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Early Detection and Quantification of Ramularia Beticola in Sugar Beets

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Early Detection and Quantification of Ramularia Beticola in Sugar Beets FACULTY OF SCIENCE UNIVERSITY OF COPENHAGEN Early detection and quantification of Ramularia beticola in sugar beets using spore traps and QPCR and the effect of early fungicide treatment on Ramularia leaf spot MSc Thesis Thies Marten Wieczorek GCH350 December 2012 Supervisor (Sciences): Lisa Munk Supervisor (AU): Lise Nistrup Jørgensen Supervisor (AU): Annemarie Fejer Justesen Supervisor (extern): Anne Lisbet Hansen NBR Nordic Beet Research Abstract Ramularia leaf spot (RLS) is one of the most important foliar diseases in sugar beets in the temperate climates. It is caused by the ascomycete Ramularia beticola and thrives best at relatively high humidity (> 95 %) and temperatures between 17 and 20 °C. If not controlled, yield losses can amount to 15 ± 20 %. The disease is typically controlled using fungicides like epoxiconazole or epoxiconazole + pyraclostrobin. Preliminary field trials have indicated a more efficient control of fungal diseases and higher sugar yield if sugar beets are treated with fungicides before visual symptoms occur. For a better determination of the optimized timing of disease control, more advanced methods are needed. This MSc thesis was divided into two parts: 1) a molecular part with the goal to develop a QPCR-based method to detect and quantify R. beticola DNA recovered from plastic tape used for the purpose of spore trapping, and 2) an agronomical part evaluating the effect of early fungicide application on RLS as well as to investigate the fungus presence in the air above sugar beet fields in Denmark using Burkard-Hirst traps. Primers for the use in QPCR were designed based on the transcribed space region 2 (ITS 2) and tested on a number of R. beticola isolates and on isolates from relative fungi (M. graminicola, R. collo-cygni and C. beticola). Primer pair Rb2f/Rb4r was found to be highly specific. DNA form tape used in the spore traps was efficiently extracted using a commercially available kit. Using QPCR it was possible to detect low amounts of R. beticola DNA. Furthermore, it was demonstrated that the developed primers can be used for the detection of fungal biomass in living tissues. Two field experiments were conducted in order to elucidate the effect of early fungicide treatments. Results showed a tendency that treatments which took place 4 to 5 weeks prior to first visible symptoms had a positive effect on yield parameters and slowed down the development of the disease in the beginning of the leaf disease season. At the end of the season, however, all fungicides treatments performed equally well. Three Burkard spore traps were operated on the Danish island of Lolland from which air samples were collected on a daily basis throughout weeks 28 - 37. Based on the molecular method developed in 1) first spores in the air samples were detected 14 and 16 days prior to first visible symptoms on untreated plants, which is in accordance with the reported latency of 2 weeks of the fungus. The spore concentration in the air varied strongly. Three massive spore releases were observed, which were influenced by low mean temperatures and RH around 95 % one week before. It is expected that the results can lead to an IPM based monitoring/warning system where spraying only takes place when a real risk has been verified based on spore trapping or weather data. I Zusammenfassung Die Ramularia Blattfleckenkrankheit ist eine der wichtigsten Zuckerrübenkrankheiten in den gemäßigten Breiten. Sie wird hervorgerufen durch den Erreger Ramularia beticola, der am besten bei hoher Luftfeuchtigkeit (> 95 %) und Temperaturen zwischen 17 und 20 °C gedeiht. Zur Bekämpfung der Krankheit steht eine Reihe von Fungiziden zur Verfügung. Erfolgt keine Behandlung der Krankheit, können Ertragseinbußen von bis zum 15 ± 20 % erreichen werden. Einleitende Feldversuche haben gezeigt, dass frühzeitige Fungizidbehandlungen, d.h. vor dem Erscheinen erster sichtbarer Symptome, zu einer besseren Kontrolle und höherem Zuckerertrag führten. Um den idealen Behandlungszeitpunkt besser bestimmen zu können, bedarf es neuer Ansätze zur frühzeitigen Erkennung von Zuckerrübenkrankheiten. Diese Masterarbeit ist in zwei Teile gegliedert. Der erste Teil behandelt die Entwicklung einer auf QPCR basierender Methode, mit deren Hilfe R. beticola DNS detektiert und quantifiziert werden kann. Zusammen mit Burkard-Hirst Sporenfallen wird sich dieser Methode bedient, um die Fluktuation von R. beticola Sporen in der Luft zu folgen. Im zweiten Teil wird der Effekt von frühzeitigen Fungizidbehandlungen auf die Entwicklung der Ramularia Blattfleckenkrankheit und den Zuckerertrag getestet. Die Primer (Rb2f/Rb4r) zur Anwendung von QPCR ZXUGHQ DXI %DVLV GHU ³WUDQVFULEHG VSDFH region 2´ ,76 HQWZLFNHOW'LHVHZLHVHQVLFKGXUFKHLQHKRKH6SH]LILWlWund Sensibilität aus. Es traten keine Kreuzreaktionen mit M. graminicola, R. collo-cygni und C. beticola auf. DNS wurde erfolgreich mittels eines kommerziellen erwerbbaren Kits extrahiert. Mit Hilfe der QPCR war es möglich niedrige Mengen von R. beticola DNS nachzuweisen. Zwei Feldversuche mit frühen Behandlungszeitpunkten wurden durchgeführt. Die Parzellen, die 4 bis 5 Wochen vor dem ersten Auftreten von Symptomen behandelt wurde, wiesen tendenziell die besten Ergebnisse hinsichtlich Erträge und Krankheitskontrolle auf. Drei Burkard-Sporenfallen wurden im Zeitraum vom KW 28-37 zur Sammlung von Luftproben eingesetzt. Mit Hilfe der Methode aus dem ersten Teil dieser Arbeit war es möglich R. beticola Sporen schon 14 bzw.16 Tage vor dem Erscheinen der ersten Symptome nachzuweisen. Die Sporenkonzentration variierte stark und wurde durch hohe mittlere Luftfeuchtigkeit und niedrigen mittlere Temperaturen eine Woche vor Masssporenabgabe beeinflusst. Mit Hilfe der QPCR war es über die Detektion von Sporen hinaus möglich die Entwicklung des Erregers in Zuckerrübenblätter zu verfolgen. Die Ergebnisse dieser Arbeiten könnten mit in die Entwicklung eines IPM - Warnsystems eingehen, mit dessen Hilfe Fungizidbehandlungen nur dann stattfinden, wenn ein erhöhtes Risiko für die Entwicklung von Blattkrankheiten nachgewiesen wurde. II Sammendrag Ramularia (pletskimmel) er en alvorlig sygdom i sukkerroer i lande med tempereret klima. Årsagen til sygdommen er svampen Ramularia beticola. Ramularia kræver en høj luftfugtighed (> 95 %) og temperaturer mellem 17 og 20 °C. Forsøg har vist at svampebekæmpelse kan medføre udbytte stigninger op til 10 ± 20 %. I de nuværende anbefalinger angående fungicidbehandling (epoxiconazole eller epoxiconazole + pyraclostrobin) tilrådes at udføre første sprøjtning ved begyndende angreb. Imidlertid har indlendende markforsøg vist at der er potentiale for forøget sukkerudbytte og mere effektiv bekæmpelse af bladsvampe ved tidlig behandling, foretaget inden synlige symptomer forekommer. På nuværende tidspunkt findes ingen effektive metoder til at monitere, hvornår svampesporer befinder sig i marken, som et grundlag for fremtidigt at udvikle en risikovurdering for angreb og en ny bekæmpelsestærskel for hvornår der i givet fald kan behandles. Formålet med specialet var 1) at udvikle en moniteringsmetode til detektion af forekomst af pletskimmelsporer baseret på den molekylær metode QPCR anvendt i forbindelse med Hirst- Burkard sporefangere til at detektere og kvantificere svampe DNA samt at følge forekomst af R. beticola sporer i luft ved hjælp af sporefangere og 2) at undersøge effekten af tidlig behandling med fungicider på udvikling af Ramularia og sukkerudbytte, samt at sammenholde resultaterne herfra med forekomst af sporer i luften detekteret i 1). Til QPCR var grundlaget for primer designet svampens ribosomale DNA region "transcribed space region 2´ ,76 3ULPHUSDUUHW5EI5EUYLVWHVLJDWY UHPHJHWVSHFLILNWGDGHWEOHY testet med andre svampe (M. graminicola, R. collo-cygni and C. beticola). Det lykkedes at ekstrahere svampe DNA fra tape ved brug af et kommercielt kit og at detektere meget små DNA mængder af R. beticola DNA. I to markforsøg blev effekten af fungicidbehandlinger ved forskellige tidspunkter undersøgt. Selvom resultaterne ikke var signifikant forskellige, viste de en tendens til at behandlingerne påbegyndt 4 til 5 uger inden synlige begyndende symptomer af Ramularia havde den højeste udbyttestigning og en bedre bekæmpelseseffekt i starten af svampesæsonen. Det var tre Burkard sporefangere der blev sat i gang for at indsamle luftprøver på døgnbasis i uge 28 - 37. Ved hjælp af den tidligere udviklede QPCR metode var det muligt at identificere R. beticola sporer i luften allerede 14 til 16 dage inden synlige symptomer af bladsvampen forekom. I hele perioden varierede sporekoncentration meget. I alt blev det konstateret tre tidspunkter hvor massive frigivelser af sporer fandt sted. Sporefrigivelsen blev sammenholdt med samtidigt registreret relativ luftfugtighed og temperatur. Udover detektion af Ramularia-sporer fra sporefælderne viste metode sig også anvendelig til detektion på selve bederoebladene. Det forventes at resultaterne kan bidrage til udvikling af et ny moniterings- / varslingssystem for i første omgang bekæmpelse af Ramularia og med deraf forbedret IPM model til bekæmpelse af sygdommen. Metoden forventes at kunne udvikles også for andre bladsvampe i bederoe III Résumé La ramulariose est une maladie, provoquée par le champignon ascomycète Ramularia beticola affectant le feuillage de la betterave sucrière. Le développement de la ramulariose se produit à des températures relativement basses, comprises entre 17 et 20 °C ainsi qu´une humidité relative dépassant 95 %. Une perte de rendement remarquable, entre
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