Screening Cotton Genotypes for Reniform Nematode Resistance

Summary

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.

Abstract

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.

Introduction

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 States^1,2,3. The nematode is an obligate, sedentary semi-endoparasite requiring a host plant to complete its life cycle^2,4. Vermiform preadult female nematodes penetrate the host root system to establish a feeding site in the stele^2,3. 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 resistant varieties hinders nematode management^2,3. Management strategies such as nematicide treatment and rotation with non-host crop species have been used to reduce soil R. reniformis population densities^5,6, 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 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 cotton^{8,9,10,11,12,13,14,15}. 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 soil^{8,11,12,14,15}. 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 roots^10,16. 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.

Protocol

1. Maintaining a Source of R. reniformis Inoculum

1. Fill terra cotta clay pots (15 cm in diameter, 13.5 cm in height) with a steam pasteurized mix of 1-part sandy loam 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.
2. Inoculate the tomato plants with vermiform reniform nematodes (see step 3.3). Maintain the plants in the glasshouse at a temperature of approximately 28 °C.

2. Planting Cotton Genotypes for R. reniformis Resistance Evaluation

1. Prepare soil by combining 2-parts fine sand with 1-part sandy loam collected from the field.
2. Steam pasteurize the soil mixture to ensure that the soil is free of nematodes and soil-borne plant pathogens.
3. Add the soil mixture to conical plastic pots (4 cm in diameter, 21 cm in height). Prior to filling the pots, place a ball of cotton in the bottom of the pot to prevent soil loss. Partially fill the pots with soil approximately 2 cm from the top.
4. Prepare a plastic stake for each pot to designate the genotype to be planted.
5. Plant seeds of the cotton genotypes selected for evaluation.
   1. Plant a single seed in each pot for the evaluation of segregating populations, in which each seed represents a unique genotype.
   2. For cotton varieties or germplasm accessions, plant 2 to 3 seeds in a single pot to insure germination of at least one plant with the other seedlings removed from the pots prior to nematode inoculation.
      Note: Alternatively, seeds can be germinated for 24 to 72 h prior to planting to minimize the number of pots with non-viable seeds.
6. Plant seeds of selected resistant and susceptible control genotypes.
   Note: Control genotypes are replicated 5 to 10 times to assess natural variations inherent to the screening methodology.
7. Fill pots with additional soil to cover the seed in each pot.
8. Place the pots in the growth chamber. Maintain a constant temperature of 28 °C, an ambient air temperature, for the growth chamber. Provide artificial lighting with a mixture of fluorescent and incandescent lamps with a 16 h photoperiod.
9. Place water emitters in each pot, and water the pots twice per day using an automatic watering system. Adjust the watering system to supply additional water as the plant grows.

3. Nematode Inoculation of Cotton Plants and Preparation of Root Samples

1. Extract vermiform reniform nematodes maintained on susceptible tomato plants (see step 1) using elutriation¹⁹ and centrifugal flotation²⁰ methodologies the day before inoculation. Store the extracted nematodes at 4 °C.
   NOTE: Baermann funnel extraction²¹ is an alternate method for nematode extraction.
2. Determine the number of nematodes extracted by counting the number of nematodes in a 100 µL subsample and prepare a suspension of 1,000 nematodes/mL in tap water for inoculations.
3. Inoculate the cotton seedlings 7 d after planting with the nematode suspension. Create a small depression in the soil next to the plant, and pipette 1 mL of the reniform nematode suspension into the depression.
4. Remove plants from the pots 28 d after inoculation for nematode evaluation.
   NOTE: At this stage, plants are approximately 15 cm tall with 4 to 6 fully expanded leaves.
   1. Remove most of the fully expanded leaves from the plants using scissors prior to removing the plants from the pots.
   2. To remove plants from the pots, squeeze the pot and slide the soil out into the hand.
   3. Gently remove the soil from the roots by agitating the root system in tap water in a 10 L container. Briefly rinse the root system in a container of clean tap water.
   4. Remove the root system from the plant approximately 1 cm below the soil line using scissors.
5. Place the root system into a 120 mL plastic, non-sterile, disposable specimen container along with the plastic stake from the pot used for identification.
   NOTE: Multiple samples are processed before proceeding to step 3.7. Proceed to step 4 for the vegetative propagation of the plant shoot.
6. Prepare a 12.5% (v/v) solution of red food coloring¹⁷ in tap water to stain the nematodes attached to the root system.
7. Add approximately 30 mL of the red food coloring solution to the root sample in the specimen container to completely cover the root system.
8. Place the specimen container in a microwave oven and heat the root sample until the staining solution starts to boil. Remove the sample from the microwave oven and allow the sample to cool at room temperature.
9. Decant the red food coloring solution from the root sample and add approximately 100 mL of tap water to the specimen container to remove excess stain. Place the cover on the specimen container and store the sample in a refrigerator at 4 °C. Proceed to step 5 for evaluation of root infection.
   NOTE: The protocol can be paused here before proceeding to step 5.

