Survival and infection of root-knot nematodes added
to soil amended with rye at different stages of
decomposition and cropped with cotton
Robert G. McBride
a,*, Robert L. Mikkelsen
a, Kenneth R. Barker
baDepartment of Soil Science, Box 7619, N.C. State University, Raleigh, NC 27695, USA bDepartment of Plant Pathology, Box 7616, N.C. State University, Raleigh, NC 27695, USA
Received 22 October 1998; accepted 4 June 1999
Abstract
The incorporation of a rye (Secale cerealeL.) cover crop into the soil prior to planting cotton (Gossypium hirsutumL.) has been shown to restrict damage caused by root-knot nematodes (Meloidogyne incognita(Kofoid and White) Chitwood). A greenhouse study was conducted to determine the duration of the effectiveness of rye decomposition in controlling root-knot nematode damage in relation to the time between rye incorporation and cotton planting. Fresh, chopped-rye foliage was mixed into pots of soil and root-knot nematode eggs were added to the rye + soil mixture or a non-amended soil at 0, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 days following rye incorporation. This resulted in a sequence of pots containing nematode eggs exposed to rye at different stages of decomposition. Cotton plants were transplanted into the pots after the addition of nematode eggs and assessed for damage after 28 days of exposure. Although the effectiveness of the rye treatment declined over the 21 days of the incubation, the root-knot nematode populations were signi®cantly reduced by the rye treatment for all planting dates. This suggests that it is not necessary to plant cotton immediately after plowing in a rye cover crop, thereby providing some ¯exibility in the cotton planting date, minimizing any associated phytotoxicity to the young cotton plants.#1999 Elsevier Science B.V. All rights reserved.
Keywords:Biocontrol;Meloidogyne incognita;Secale cereale
1. Introduction
When organic materials such as fresh rye are added to soil, these materials are degraded by microbes and rapidly depleted of readily decomposable organic substances such as sugars, starches, and simple pro-teins (Paul and Clark, 1996). Breakdown of these
readily decomposable substances are followed by that of more resistant compounds such as crude proteins, hemicelluloses, fats, waxes, and lignin (Killham, 1994). By-products from this degradation process include CO2, NH3, H2S, organic acids, and other
incompletely oxidized substances (Stevenson, 1994). As rye matures, the ratio of resistant to non-resistant materials increases, as does the C : N ratio in the plant. These changes in plant composition affect the length of time required for degradation, while the *Corresponding author.
E-mail address: rgmcbrid@unity.ncsu.edu (R.G. McBride)
speci®c plant species and its age, soil microbe popula-tions, and environmental conditions all in¯uence the production of speci®c degradation products. A pro-blem encountered when rye is used as a green manure crop prior to planting cotton is that metabolites pro-duced during early stages of decomposition are often phytotoxic, resulting in a poor seedling emergence and growth. (Tang, 1986).
Root-knot nematodes enter roots as juveniles, with the females remaining inside the root through the remainder of their life cycle. The infecting nematodes induce changes in the root resulting in galls. Incor-poration of rye prior to planting cotton has been shown to limit the damage caused by root-knot nematodes (McSorley and Dickson, 1995). However, the duration of the suppressive effect of rye on nematode damage is unknown.
The purpose of this greenhouse experiment was to determine the effectiveness of rye decomposition in controlling root-knot nematode damage in relation to the time between rye incorporation and cotton plant-ing.
2. Materials and methods
2.1. Soil
Steam-sterilized loamy sand soil (Norfolk series, ®ne-loamy, siliceous, thermic Typic Paleudult) was mixed 50/50 with sterilized sand. The soil was stored for over six months prior to the greenhouse study. A coarse-textured soil was used for this experiment due to the nematodes' af®nity for well-aerated soils (Van Gundy, 1985).
2.2. Rye
Rye (variety Abruzzi) was grown in a greenhouse in clay pots. The soil used was a 50/50 sterilized mixture of loamy sand (85% sand, 9% silt, and 6% clay) and coarse sand. Sterilized soil was used to eliminate background nematode populations. The rye was ferti-lized weekly with N, P, and K in the irrigation water and periodically with a controlled-release fertilizer. The rye was harvested 10 weeks after emergence and cut into 2±3-cm segments before being used in the nematode experiment.
