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

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

Sep 07, 2023 172 likes •451 views

Emerging phytoplasma diseases: research of the insect vectors Alberto Alma and Rosemarie Tedeschi DIVAPRA Entomologia e Zoologia applicate allAmbiente C. Vidano University of Torino - Italy In the last years: Discovery of new

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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

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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

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Europe

‘Candidatus Phytoplasma pini’

(16SrXXI, subgroup A)

Poland, Lithuania, Germany, Spain Vectors: unknown

Kamìnska & Berniak, 2011 Valiunas et al., 2010 Kamìnska et al., 2011

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Maize redness – Stolbur phytoplasma

(16SrXII-A)

Serbia, Bulgaria, Romania, Hungary, Italy Vectors: Reptalus panzeri

Jović, 2010

Europe

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Mediterranean Basin and Middle East

‘Candidatus Phytoplasma phoenicium’

(16SrIX)

Lebanon, Iran Vectors: unknown

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The insect vectors

New phytoplasma diseases New outbreaks Unknown Known Possible new vectors

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Knowledge of the insect vectors Well timed and efficient control strategies

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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

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The Malaise traps

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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

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The sticky traps

A good support to the Malaise traps for a

preliminary screening

Very good to study the population dynamics

  • f a selected group of possible vectors

‼ No direct association with the crop or the wild

vegetation

‼ No alive specimens

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The beating tray

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The sweep net

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Very cheap Allow to investigate plant species selectively

The beating tray and the sweep net

Allow to collect alive insects

Host plant association Molecular analyses, laboratory rearings, transmission trials

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The vacuum insect collector (D-VAC)

Very useful to sample dense vegetation or the soil surface Collection of alive insects

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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

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The identification of collected insects

Order Hemiptera

(Feeding using piercing and sucking mouth parts)

Sternorrhyncha

(whiteflies, scale insects, aphids, psyllids)

Auchenorrhyncha

Leafhoppers and planthoppers, spittlebugs etc

Heteroptera

(True bugs)

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Use morphological tools following

dichotomous keys.

Good expertise is required Appropriate grounding of the technicians

The identification of collected insects

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The identification of collected insects The dissection

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In the case of criptic species The identification of collected insects

Molecular tools for species discrimination

They are very useful also for females and young stages

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The identification of collected insects

  • R. q.
  • R. c.
  • R. p.
  • R. m.

M 600bp 300bp 100bp R.q.=R. quinquecostatus R.c.=R. cuspidatus R.p.=R. panzeri R.m.=R. melanochaetus;

Bertin et al., 2010. Bulletin

  • f Entomological Research

Reptalus spp.

  • R. q.
  • R. c.
  • R. p.
  • R. m.

M 1600bp 1000bp

  • R. c.
  • R. p.

M 900bp 400bp 200bp

COI-PCR + AluI ITS2- PCR

  • TaqI
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The identification of collected insects

M

  • H. scotti
  • H. luteipes
  • H. obsoletus

600bp 300bp

COI-PCR + AluI

  • H. s.
  • H. l.
  • H. o.

ITS2-PCR

  • TaqI

M 1600bp 500bp

  • H. s.
  • H. o.

M

800bp 200bp

Hyalesthes spp.

Bertin et al., 2010. Annals of Applied Biology

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The identification of collected insects

MEL_fw/MEL_rev AFF_fw/AFF_rev

DUPLEX-PCR

Mitochondrial Control Region (CR)-PCR

Cacopsylla spp.

  • C. melanoneura
  • C. affinis

Tedeschi & Nardi, 2010. Bulletin

  • f Entomological Research.
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Preliminary screening to assess the presence

  • f phytoplasmas

Molecular analyses to ascertain the presence of phytoplasmas in the insects Positive insect

≠ ≠ ≠ ≠

vector

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They are the only prove of vector ability

Transmission trials

Infected plants Acquisition access period (AAP) Healthy plants Healthy plants Latent period (LP) Inoculation access period (AAP) Plants tested for phytoplasma presence by molecular tools

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Transmission trials

Very laborious procedure that requires big ammounts

  • f healty and infected insects and healthy plants

Sometimes we do not know the host plants

Transmission trials to an artificial feeding medium

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Transmission trials to an artificial feeding medium

Feeding solution covered by parafilm Breeding chamber Cotton for air source