines in France

Kbp M IP HHo SHo 1 2 3 4 5 6 7 8 9 10 11

0.5 —

Fig. I Results of DNA amplification with primers STOLI1 f2;rj usinglemplalc DNA prepared Irom wild specimens oi Hyak'sihe.\ oh^uleiuscaptured in 1996 and ieaftiopper^ either from lahoralory rearings orcollected in experimental blocks in Rhone valley in 1995. M, molecularweight marker (Ikb ladder BRLi; IP. periwinkle infected with strainSTOL C; HHo, laboralory-reared healthy IL ohsokius: SHo. lab-oratory-reared stolbur-infecied //. ob^oklus'. 1 ^ . slolbur-infectedaduits of//, vhsoleiu.s from fields stored in different conditions; 1. fresh:2. glued and stored 4 months on sticky trap: 3. stored i month on thebench: 4. stored 4 months in 1(}% ethanoi: 5. fresh nymph of fifthinstar of / / . oh-wk'ius captured ois root of symptomatic bindweed: 6-9:laboratory-reared leallioppers used for CKperimenta! stoibur trans-missions trials: 6. heallhv Scapfuiideus nlanus: 7. .S'. filanus after a 3-week acquisition period on Chardonnay with bois noir symptoms anda 2-week inoculation period on healthy vine: 8. hei Uhy Eusrelidhurarie^aius: 9. £. varicfiaius after a 2-week acquisition period on P. -1 periwinkle (see Results) and 2-week inoculation period on healthyperiwinkle: Hi and I i. wild leafhopper MocrcHa vroceu individuals

1996) and the last adults were caplufed in August (week32 in ! 995 atid week 33 in 1996). Thus, adults were presentfor at least 1--9 weeks. The same results were obtainedvvith yellow water-bowls (data not shown). A strikingcontrast in the regularity of captures and in the numberof individuals appeared between F3' (Fig. 2C) and theother blocks (Fig. 2A. 2B). in 1996. the captures wereerratic in V and F blocks (Fig,2A. 2B). In F3'. adultswere captured weekly. Numerous nymphs of fourth andfifth instarcouid also be observed in F3' tn May and Juneon bindweed and hoary cress which were frequent on iheblock; during the same period much smaller numbers ofspecimens could be found also on these plants in V3.

The inoculation period was determined by bait plantexperiment. All the bait plants exhibiting phytoplasmasymptoms in 1994, 1995 and 1996, turned out to be stoi-bur positive in the PCR assays. Neither symptoms norphytoplasmas were detected in the control plants. Exam-ples of PCR results on bait plants are given in Fig. 3 (lane8 and 10) and Fig.4 (lane 9 and 10). In 1994. none ofthe 100 tomato plants became infected; out of the 100periwinkles, two plants which had been exposed from 21June to 7 July (weeks 25^27) were infected and referredto as P3-1 and P8-1. In 1995, out of 506 bait plants, otiethorn apple exposed from 29 May to 12 June (weeks 22-24) and four periwinkles exposed from 15 June to 11 July(weeks 24-28) were infected. In 1996, out of the 453 baitplants, 17 were infected by a SP, i.e. four thorn apples.10 periwinkles and three grapevines (Chardonnay andPinot noir).

Figure 5 shows both the results in 1996 of stoibur infec-tion in bait plants from week 24 to week 31, and thepresence oi H. obsokfm expressed as the total number ofadults captured by sticky traps in the experimental V and

a.

1• •

"o

c

2 •

0 1

A VI, V2. V3 1

"5

c 0?

So,

n

-f|-|

iu1

22 23 24 2S 2G 27 2a 2S 30 3i 32 23 34

1 ESii;:ky traps 1SS5J

—•—D-VAC 1S96 I

Fin. 2 Trappmg of Hralesihe.s iihsoleius in 1995 and 1996 in vineyardblocks (Vi,V2 and V3)and in fallow land blocks (Fl . F2. F3 and F3')in the Rhone valley, vvith two diflerent methods of capture. .\: totalvveeklv capture bv yellow sticky traps (nine traps per week) in V blocksin 1995 and 1996: B: total weekly capture by yellow sticky traps (ninetraps per week) in F blocks in 1995 and 1995: C: comparative capturesin 1996 in F3' between D-vac suetion (Smin trapping every 2 weeks)and sticky traps (three traps per week). No adults were captured fromweek 14 to week 24 (week 23 for C) and from week 33 (week 34 for B)to week 40

F blocks during the same period. Percentages of stolbur-infected bait plants were 14% (7 infected;51 exposed) forweek 24 and 25. 8% (4/51) for week 26 and 27, 6% (3/51)for week 28 and 29 and 6% (3/51) from week 3 0 - 3 1 .