2.3. Meloidogyne egg inoculum
A 2-cm section of root with observable egg masses was isolated from a tomato plant (Lycopersicon escu-lentumMill.) infected with root-knot nematodes (race 3 ofM. incognitafrom North Carolina). The root segment was buried in the center of a 15-cm clay pot containing a 50/50 mixture of sterilized loamy sand soil (85% sand, 9% silt, and 6% clay) and sterilized sand. A tomato seedling (variety Rutgers) was then transplanted into the inoculated soil to serve as a host plant. Tomatoes are very susceptible to root-knot nematode infestation and accordingly make an excellent host for the organ-ism (Sasser, 1990). The infected tomato plants were watered daily and fertilized weekly with N, P, and K in the irrigation water and periodically with a con-trolled-release fertilizer. It takes30 days for the eggs to hatch and complete their life cycle (Heald and Orr, 1984). After 3±4 life cycles, the root-knot nematode eggs were extracted as needed by means of the NaOCl-extraction method (Barker, 1985).
2.4. Setup
Fifteen hundred milliliters of the sterilized soil + sand mix were thoroughly mixed with 53 g (equivalent to 30 000 kg dry rye/ha) of the fresh chopped rye and packed in 15-cm diameter clay pots in a greenhouse. The soil + sand mix without the addition of rye was used as a control.
2.5. Addition of eggs
2.6. Cotton
Eight pre-germinated cotton seeds (variety Delta Pine 50) per pot were planted into the rye-amended and non-amended soil 48 h after the addition of the nematode eggs. The radicle (1±4 cm in length) was buried with the cotyledons left exposed at the surface. The cotton was watered daily and fertilized weekly with N, P, and K in the irrigation water. An effort was made to carefully water the transplanted cotton plants and yet-unplanted pots to avoid leaching of the soils and to maintain aerobic conditions. Fourteen days after transplanting, three of the cotton plants were removed with the root system intact and weighed, and the remaining plants were thinned to three. Twenty-eight days after transplanting, the remaining three plants were removed. The roots were removed intact by submerging the soil and root ball in water and gently rinsing away the soil.
2.7. Analysis
The roots of the cotton plants that were collected after 28 days were given a visual rating for nematode damage and surface necrosis. To maintain consistency, the same person did all the visual assessments. The weights of the shoots were recorded for the cotton plants that were collected at 14 days and 28 days after transplanting.
2.8. Experimental design
The collected data were analyzed using a rando-mized complete block design with six blocks (sections of greenhouse bench) and a factorial arrangement of treatments: 2 rye treatments (0, and 53 g rye) and 12 incorporation dates (number of days after the addition of rye that nematode eggs were incorporated). The model Yijk= u + Ri+ Tj+ Sk+ (TS)jk+ eijk was uti-lized where Rirepresents reps, Tjthe rye treatments, and Skthe incorporation dates for each sampling date.
3. Results and discussion
3.1. Visual gall rating
Signi®cantly fewer root galls were observed on the 28-day-old cotton plants in the rye-amended
soils (p< 0.05) than on the control plants (Fig. 1). The root galls in the rye-amended soil averaged 14% over the course of the experiment, while the roots grown in the non-amended soil averaged 24% galled roots. Although visually rating the root systems for nematode induced damage may be somewhat subjective, this method proved to be the best indicator of plant damage of three root rating systems used in this experiment (McBride et al., 1999). The suppressive effect of the rye on nematode damage was evident throughout the experiment, with statistically signi®cant difference occurring on 9 of the 12 sampling dates. This indicates the rye decom-position can effectively reduce nematode damage for a prolonged period following incorporation into the soil.
3.2. Necrosis
Roots from the unamended soil, even with severe nematode infections, maintained a smooth, unbroken appearance and light color. In contrast, roots from soils with the rye treatment were darker, and slightly exfoliating with signi®cant differences occurring for the latter six rye-treatment dates (Fig. 2). The condi-tion did not appear to adversely affect the growth of the cotton plants. The visual surface necrosis rating data were transformed by taking the square root of the data to stabilize variance.
3.3. Plant growth
The cotton plants growing in the soil that received the rye amendment were signi®cantly larger (p< 0.05) than those plants growing in the non-amended soil as
measured by fresh shoot weights at both sampling dates (Fig. 3(A) and (B)). The 14-day-old shoot weights were transformed by taking the square root of the data to stabilize variance. The 28-day-old plants grown in the rye-amended soil were on average 8.8 g larger than the plants grown in the non-amended soil with a standard error of 0.87.