4. Vegetative Propagation to Recover Plants for Seed Production

1. Place a ball of cotton in the bottom of a conical plastic pot (see step 2.3) and partially fill the pot with peat moss potting media. Then, place the vegetative shoot in the pot and firmly add potting media to fill the pot. Place a new labeled plastic stake in each pot to designate the cotton genotype.
2. Place the tray of pots in a plastic container (73.6 cm length x 45.7 cm width x 15.2 cm height) with water and briefly water the plants to moisten the potting media. Place the pots in a growth chamber with a constant temperature of 28 °C using a 16 h photoperiod. Add additional water to the plastic container as needed to maintain soil moisture.
3. Transplant the plants to larger pots for seed production after approximately 30 d. Partially fill a 6 L plastic pot with potting media (see step 4.1), remove the plant from the small pot, place the plant into the 6 L pot, and firmly add potting media to fill the pot.
4. Place the plants in a glasshouse and add water to moisten the potting media. Maintain the temperature in the glasshouse at approximately 28 °C (artificial lighting is not required).
   1. Water plants by hand as needed for approximately 30 d.
   2. When approximately 75% of the plants require daily watering, water plants daily using an automatic watering system. Adjust the automatic watering system to water more frequently as needed for plant growth.
   3. Add approximately 10 g of a slow-release fertilizer to each pot prior to the start of floral initiation.
5. Harvest plants at maturity and process the cotton seed samples to obtain seeds for further evaluation.
   1. To harvest cotton seeds, remove the cotton from the open bolls on the plant by hand and place it in a labeled paper bag. The seeds are attached to the cotton fibers, which are removed in the following steps.
   2. Remove lint fibers from the seed samples using a 10-saw laboratory gin.
   3. Remove fuzz fibers from the seed samples using concentrated sulfuric acid. Neutralize seed samples in a 15% (v/v) solution of sodium carbonate, rinse the samples with tap water, and dry the samples in a forced air drier.
   4. Place the seed samples in labeled envelopes for storage.

5. Evaluation of R. reniformis Root Infection

1. Remove the root sample from the specimen container and count the number of female nematodes attached to the root system using a stereomicroscope (20X magnification).
   NOTE: Only female reniform nematodes are capable of infecting plant roots.
2. Place the root system on paper towels for approximately 10 min to remove excess moisture. Weigh the root system to determine the fresh root weight.
3. Enter the nematode count and fresh root weight data into a computer spreadsheet program and calculate the number of females per gram of root.

Results

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 ...

Discussion

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 soil^8,11,12,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ray Leach Cone-tainer	Stuewe and Sons Inc.	SC10U
Cone-tainer tray	Stuewe and Sons Inc.	RL98
Sand	various
Cotton balls	various
Pylon 4 inch plant labels (4 in L x 5/8 in W)	Pylon Platics	L-4-W	Any brand or vendor is acceptible.
4 oz. specimen containers	Fisher Scientific	16-320-731	Any brand or vendor is acceptible.
Red food coloring	McCormick & Co., Inc.
1 mL Pipet tips	various
10 L container	various		Inexpensive buckets work well.
6 L pots	Nursery Supplies Inc.	Poly-Tainer-Can No2A	Any brand or vendor is acceptible. Different size pots can be used
Potting media	Sun Gro Horticulture	Metro-Mix 360	Any brand or vendor is acceptible.
Fertilizer	Everris NA Inc.	Osmocote Plus	Any brand or vendor is acceptible.
Plastic container (73.6 cm L x 45.7 cm W x 15.2 cm D)	Rubbermaid	3O29	Any brand or vendor is acceptible.