Methods for extracting plant pathogenic nematodes from Brachiaria seed
Abstract: ABSTRACT A commonly used forage grass in Brazil is Brachiaria spp. These grasses are used as feed for beef and cattle production and are grown on approximately 70 million hectares in Brazil. A pasture with Brachiaria spp. can last for 20 years and is relatively free from diseases. Two of the pathogens that do attack Brachiaria spp. are the nematodes Aphelenchoides besseyi and Ditylenchus dipsaci. These nematodes are seed-borne and affect the host plant by eating of the plants growing parts of stems and leaves. As the plant matures, the nematodes move to the seeds where they overwinter or stay until the seeds get in contact with moisture. Both A. besseyi and D. dipsaci can survive in the seeds in a dry state for several years. Both of these nematodes are classified as quarantine pests due to the damage they cause on their host plants and economically. To stop the spreading of A. besseyi and D. dipsaci, chemical or hot water treatments are used to control the nematodes. In Sweden, a company called Incotec Sweden AB has developed a method for treating seedborne diseases, named ThermoSeed. This treats the seeds with different intensities of steam. To evaluate their treatment, a method to extract nematodes from Brachiaria seed is needed to be able to guarantee seeds without living nematodes. The purpose of this master thesis was to find a method for extracting nematodes from Brachiaria ruzizensis seed that is fast, reliable and cost effective. Four different methods were tested: the Baermann funnel method, a method developed by Zuckermann et al., a method developed by Coolen and D’Herde and The simple method developed by Hoshino and Togashi. To test the methods, three types of treated seeds were used. Untreated seed (positive control), treated seeds with Incotec treatment and negative control (seeds that have been treated multiple times with high steam intensity). The methods were analyzed by counting the recovered nematodes from each of the methods. The data collected were analyzed statistically with ANOVA and a following post hoc Tukey test. From these analyses it was concluded that the Baermann funnel was the method which extracted the most nematodes from the untreated seeds compared to the other methods. The Baermann funnel method also gave clear samples and was easy to conduct.
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