Entwicklung und Anwendung von Methoden für einen frühen Nachweis der Flugbranderreger Ustilago nuda und U. tritici in Gerste und Weizen

  • Development and implementation of early detection methods of the loose smut pathogens Ustilago nuda and U. tritici in barley and wheat

Wunderle, Jan Ulrik; Slusarenko, Alan (Thesis advisor)

Aachen : Publikationsserver der RWTH Aachen University (2013)
Dissertation / PhD Thesis

Aachen, Techn. Hochsch., Diss., 2013

Abstract

Infestation of barley (Hordeum vulgare) and wheat (Triticum aestivum) with loose smut (pathogen: Ustilago nuda and U. tritici, resp.) is not visible until the appearance of the smutted head. Both the development of seed treatment products for loose smut control and the breeding for loose smut resistance could be accelerated if it were possible to earlier detect and quantify an infestation with loose smut. In the present work the development of the fungus in its host plant was monitored by fluorescence microscopy with the aim to test and establish such methods. Mycelium which invaded the shoot apical meristem (SAM) and the leaf primordia could be observed in hand sections stained with the fluorophore Blankophor, within a few days after sowing. At the one node stage the ear primordia were already heavily affected and the fungus was regularly present in the nodes as well. Furthermore, with a polyclonal antibody-based DAS-ELISA it was possible to detect the fungus inside the host plant. U. nuda protein content in the area of the SAM could be reliably quantified in spring barley already at the three leaf stage (10-14 days after sowing), and future ear symptoms could be predicted. For winter wheat a sufficient prediction was not possible until the one node stage. After application of efficient seed treatments (chemical treatment with Sedaxane or Raxil [active ingredient: tebuconazole] and warm or hot water treatment, resp.) amounts of the U. nuda protein were generally below 500 ng/ml and thus in a similar range as in plants grown from healthy grains. Microscopic analyses performed in parallel suggested that the mycelium, depending on the method applied, died off rapidly (seed treatment with Sedaxane) or temporarily persisted in a damaged condition (seed treatment with tebuconazole, hot water treatment). After seed treatment with extracts of rhubarb (Rheum rhabarbarum) root or butterbur (Petasites officinales) at the three leaf and one node stage partially lower amounts of U. nuda resp. U. tritici protein were determined than in the controls, suggesting an activity of these agents. However, the treatment had no or only little effect on the incidence of smutted heads. Moreover, a protocol for specific amplification and quantification of U. nuda resp. U. tritici DNA (real-time PCR) was established. DNA was extracted from infected plants and amplified with fungus-specific primers. Also with this method a prediction of an infestation with loose smut was possible already at the three leaf stage. Overall, there were good matches concerning the results of microscopy, DAS-ELISA and real-time PCR. The latter, however, was more sensitive by a factor of 250 than the immunological approach. The results of this work showed that the development of early test systems for predicting loose smut infestation is possible with various methods, indeed. Though, for routine use optimizations are necessary, as in the case of DAS-ELISA, a safer assess of the U. nuda protein content (threshold), at which an ear infestation is to be expected or, when using all of the methods, the use of seed stocks that are germinating unabatedly well in spite of high infection rates.

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