Name: screening cotton genotypes for reniform nematode resistance
Uploaded: 2019-05-02T12:30:00
Description: Watch this Scientific Journal Video about Screening Cotton Genotypes for Reniform Nematode Resistance at JoVE.com

This research was funded by the United States Department of Agriculture, Agricultural Research Service. Mention of trade names and commercial products in this article are solely for the purpose of providing specific information and do not imply recommendations or endorsements by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. The authors have no conflict of interest to declare. Technical assistance was provided by Kristi Jordan.

1. Maintaining a Source of R. reniformis Inoculum
<ol>
	<li>Fill terra cotta clay pots (15 cm in diameter, 13.5 cm in height) with a steam pasteurized mix of 1-part sandy loam&#160;and 2-parts sand. Plant a susceptible tomato (Solanum lycopersicon) variety in each pot and place the pots in a glasshouse. 
	NOTE: Other susceptible plant varieties such as cotton can be used instead of tomato.</li>
	<li>Inoculate the tomato plants with vermiform reniform nematodes (see step 3.3). Maintain th...

<ol>
	<li>Heald, C. M., Robinson, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+current+distribution+of+Rotylenchulus+reniformis.+in+the+United+States.">Survey of current distribution of Rotylenchulus reniformis. in the United States.</a> Journal of Nematology. 22 (4), 695-699 (1990).</li><li>Koenning, S. R., Wrather, J. A., Kirkpatrick, T. L., Walker, N. R., Starr, J. L., Mueller, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-parasitic+nematodes+attacking+cotton+in+the+United+States:+old+and+emerging+production+challenges.">Plant-parasitic nematodes attacking cotton in the United States: old and emerging production challenges.</a> Plant Disease. 88 (2), 100-113 (2004).</li><li>Robinson, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reniform+in+U.S.+cotton:+when,+where,+why,+and+some+remedies.">Reniform in U.S. cotton: when, where, why, and some remedies.</a> Annual Review of Phytopathology. 45, 263-288 (2007).</li><li>Robinson, A. F., Inserra, R. N., Caswell-Chen, E. P., Vovlas, N., Troccoli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotylenchulus+species:+identification,+distribution,+host+ranges,+and+crop+plant+resistance.">Rotylenchulus species: identification, distribution, host ranges, and crop plant resistance.</a> Nematropica. 27 (2), 127-180 (1997).</li><li>Davis, R. F., Koenning, S. R., Kemerait, R. C., Cummings, T. D., Hurley, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotylenchulus+reniformis+management+in+cotton+with+crop+rotation.">Rotylenchulus reniformis management in cotton with crop rotation.</a> Journal of Nematology. 35 (1), 58-64 (2003).</li><li>Starr, J. L., Koenning, S. R., Kirkpatrick, T. L., Robinson, A. F., Roberts, P. A., Nichols, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+nematode+management+in+cotton.">The future of nematode management in cotton.</a> Journal of Nematology. 39 (4), 283-294 (2007).</li><li>Weaver, D. B., Lawrence, K. S., van Santen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reniform+nematode+resistance+in+upland+cotton+germplasm.">Reniform nematode resistance in upland cotton germplasm.</a> Crop Science. 47 (1), 19-24 (2007).</li><li>Robinson, A. F., Cook, C. G., Percival, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+Rotylenchulus+reniformis+and+Meloidogyne+incognita+race+3+in+the+major+cotton+cultivars+planted+since+1950.">Resistance to Rotylenchulus reniformis and Meloidogyne incognita race 3 in the major cotton cultivars planted since 1950.</a> Crop Science. 39 (3), 850-858 (1999).</li><li>Carter, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+and+resistant+reaction+of+Gossypium+arboreum+to+the+reniform+nematode,+Rotylenchulus+reniformis.">Resistance and resistant reaction of Gossypium arboreum to the reniform nematode, Rotylenchulus reniformis.</a> Journal of Nematology. 13 (3), 368-374 (1981).</li><li>Erpelding, J. E., Stetina, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+reniform+nematode+resistance+in+Gossypium+arboreum+germplasm+line+PI+529728.">Genetics of reniform nematode resistance in Gossypium arboreum germplasm line PI 529728.</a> World Journal of Agricultural Research. 1 (4), 48-53 (2013).</li><li>Robinson, A. F., Bridges, A. C., Percival, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+sources+of+resistance+to+the+reniform+(Rotylenchulus+reniformis)+and+root-knot+(Meloidogyne+incognita)+nematode+in+upland+(Gossypium+hirsutum+L.)