244 R.G. McBride et al. Applied Soil Ecology 15 2000 243–251
control resulting from the use of organic amendments has been attributed to several factors, including toxic
effects of nitrogen, predatory fungi, nematodes, in- sects, and mites, as well as organic acids and their
interactions. The contribution of organic acids from decomposing organic matter has been assumed to be
the causative factor for many years Stephenson, 1945; Johnston, 1959; Sayre et al., 1964; Badra et al., 1979.
However, no research has been done to measure organic acids produced in situ and their subsequent
influence on nematode infection.
Low molecular weight organic acids have been re- ported to be present in anaerobic soils following the
addition of plant material Gotoh and Onikura, 1971; Chandrasekaran and Yoshida, 1973. In well-aerated
soils, these acids generally exist in low concentrations and only for relatively short periods of time, being
rapidly broken down and utilized by bacteria and fungi Schwartz et al., 1954; Hollis and Rodriguez-Kabana,
1966; Lynch, 1991. These soluble organic products are generated as metabolic by-products from soil or-
ganisms, such as bacteria and fungi, and are also ex- creted from plant roots McLaren and Peterson, 1967.
The limited quantities and transient nature of low molecular weight organic acids in aerobic soils ap-
pear contradictory to the nematicidal merit these acids have received. Our objective was to quantify the per-
sistence and quantity of the five most cited low molec- ular weight organic acids in the soil solution after the
addition of fresh rye. We also sought to determine any associated change in the root-knot nematode pop-
ulation, a worldwide pest responsible for damage to nearly every food crop.
In the first experiment, the effects of three applica- tion rates of rye on the root-knot nematode infection
and the concentration of five low molecular weight or- ganic acids in the soil solution were evaluated. A sec-
ond experiment quantified the rate at which five low molecular weight organic acids were removed from
the soil solution over a 10 h period.
2. Materials and methods
2.1. Experiment 1: rye addition 2.1.1. Soil
A sandy loam surface horizon was collected near Clayton, NC Goldsboro, fine-loamy, siliceous,
thermic Aquic Paleudult. This coarse-textured, well-drained soil was selected for this experiment
due to the nematode’s affinity for well-aerated soils Van Gundy, 1985 and the necessity of collecting a
leachate sample. After collection, the soil was stored moist at 25
◦
C for several weeks to maintain micro- bial populations. The soil was analyzed for nematode
population by the semi-automatic elutriator method Barker, 1985a and found to contain 590 root-knot
Meloidogyne spp., 550 stunt Tylenchorhynchus spp., 10 ring Criconemella spp., and 10 spiral Helicoty-
lenchus spp. nematodesl.
2.1.2. Rye Rye Secale cereale L. var. Abruzzi was grown in
15 cm diameter clay pots containing a 5050 mixture of sterilized loamy sand soil and coarse sand. The rye
was fertilized weekly with N, P, and K in the irrigation water. The rye shoots were harvested approximately 10
weeks after planting and were cut into 2.5 cm segments before being used in the experiment.
2.2. Meloidogyne egg inoculum A mature tomato plant Lycopersicon esculentum
infected with a North Carolina population of root-knot nematodes Meloidogyne incognita race 3 was uti-
lized as a nematode source. To increase this nema- tode, 3 cm long pieces of tomato root with observable
egg masses were removed. Each piece of infected root was buried in the center of a 15 cm diameter clay pot
containing a 5050 mixture of sterilized loamy sand soil and coarse sand. Tomato seedlings Rutgers vari-
ety, were transplanted into the inoculated soil to serve as host plants. Tomatoes are generally quite suscepti-
ble to root-knot nematode infestation and accordingly make an excellent host Sasser, 1990. The plants were
fertilized weekly with N, P, and K in the irrigation water. It takes approximately 30 days for the eggs to
hatch and complete their life cycle Heald and Orr, 1984. After three to four life cycles, the developing
root-knot nematode eggs were extracted by means of the NaOCl-extraction method Barker, 1985b.
2.3. Experimental set up The fresh-collected field soil was added to 15 cm
diameter clay pots, 1450 g of soil oven dry weight
R.G. McBride et al. Applied Soil Ecology 15 2000 243–251 245
per pot. The drain hole in the pot was covered with pieces of woven fiberglass cloth to retain the soil in
the pot, yet allow drainage.
2.4. Organic acid analysis Three application rates 0, 23, and 34 g dry weight
of ryepot were established by thoroughly mix- ing fresh rye shoots into the soil in the pot. These
very high rye application rates equivalent to 16 and 23 tacre, respectively were used to ensure that the de-
sired processes would take place and that organic acid concentrations would be well above minimum analyt-
ical detection limits. Root-knot nematode eggs were added to each pot at a rate of 20,000 eggspot. Eggs
gathered by the NaOCl-extraction method Barker, 1985b were suspended in water. The eggs were
added to the pots by removing the top 2 cm of soil, pouring the eggs suspended in 30 ml of water over
the surface of the exposed soil, and then replacing the soil.
The first phase of the experiment was conducted in a temperature-controlled incubator where the temper-
ature was maintained at 28
◦
C. Soil moisture content was brought up to approximately 80 of saturation
each day with distilled water. A leachate sample was obtained by slowly adding distilled water to the sur-
face of the soil. When water was first observed drip- ping from the bottom of the pot, an additional 200 ml
was added and allowed to drain into a glass jar. The pH of each sample was measured and subsequently
adjusted to pH 5 with NaOH or HCl and stored at 1
◦
C in glass containers with Teflon-lined lids. When drainage ceased, the pots were immediately returned
to the incubator to maintain the constant environmen- tal conditions.
2.5. Chemical analysis Prior to analysis of the leachate, the samples were
allowed to warm to room temperature, the pH mea- sured, and He was bubbled through each sample for
approximately 20 min to eliminate carbonate in solu- tion. When left untreated, the carbonate in the samples
resulted in a negative peak during analysis, which obscured the positive propionic and butyric acid
peaks. The samples were analyzed for formic, acetic, pro-
pionic, butyric, and valeric acids by means of ion ex- clusion chromatography Rocklin et al., 1986. Indi-
vidual organic acids were separated and quantified by ion chromatography exclusion ICE using a Dionex
model DX100 ion chromatograph equipped with a Dionex lonpac ICE-AS1 column, