Emerging phytoplasma diseases: research of the insect vectors - PowerPoint PPT Presentation

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

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

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

Europe � Maize redness – Stolbur phytoplasma (16SrXII-A) � Serbia, Bulgaria, Romania, Hungary, Italy � Vectors: Reptalus panzeri Jovi ć , 2010

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

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

Knowledge of the insect vectors Well timed and efficient control strategies

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

The Malaise traps

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

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

The beating tray

The sweep net

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

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

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

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

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

The identification of collected insects The dissection

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

The identification of collected insects Reptalus spp. R. q. R. c. R. p. R. m. M COI-PCR + Alu I R.q.= R. quinquecostatus R.c.= R. cuspidatus R.p.= R. panzeri R.m.= R. melanochaetus ; 600bp 300bp Bertin et al., 2010. Bulletin 100bp of Entomological Research R. p. R. q. R. c. R. m. R. c. R. p. M M 900bp 400bp 1600bp 200bp 1000bp Taq I ITS2- PCR �

The identification of collected insects H. obsoletus H. luteipes H. scotti M Hyalesthes spp. 600bp Bertin et al., 2010. Annals of 300bp Applied Biology COI-PCR + Alu I H. s. H. o. M H. s. H. l. H. o. M 800bp 1600bp 200bp 500bp Taq I ITS2-PCR �

The identification of collected insects Cacopsylla spp. C. melanoneura C. affinis � MEL_fw/MEL_rev DUPLEX-PCR � AFF_fw/AFF_rev Mitochondrial Control Region (CR)-PCR Tedeschi & Nardi, 2010. Bulletin of Entomological Research.

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

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

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

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