Transmission trialsExperimental transmission of SP to grapevine, periwinkleand thorn apple was obtained only with wild H. ohsoletiis.All transmission trials with all other wild Hemiptera spec-ies failed. The acquisition process of SP by rearedleafhoppers, S. titamis (0/69) on grapevine with symp-toms which had tested SP positive, or by E. rariegalux(0/25) on infected periwinkle, did not result in infectedinsects (Fig. 1; lane 6-9).

Hvalcsthes ahsoletus transmitted SP in 1995 to grape-vine (8/3!) and to periwinkle (1/1). and in 1996 to grape-

554 S)-ORZ.A et al.

Kbp ••• i: I " 1 2 3 4 5 6 7 8 9 10 II 12 13

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}IL! M\) Lo!lLi_t,.J '11 1 !!kn\ 1 iuU 2 h i i d u ^ ^ d n u n ^ •nputniN coll i.ti.Jlii lalli \\ i hid witii n m p l i s ot / / c/ ^ ' i-^ iMi u t5t> " h t i l ' iu C h rd o n n n 4 " \ m c ^ . . •^^ ib iun-nhxlopi INMI i s\ '>ip'i m^ 4 C h tulu i i\• GrLna thc '• p!n">[noh " C iiis lut i \ \ t li- b<ndi ^ b i n p h u i p ^ i i\\!i"\kic t WUh ^^P3ptO Ti^ O\ \^\\K\\^ ^\pi. M «J ill 1 I'lL*. K! "^loi-k slOni 2^lunc irt " luK IP i^^h --> liL Llt^n S . L J I i i j l^n-n mpl i / ) m•^'} n >i uni) !0 bus p ' i P l ) horn tnp iL iu !h \ m p t o ii ^\poM.d ii \\ [ n c \ 1 ct hloLi fi • 1 in lunt *i 2 " U iv " i' '-'fi II ] uus t \ l i i b u i i_s p i P t o n s 'it*. c\pi-[ ^Kiit l i t n-^m-vs ' 11 t'! ^tt Ibui p] \ It pi isPi a '">

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Fig.-^ 7nA'i digjstu^n oi tht nc?->ti,-J P( B p ioduL ' s k\uh pIinlL]^ Fl P "and tT_ ^ iL ^ lisnii: ttrmplatc DN-X tiiini u i JJ plants LoilcetL'd m c-.pciimemai bloLl s a n d 111 in bai l pijnt% in Rhnrpc . j l l e \ in UWd \1 iTmieL-ular weight marLei ( !kb laddL* BRLi H P h^\iitln [ i t i i u m t j c(Calhiiumllni-, uiMif,! IP p^nv.inUi.'li '. '^xIcU with a i a i n S T O L C 1symptomk-ss hciar\ ^ics-. ' < auhina Jfuhuf 2 ^viripUimatK hc-au Liess3-10 i\i!d s h i u b - and t rc j^ s.cflL'cled in iaUcv land biuLk^ ^ ^ \ m p -tomless cherr*. iPiunu- sp } 4 diseased SHLLI cheirv iPiunu^ sp ). 5sympiomjcss jig tsee {fuir ctiran f sur-plumatiL iisi tret.. " s^mp-tomal ie ]l]ae ! S I f//;ec/F 2//[,'i!f/^) N s \ n i p t o m j t K elm i t//iiiis sp f ' Kibait p lants e\pt<scU m \ j n e \ a i d h lo t l s t i um ]0 iunt. to 26 Uuic >s y m p t o i n k s s C h a i d o n n a v 10 rha id< i i ina \ u i t h ' - \ m p i o m i ol \ c ! k n \ s

vine (17/61), to periwinkle (16/27) and to ihorn apple(3/4). Successful transmissions were realized as well withthe first adults emerging in June as with specimens cap-tured in July and August (data not shown). The SP infec-tion was confirmed by PCR and examples of assays aregiven in Fig. 3 (lane 11-13).

