Putting a historic view in molecular perspective Potato wart - - PowerPoint PPT Presentation

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Putting a historic view in molecular perspective Potato wart - - PowerPoint PPT Presentation

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Putting a historic view in molecular perspective Potato wart disease workshop 26-6-2019 Presentation outline Spread of potato wart disease in the last century Putting the historical collection to use Towards molecular

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Putting a historic view in molecular perspective

Potato wart disease workshop 26-6-2019

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Presentation outline

▪ Spread of potato wart disease in the last century ▪ Putting the historical collection to use ▪ Towards molecular characterization of the Dutch isolates ▪ Optimization of zonal centrifuge for the extraction of spores from

soils and composts

▪ DNA/RNA extraction and quality control ▪ Sequencing and haplotyping

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Potato wart disease was first found in 1915

▪ Found in Winschoten (Groningen) in a private garden on “‘t Zandpad” ▪ Found by Mr. Uil who was a teacher at an agricultural school ▪ Poverty-stricken part of town: home-grown food for own use

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Stichting Oud-Winschoten, ‘t Zandpad ~1930

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Timelapse 1915 - 1927

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Rapid spread in 1926 – 1927 along the course of the river Meuse following massive floods

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Beneden Leeuwen 1926 Gennep 1926 Roermond 1926 Tegelen 1926

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First

  • utbreaks

vs current risk zones

Current zones of increased vigilance are shown in red

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Brief period of absence and rediscovery

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▪ Strict phytosanitary measures led to a brief reported absence of the

pathogen in 1970

▪ Re-discovery of the pathogen in 1973 in the north of the Netherlands

  • Before this was pathotype 1(D1), new findings were 2(G1)

▪ In 1990, pathotype 6(O1) was reported from north-east Netherlands ▪ In 2001, pathotype 18(T1) was reported from N-E Netherlands ▪ In 1990 also rediscovery of the pathogen in south-east Netherlands

  • In contrast to northern provinces, only pathotype 1(D1) was

found

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Compost collection at NVWA

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Collection (183 pot ID) on tour

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Hendrickx zonal centrifuge at PPO-AGV

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Schematic side view of the Hendrickx centrifuge in operation. 1: separation fluid (CaCl2 1.4 SD ), 2: water, 3: soil suspension, 4: kaolin suspension, 5: overflow A: sediment from soil, B: kaolin, C: CaCl2, D: fluid with sporangia, E: water, F: hollow drive tube, G: bottle with supernatant 15.000 x g

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Optimization of ZC method

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Washing & concentrating fluid with sporangia

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Yield of spores on 20 um Nylon net filter

Filter funnel combined with nylon net

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Differences in yield due to different composts

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Pot ID 100 Larger pieces after ZC Wart material containing ws

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Remarkable things

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Pot ID 83 and 96

extra sieving 40uM Spore suspension is very difficult to filtrate through 20uM nylon sieve

Many ws are encapsulated In starch?

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After ZC nicely cleaned up WS

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Preparing for nucleic acid extraction

▪ Total of 183 pot ID ▪ Oldest is from the 80s ▪ At least 83 pot ID registered

as NL

▪ 11 pot ID < 100 gr compost ▪ 54 pot ID too low yield of ws

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DNA-RNA extraction (cat no 80204)

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DNA-RNA extraction (cat no 80244)

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Molecular assays

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Comparison kits: DNA samples (60ul)

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AllPrep power fecal AllPrep Avg Ct = 22.31 Sendo Avg Ct = 32.83 COX dCt = 10.52 Avg Ct = 24.69 Sendo Avg Ct = 31.74 COX dCt = 7.05 Sendo COX

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Results of 203 composts in TaqMan assays using MB42 DNA as standard curve

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Sendo COX

Pot ID 1 - 104 Pot ID 105 - 203

Sendo COX

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Selected isolates for sequencing

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potID 1 9 17 25 42 49 57 73 81 89 97 2 10 18 27 43 50 65 74 82 90 98 3 11 19 28 47 51 68 75 83 91 99 4 12 20 31 MQ 52 69 76 86 92 100 5 13 21 32 MQ 53 71 77 88 93 101 6 14 22 34 MQ 54 72 78 MQ 94 102 7 15 23 37 MQ 55 MQ 79 MQ 95 104 8 16 24 38 MQ 56 MQ 80 MQ 96 MB42 ng/ul 0.4 0.1 0.2 0.3 0.1 0.4 1.4 0.6 0.2 0.5 0.2 0.1 0.2 0.3 0.1 0.1 0.2 0.6 0.4 0.4 1.1 0.5 0.4 0.1 0.3 0.4 0.1 0.2 0.3 0.2 0.2 0.5 0.8 0.1 0.1 0.3 0.6 0.1 0.3 0.3 0.1 1.3 0.2 0.6 0.1 0.1 0.4 0.1 0.1 0.3 0.3 0.7 0.1 0.2 0.6 0.1 0.1 0.1 0.1 0.1 0.3 0.2 0.2 0.1 0.3 0.1 0.1 0.1 0.2 0.2 0.1 0.6 0.1 0.2 0.1 0.3 1.2 0.1 0.2 0.2 0.2 0.1 0.1 0.1 1.3 0.1 0.5 1.2

Picogreen measurement: Very low DNA concentration

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Different mitochondrial lineages?

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Use of historical collection

▪ 183 pot ID (isolates) ▪ Many of them are not viable ▪ Volume reduction by storing collected ws dry ▪ 129 isolates DNA/RNA extracted ▪ 118 are of enough quality ▪ Sequencing ▪ Hope to build a new network and understand the spread

and epidemiology

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Co-operation

▪ Wageningen UR, Biointeractions and Plant Health

  • Marga van Gent-Pelzer
  • Theo van der Lee

▪ Dutch National Plant Protection Organization

  • Bart van de Vossenberg
  • Patricia van Rijswick
  • Gerard van Leeuwen

▪ Wageningen UR, Field Crops

  • Olaf Hartsema
  • Andre Ramaker

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