Alberto Alma and Rosemarie Tedeschi DIVAPRA Entomologia e Zoologia applicate all Ambiente «C. Vidano» University of Torino - Italy

Transcription

1 Emerging phytoplasma diseases: research of the insect vectors Alberto Alma and Rosemarie Tedeschi DIVAPRA Entomologia e Zoologia applicate all Ambiente «C. Vidano» University of Torino - Italy

2 In the last years: Discovery of new phytoplasma diseases Outbreaks of already known phytoplasma diseases Europe Mediterranean Basin and Middle East Impact on EU Countries

3 Europe Candidatus Phytoplasma pini (16SrXXI, subgroup A) Poland, Lithuania, Germany, Spain Vectors: unknown Valiunas et al., 2010 Kamìnska & Berniak, 2011 Kamìnska et al., 2011

4 Europe Maize redness Stolbur phytoplasma (16SrXII-A) Serbia, Bulgaria, Romania, Hungary, Italy Vectors: Reptalus panzeri Jović, 2010

5 Mediterranean Basin and Middle East Candidatus Phytoplasma phoenicium (16SrIX) Lebanon, Iran Vectors: unknown

6 The insect vectors New phytoplasma diseases Unknown New outbreaks Known Possible new vectors

7 Knowledge of the insect vectors Well timed and efficient control strategies

8 How to optimize the search of the vectors Choise of the best sampling method/s Do not restrict the search only to the crop of interest, but extend it also to the surrounding weeds and shrubs

9 The Malaise traps

10 The Malaise traps Very good when nothing is know in a certain area High number of insects belonging to different species!! No direct association with the crop or the wild vegetation!! No alive specimens

11 The sticky traps A good support to the Malaise traps for a preliminary screening Very good to study the population dynamics of a selected group of possible vectors!! No direct association with the crop or the wild vegetation!! No alive specimens

12 The beating tray

13 The sweep net

14 The beating tray and the sweep net Very cheap Allow to investigate plant species selectively Host plant association Allow to collect alive insects Molecular analyses, laboratory rearings, transmission trials

15 The vacuum insect collector (D-VAC) Very useful to sample dense vegetation or the soil surface Collection of alive insects

16 Handling of the collected insects If molecular analyses, laboratory rearings, transmission trials are required Good practices to be followed Place collected insects in vials containing a leaf or a twig Use a cool box for transfers Preserve the samples in pure ethanol or at -20 C

17 The identification of collected insects Order Hemiptera (Feeding using piercing and sucking mouth parts) Sternorrhyncha (whiteflies, scale insects, aphids, psyllids) Heteroptera (True bugs) Auchenorrhyncha Leafhoppers and planthoppers, spittlebugs etc

18 The identification of collected insects Use morphological tools following dichotomous keys. Good expertise is required Appropriate grounding of the technicians

19 The identification of collected insects The dissection

20 The identification of collected insects In the case of criptic species Molecular tools for species discrimination They are very useful also for females and young stages

21 The identification of collected insects R. q. R. c. R. p. R. m. M COI-PCR + AluI 600bp Reptalus spp. R.q.=R. quinquecostatus R.c.=R. cuspidatus R.p.=R. panzeri R.m.=R. melanochaetus; 300bp 100bp Bertin et al., Bulletin of Entomological Research R. q. R. c. R. p. R. m. M R. c. R. p. M 900bp 400bp 1600bp 1000bp 200bp ITS2- PCR TaqI

22 The identification of collected insects H. obsoletus H. luteipes H. scotti M 600bp Hyalesthes spp. 300bp Bertin et al., Annals of Applied Biology COI-PCR + AluI H. s. H. l. H. o. M H. s. H. o. M 800bp 1600bp 500bp 200bp ITS2-PCR TaqI

23 The identification of collected insects MEL_fw/MEL_rev AFF_fw/AFF_rev DUPLEX-PCR Cacopsylla spp. C. melanoneura C. affinis Mitochondrial Control Region (CR)-PCR Tedeschi & Nardi, Bulletin of Entomological Research.

24 Molecular analyses to ascertain the presence of phytoplasmas in the insects Preliminary screening to assess the presence of phytoplasmas Positive insect vector

25 Transmission trials They are the only prove of vector ability Infected plants Healthy plants Healthy plants Acquisition access period (AAP) Latent period (LP) Inoculation access period (AAP) Plants tested for phytoplasma presence by molecular tools

26 Transmission trials Very laborious procedure that requires big ammounts of healty and infected insects and healthy plants Sometimes we do not know the host plants Transmission trials to an artificial feeding medium

27 Transmission trials to an artificial feeding medium Feeding solution covered by parafilm Cotton for air source Breeding chamber