Decreased plant growth results as second-stage juvenile root-knot nematodes enter roots and begin feeding on parenchyma cells. Some of the plant cells become hypertrophied and multinucleate, forming `giant cells' which serve as feeding stations for the rapidly developing nematode (Huang, 1985). These cells also draw resources away from other parts of the plant. Therefore, the infected plant not only suffers from an impaired root system, but also expends energy supporting the parasitic nematodes. This process may explain why the highly infected plants growing in the non-amended soil were smaller than the less infected plants growing in the rye-amended soil.
The variation in root damage of plants between planting dates is likely a result of environmental
Fig. 2. Visually rated necrosis of 28-day-old cotton roots grown in soil with and without the addition of fresh rye shoots. Cotton plants were planted two days after the egg addition. Data points represent an average of six pots. Treatments within each date with the same letters are not different when the means are compared by a pairwise
t-test at the 0.05 level (standard error of the transformed means = 0.24).
¯uctuations during the growing period. The tempera-ture and solar radiation in the greenhouse varied over the course of the experiment, which can affect plant growth and nematode hatch. Additionally, a separate extraction of fresh nematode eggs was used for every application date, which may introduce some variabil-ity.
Although the rye treatment was most effective as a nematode control agent when cotton was planted within 11 days following rye addition, this bene®t was still observed for the last three planting dates (as measured by visual rating). This suggests that it is not necessary to plant cotton immediately after the incor-poration of a rye cover crop to bene®t from the amendment. It may be necessary in some instances, such as following incorporation of an abundant rye crop, to allow some time to pass after the rye incor-poration prior to planting cotton in order to avoid phytotoxicity problems (Tang, 1986). Presumably, the metabolites from the readily decomposed materials and the associated microorganisms are partially responsible for the reduction in root-knot nematode damage.
4. Conclusions
The addition of fresh rye to soil is effective at limiting root-knot nematode damage on cotton as measured by root gall ratings and by plant growth. This inhibitory effect persisted even when planting was delayed for up to 28 days following rye addition and resulted in signi®cant suppression of root-knot nematode population and enhanced plant growth. This ®nding suggests that it is not necessary to plant cotton immediately after incorporating a rye cover crop into soil, thereby providing some ¯exibility in the planting date. Allowing the rye to decompose for a short period
would also make it possible to avoid potential phy-totoxicity problems.
References
Barker, K.R., 1985. Nematode extraction and bioassays. In: Barker, K.R, Carter, C.C., Sasser J.N. (Eds.), An advanced treatise on
Meloidogyne: vol. II. Methodology. North Carolina State University Graphics, Raleigh, North Carolina, 28 pp. Heald, C.M., Orr, C.C., 1984. Nematode parasites of cotton. In:
Nickle, W.R. (Ed.), Plant and insect nematodes. Marcel Drekker, Inc., New York, New York, pp. 147±166.
Huang, C.S., 1985. Formation, anatomy and physiology of giant cells induced by root-knot nematodes. In: Sasser, J.N., Carter, C.C. (Eds.), An advanced treatise on Meloidogyne: vol. I. Biology and Control. North Carolina State University Graphics, Raleigh, North Carolina, pp. 155±164.
Killham, K., 1994. Soil ecology. Cambridge University Press, Cambridge, UK, 242 pp.
McBride, R.M., Mikkelsen, R.L., Barker, K.R., 1999. A compar-ison of three methods for determining root-knot nematode damage on cotton roots. Nematropica (in press).
McSorley, R., Dickson, D.W., 1995. Effect of tropical rotation crops on Meloidogyne incognita and other plant-parasitic nematodes. J. of Nematol. 27, 535±544.
Paul, E.A., Clark, F.E., 1996. Soil microbiology and biochemistry, second ed., Academic Press, San Diego, California, 273 pp. Sasser, J.N., 1990. Plant-parasitic nematodes: the farmers hidden
enemy. North Carolina State University Graphics, Raleigh, North Carolina, 115 pp.
Stevenson, F.J., 1994. Humus chemistry: genesis, composition, reactions, fourth ed., John Wiley & Sons, Inc. New York, New York, 443 pp.
Tang, C.S., 1986. Allelopathic activity of rye (Secale cerealeL.). In: Barnes, J.P., Putnam, A.R., Burke, B.A. (Eds.), The science of allelopathy. John Wiley & Sons, Inc. New York, New York, pp. 271±286.
Van Gundy, S.D., 1985. Ecology of Meloidogyne spp. ± emphasis on environmental factors affecting survival and pathogenicity. In: Sasser, J.N., Carter, C.C. (Eds.), An advanced treatise on