+and+sea+island+(G.+barbadense+L.)+cotton.">New sources of resistance to the reniform (Rotylenchulus reniformis) and root-knot (Meloidogyne incognita) nematode in upland (Gossypium hirsutum L.) and sea island (G. barbadense L.) cotton.</a> Journal of Cotton Science. 8 (3), 191-197 (2004).</li><li>Robinson, A. F., Percival, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+Meloidogyne+incognita+race+3+and+Rotylenchulus+reniformis+in+wild+accessions+of+Gossypium+hirsutum+and+G.+barbadense+from+Mexico.">Resistance to Meloidogyne incognita race 3 and Rotylenchulus reniformis in wild accessions of Gossypium hirsutum and G. barbadense from Mexico.</a> Journal of Nematology. 29 (4), 746-755 (1997).</li><li>Stetina, S. R., Young, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparisons+of+female+and+egg+assays+to+identify+Rotylenchulus+reniformis+resistance+in+cotton.">Comparisons of female and egg assays to identify Rotylenchulus reniformis resistance in cotton.</a> Journal of Nematology. 38 (3), 326-332 (2006).</li><li>Usery, S. R., Lawrence, K. S., Lawrence, G. W., Burmester, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+cotton+cultivars+for+resistance+and+tolerance+to+Rotylenchulus+reniformis.">Evaluation of cotton cultivars for resistance and tolerance to Rotylenchulus reniformis.</a> Nematropica. 35 (2), 121-133 (2005).</li><li>Yik, C. -. P., Birchfield, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistant+germplasm+in+Gossypium+species+and+related+plants+to+Rotylenchulus+reniformis.">Resistant germplasm in Gossypium species and related plants to Rotylenchulus reniformis.</a> Journal of Nematology. 16 (2), 146-153 (1984).</li><li>Stetina, S. R., Erpelding, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gossypium+arboreum+accessions+resistant+to+Rotylenchulus+reniformis.">Gossypium arboreum accessions resistant to Rotylenchulus reniformis.</a> Journal of Nematology. 48 (4), 223-230 (2016).</li><li>Thies, J. A., Merrill, S. B., Corley, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+food+coloring+stain:+new,+safer+procedures+for+staining+nematodes+in+roots+and+egg+masses+on+root+surfaces.">Red food coloring stain: new, safer procedures for staining nematodes in roots and egg masses on root surfaces.</a> Journal of Nematology. 34 (2), 179-181 (2002).</li><li>Erpelding, J. E., Stetina, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+characterization+of+reniform+nematode+resistance+for+Gossypium+arboreum+accession+PI+417895.">Genetic characterization of reniform nematode resistance for Gossypium arboreum accession PI 417895.</a> Plant Breeding. 137 (1), 81-88 (2018).</li><li>Byrd, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+semi-automatic+elutriators+for+extracting+nematodes+and+certain+fungi+from+soil.">Two semi-automatic elutriators for extracting nematodes and certain fungi from soil.</a> Journal of Nematology. 8 (3), 206-212 (1976).</li><li>Jenkins, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+centrifugal-flotation+technique+for+separating+nematodes+from+soil.">A rapid centrifugal-flotation technique for separating nematodes from soil.</a> Plant Disease Reporter. 48 (9), 692 (1964).</li><li>Robinson, A. F., Heald, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+dioxide+and+temperature+gradients+in+Baermann+funnel+extraction+of+Rotylenchulus+reniformis.">Carbon dioxide and temperature gradients in Baermann funnel extraction of Rotylenchulus reniformis.</a> Journal of Nematology. 23 (1), 28-38 (1991).</li><li>Williams, C., Gilman, D. F., Fontenot, D. S., Birchfield, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+technique+for+screening+soybeans+for+reniform+nematode+resistance.">A rapid technique for screening soybeans for reniform nematode resistance.</a> Plant Disease Reporter. 63 (10), 827-829 (1979).</li><li>Schmitt, D. P., Shannon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiating+soybean+responses+to+Heterodera+glycines+races.">Differentiating soybean responses to Heterodera glycines races.</a> Crop Science. 32 (1), 275-277 (1992).</li></ol>

An effective screening protocol is required for 1) the identification of R. reniformis resistant cotton genotypes in order to evaluate the genetics of resistance and 2) the breeding of resistant varieties. Most protocols assess R. reniformis population densities or reproduction rates by extracting vermiform nematodes or eggs from the cotton root system or potting soil8,11,12,<sup cl...