The survival of adults of wild H. obxoleliix caged ongrapevine or bindweed were compared. Starting with 53adults on the grapevine, 80% of mortality was obtainedin the first 3 days and none exceeded 7 days. However,survival on bindweed was much longer, starting with 80adults, 75% (60) were still alive after 7 days, 25% (20)after 21 days. 12% (10) after 35 days, and the last onewas observed at day 56.

PCR assays on plant material with symptoms from the fieldsFigures 3 atid 4 present some results of PCR assays ongrapevine, bait plants, and naturally infected plants col-lected in the fields (Fig. 3. lane 1-12: Fig. 4, lane 1-8). Ingrapevine, positive assays with STOL J1 O/rl (Fig. 3.lane 4-7) were obtained from Chardotinay (20 inlected/29tested). Grenache (1 2). Pinoi tioir (1:2) and Cinsaul (3/3)collected in the experimental blocks and surroundingvineyards. This showed that BN was prevalent in theregioti, in agreement with other reports (Daire etal..1997b).

SP was detected in bindweed with STOL ! I O-rl (31infected 61 tested) (Fig. 3. lane 1-2). Nested PCR wassometimes necessary to detect SP in other plants, i.e.hoary cress (3 7). sweet cherry (16), plum (i 1). lilac(1 ' 11, fig tree (I 3) and elm (1 2) as shown in Fig. 4 (lane1 -8). Symptoms on hoary cress were dwartism. reddeningofthe leaves and an absence of flowers; a general declineatid a yd lowing of leaves were noticed on sweet cherryand elm. a decline and a shrivellitig of leaves on plumand lilac, and decline on fig tree.

DiscussionThe results of ihis study show for the first time the majorrole of H. absolelus iti BN disease occurrence in France,in this study, it was also found to be an efticient vectorof SP to grapevine in spite of its poor survival when itwas caged on this plant. Results for the percentage ojinfected //. obsolelus among the populations were inagreement with previous studies (Fos etal., 1992; Maix-tier etal., 1995). As BN and VK have closely related, iftiot similar, pathogenic agents and have the same itisectvector, it appears that they are sitnilar diseases occurritigin different geographical areas on different cultivars.

Hvale.slhes obsoletus was the major species found to beslolbur-infecled. Although wild leallioppers, namely M.crocea and E. lineolatus, were occasionally found to becarrying SF, no transmission was obtained with thesespecies. In France, Fos etai. (1992) detected SP in threeother leafhopper genera, namely Aphrodes sp., Neo-aliturus sp., and Psammotettix sp. The latter species wasalso found infeeted by a phytoplasma in Italy (Albaneseet al., !997). In the present study high numbers of adultsamong these genera were tested without detecting SP.This might suggest an occasional acquisition by thesegenera. Their ability to transmit should be demonstrated.In addition, the inability of feeding access acquisition byreared S. titanus and £. variegatus strongly suggested thatSP transmission is specific.

Detection in plants from the fieid showed that bind-weed and hoarv eress, but also fie tree, lilac, elm, and

Role of Hyalesthes ahsoletus in Bois noir Occurrence of Grapevines in France 555

60

2G+3P-I-2T

• •

1G+3P+1T

il0G+2P+1T

l l

^ Nb of stolbur-infected bait plants

0G+2P+1T

M m^ ^— ^^22 23 24 25 26 27 26

Week

29 30 31 32 34

Fig. 5 Comparison between the number of stolbur-infected bait plants and the number of Hyalesthes ohsolelus captured in the experimental blocksin Rhone valley in 1996. The bars give the total number of adults captured during 1 week by 21 sticky traps in all the blocks. The stolbur-infectedbait plants were obtained during a 2-week period among the 51 bait plants exposed in all the blocks. All plants with symptoms were PCR testedand turned out to be stoibur positive. No adults were captured from week 14 to week 23 and from week 34 to week 40; no stolbur-infected baitplants were obtained in the same period. G. grapevine: P. periwinkle: T. thorn apple

