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 mature....

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.....

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.