Rotylenchulus reniformis (Linford and Oliveira), commonly referred to as the reniform nematode, is one of the major parasitic nematode species present in soils of the southeastern United States1,2,3. The nematode is an obligate, sedentary semi-endoparasite requiring a host plant to complete its life cycle2,4. Vermiform preadult female nematodes penetrate the host root system to establish a feeding site in the stele2,3. As the nematode feeds and matures, the posterior portion remaining outside of the host root will swell upon egg production, forming a characteristic kidney shape (Figure 1). Rotylenchulus reniformis is capable of feeding on the root system of more than 300 plant species, including cotton4. Upland cotton (Gossypium hirsutum L.) is widely cultivated in the southeastern United States, but the lack of R. reniformis&#160;resistant varieties hinders nematode management2,3. Management strategies such as nematicide treatment and rotation with non-host crop species have been used to reduce soil R. reniformis population densities5,6, but seed cotton yield losses can commonly range from 1 to 5%2. Symptoms of R. reniformis infection can include plant stunting, suppressed root growth, nutritional deficiencies, fruit abortion, and delayed maturity2. However, symptoms may not be apparent due to the uniformity of symptoms across the field; therefore, approaches to assess R. reniformis infection are needed to identify and develop resistant upland cotton varieties. Evaluation of R. reniformis resistance in cotton is considered difficult7, because the infected root system may appear normal even though the plant may show symptoms of infection8.
An effective nematode screening protocol is required for the identification of R. reniformis&#160;resistant accessions from the cotton germplasm collection, and for the determination of the resistance genetics for these accessions. Such a protocol will aid in the transfer of resistance genes to upland cotton. Various bioassay methods have been used to assess R. reniformis infection in cotton8,9,10,11,12,13,14,15. In general, two major approaches have been used for the identification of R. reniformis resistant cotton genotypes. The most frequently used approach involves extracting eggs and/or vermiform nematodes from infected plants or soil8,11,12,14,15. The general methodology for this approach involves planting seeds for the individual cotton genotypes in separate pots, allowing the seedlings to develop for 7 to 14 days, inoculating the seedlings by adding a mixture of vermiform stages of R. reniformis to the soil, and allowing the nematodes to infect the root system for 30 to 60 days. Next, vermiform nematodes and/or eggs are extracted from the infected root system of each plant or from the potting soil. The number of extracted nematodes or eggs is then determined to estimate the population density and reproduction rate, which are compared to control genotypes in order to identify resistant genotypes.
An alternative approach, as described here, involves microscopically examining the cotton root system that has been infected with nematodes to determine the number of female nematodes parasitizing the roots10,16. Similar to other approaches, cotton genotypes are planted in separate pots and inoculated with vermiform nematodes approximately 7 days after planting. Within 30 days after inoculation, the root system is removed from individual plants and the soil is rinsed from the roots. Next, the nematodes attached to the root system are stained with red food coloring17, and roots are microscopically examined to determine the number of infection sites with resistant cotton genotypes (identified based on the number of nematodes per gram of root) compared to a susceptible control16. This second approach has the advantage of increased throughput by reducing the number of days required for evaluation and increasing the number of individual genotypes evaluated in a single experiment. Screening methodologies that evaluate population density or reproduction rate are often more time-consuming than those based on visual observations of infection signs7. However, one limitation of this approach is that host-plant resistance that hinders nematode reproduction as determined by egg production is not assessed13.
Screening protocols for R. reniformis resistance often destroy the root system during evaluation7 and involve the vegetative shoot being discarded. To overcome this limitation, a method of vegetative propagation has been developed to allow the recovery of plants for seed production18. After removal of the root system for nematode evaluation, the vegetative shoot is planted in potting soil to allow the root system to regrow. This method has broad applications for most R. reniformis screening protocols. A simple and rapid method of vegetative propagation is of critical importance for breeding R. reniformis resistant upland cotton varieties, where the recovery of the progeny is required to advance resistant genotypes to the next generation.
A protocol is presented for the large-scale screening of cotton genotypes for reniform nematode resistance. The goal is to develop a simple and rapid non-destructive screening method to evaluate cotton breeding populations for nematode resistance in order to aid in the breeding of resistant upland cotton varieties. Using this protocol, data are typically obtained within 35 days, with more than 300 genotypes evaluated in a single experiment. Data are presented for resistant and susceptible genotypes to illustrate the variation commonly observed using these methods.