sweet cherry could be infected by SP. The presence of SPin bindweed was suspected by Cousin et al. (1969) andconfirmed by Fos et al. (1992) and Maixner et al. (1995).In the present study, symptomatic bindweed were oftenfound in vineyards and fallow lands, and SP was detectedin a high proportion of samples, using direct PCR withSTOLI 1 f2 r l . The results on hoary cress are new inter-esting data, as this plant was known to be a host of H.ohsoletus in Eastern Europe (Guclu and Ozbek, 1988).Thus, hoary cress might be a reservoir for the SP inocu-lum. As detection on this species was obtained only bynested PCR, it might be a low titre host for the SP, whichcould explain why it was not reported in previous studiesusing other less sensitive detection methods. The resultsof SP infection in lilac, elm, and fig tree were unexpecteddata. However, the DNA fragments amplified wereclearly visible and negative results were obtained from anumber of samples from the same species. The occurrenceof SP in such plant species could result from occasionalinoculation during feeding probing.

The study has shown the role of hoary cress and con-firmed the role of bindweed as host-plants of//, ohsotetusin France. Thus, together with lavender, three plant spec-ies are now known in France both as host plants andstoibur reservoirs (Leclant, 1968; Fos etal., 1992). Hoarycress and bindweed were numerous in fallow lands, suchas F3'. As H. ohsoletus was regularly captured in F3', thelatter site should be considered as a source of inoculumfor the disease. From such a site, aduits could transportSP from these hosts and occasionally inoculate grapevine,shrubs and trees. The poor survival on grapevine sup-ports the assumption that feeding on this plant isoccasional. Effective transmission was nevertheless poss-ible on grapevine cuttings as well in controlled conditionsas in the open air. In order to control BN disease, special

attention should be devoted to fallow lands presentingcharacteristics of stoibur inoculum seats near the vine-yards.

It was shown that the inoculation period of bait plantsoccurred during the activity of adults. The threat to agrapevine would be constant during a 3-month period inthe Rhone valley and in other viticultural areas wherepositive //. ohsoletus were found. In a previous study ontomato stoibur. Gamier et al. (1990) used only periwinkleas bait plants. To ensure a qualitative approach of plantspecies susceptible to SP inoculation, a greater diversityof plants was used in the present study. It was shownthat inoculation could occur from beginning of June toAugust.

This is the first report of the experimental inoculationof SP by H. ohsoletus to thom apple. The species hadalready been reported as naturally infected (Valenta et al.,1961) and dodder inoculated (Marchoux etal., 1970a).At the earliest period of inoculation (beginning of June),SP could be inoculated to grapevine, periwinkle and tothorn apple with wild H. obsoletus from the first captures.Thus, It can be assumed that an acquisition process takesplace at nymphal stage on roots of infected host plants.This is supported by SP detection on the fifth instar foundon diseased bindweed. No studies have been conductedup to now on the route and multiplication of phyto-plasmas in planthoppers. On the basis of the first dataobtained on experimental acquisition of SP by H. obso-letus in the laboratory, it can be assumed that SP have asimilar cycle in the insect body as described in leafhoppervectors for other phytoplasmas which persist and mul-tiply in the organs of the vector (Maillet and Gouranton,1971; Lherminieretal., 1989; Lefoletal,, 1994).

Further data on the ethology of H. ohsoletus and SPtransmission will be obtained with the use of exper-

556 S F O R Z A et al.

imentally reared colonies of this species, in studies cur-rently being undertaken,

AeknowledgemoiK

This study was supported by Association Nationale de la RechercheTechnique and grapevine industry in Rh6ne-AI[>es. The authors aregratefu! to Dr T. Bourgoin for his scientific advice, to M. Collin, B.Alixant, Ph, Lecomte and Ph. Toussaint for their technical supply. Theauthors also thank Dr M. F. Clark and Dr E. Herrbach for criticalreading of the manuscript.

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