Location: Crop Genetics ResearchTitle: Experimental protocol for evaluating reniform nematode (Rotylenchulus reniformis) resistance in cotton Author
Submitted to: Journal of Visualized Experiments
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/19/2018
Publication Date: N/A
Interpretive Summary: Reniform nematode is a microscopic worm that is commonly found in soils of the southeastern United States. This nematode infects the roots of more than 300 plant species including upland cotton, which is a major crop in this region of the United States. Resistant cotton varieties are needed to reduce yield losses; however, resistance is lacking in upland cotton varieties. Related cotton species have been identified as a source of resistant genes, but transferring resistance to upland cotton requires specialized breeding methods. Thus, a simple and rapid method to screen for reniform nematode resistance was developed to identify resistant varieties from the cotton germplasm collection, to determine the genetics of resistance for these varieties, and to aid in the transfer of resistance to upland cotton. The method that was developed is non-destructive in order to recover plants from breeding populations to produce seeds for the development of resistant varieties. For the protocol, cotton seedlings were infected with nematodes 7 days after planting with the root systems removed from these plants 28 days later. The nematodes infecting the roots are stain with red food coloring and counted. The number of nematodes per gram of root was determined for each cotton variety with resistant varieties typically showing fewer than 10 nematodes per gram of root; whereas, susceptible varieties will have more than 30 nematodes per gram of root. This protocol increases the efficiency of screening for nematode resistance resulting in more plants evaluated in a single year. Additionally, the plant shoots with the roots removed were vegetatively propagated to allow new roots to form and the plants were grown to maturity to produce seeds. These protocols will enable cotton breeders to quickly identify and transfer resistance to reniform nematode to develop new varieties.
Technical Abstract: Sources of resistance to reniform nematode (Rotylenchulus reniformis) are needed to develop improved upland cotton (Gossypium hirsutum) varieties for nematode management. Resistance is lacking in upland cotton, which has required the evaluation of related cotton species. The diploid species such as Gossypium arboreum are a rich source of resistant genotypes, but transferring resistance to upland cotton is hindered by hybridization barriers. Thus, a rapid nematode screening protocol is needed for the identification of resistant genotypes, assessing the genetics of resistance, and for the successful transfer of resistance to upland cotton. Most protocols involve extracting vermiform nematodes or eggs from the cotton root system or from the potting soil to determine population density or reproduction rate. These approaches are generally time-consuming with a small number of genotypes evaluated. An alternative approach is to visually examine the root system of nematode infected plants to determine the number of reproductive female nematodes. The protocol involves inoculating cotton seedling 7 days after planting with vermiform nematodes and determining the number of females attached to the root system 28 days after inoculation. Data are expressed as the number of females per gram of fresh root weight. The protocol provides an excellent method for evaluating host-plant resistance associated with the ability of the nematode to establish an infection site, but resistance that hinders nematode reproduction is not assessed. As with other screening protocols, variation is commonly observed for nematode infection for individual genotypes within and between experiments. Nonetheless, the protocol provides a simple and rapid method to evaluate host-plant resistance. The protocol has been successfully used to identify resistant accessions from the G. arboreum germplasm collection and to evaluate segregating populations of more than 300 individuals to determine the genetics of resistance. A vegetative propagation method for recovering plants for resistance breeding was also developed.