<table border="0" cellpadding="0" cellspacing="0" style="width:928px;" width="928">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Ray Leach Cone-tainer</td>
			<td style="width:155px;">Stuewe and Sons Inc.</td>
			<td style="width:155px;">SC10U</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Cone-tainer tray</td>
			<td>Stuewe and Sons Inc.</td>
			<td style="width:155px;">RL98</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Sand</td>
			<td style="width:155px;">various</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Cotton balls</td>
			<td style="width:155px;">various</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Pylon 4 inch plant labels (4 in L x 5/8 in W)</td>
			<td style="width:155px;">Pylon Platics</td>
			<td style="width:155px;">L-4-W</td>
			<td>Any brand or vendor is acceptible.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">4 oz. specimen containers</td>
			<td style="width:155px;">Fisher Scientific</td>
			<td style="width:155px;">16-320-731</td>
			<td>Any brand or vendor is acceptible.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Red food coloring</td>
			<td style="width:155px;">McCormick &#38; Co., Inc.</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">1 mL Pipet tips</td>
			<td style="width:155px;">various</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">10 L container</td>
			<td style="width:155px;">various</td>
			<td style="width:155px;"></td>
			<td>Inexpensive buckets work well.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">6 L pots</td>
			<td>Nursery Supplies Inc.</td>
			<td>Poly-Tainer-Can No2A</td>
			<td>Any brand or vendor is acceptible. Different size pots can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Potting media</td>
			<td>Sun Gro Horticulture</td>
			<td>Metro-Mix 360</td>
			<td>Any brand or vendor is acceptible.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Fertilizer</td>
			<td>Everris NA Inc.</td>
			<td>Osmocote Plus</td>
			<td>Any brand or vendor is acceptible.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic container (73.6 cm L x 45.7 cm W x 15.2 cm D)</td>
			<td style="width:155px;">Rubbermaid</td>
			<td style="width:155px;">3O29&#160;</td>
			<td>Any brand or vendor is acceptible.</td>
		</tr>
	</tbody>
</table>

screening cotton genotypes for reniform nematode resistance

A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium hirsutum) varieties to improve nematode management. Most protocols involve extracting vermiform nematodes or eggs from the cotton root system or 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 described here in which the root system is visually examined for nematode infection. 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 to adjust for variation in root growth. The protocol provides an excellent method for evaluating host-plant resistance associated with the ability of the nematode to establish an infection site; however, resistance that hinders nematode reproduction is not assessed. As with other screening protocols, variation is commonly observed in nematode infection among individual genotypes within and between experiments. Data are presented to illustrate the range of variation observed using the protocol. To adjust for this variation, control genotypes are included in 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 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. After removal of the root system for nematode evaluation, the vegetative shoot is replanted to allow the development of a new root system. More than 95% of the shoots typically develop a new root system with plants reaching maturity.

Rotylenchulus reniformis infection of the root system for two varieties is presented in Figure 1. Relatively fewer female reniform nematodes are able to establish a feeding site for the resistant cotton genotype compared to the susceptible genotype. Variation in root growth is common between accessions, as illustrated in Figure 2. This variation as measured by fresh root weight can also be observed between plants of the ...

Watch this Scientific Journal Video about Screening Cotton Genotypes for Reniform Nematode Resistance at JoVE.com

Screening Cotton Genotypes for Reniform Nematode Resistance

Here, a protocol is presented for the rapid non-destructive screening of cotton genotypes for reniform nematode resistance. The protocol involves visually examining the roots of nematode-infected cotton seedlings to determine infection response. The vegetative shoot from each plant is then propagated to recover plants for seed production.

A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium ...

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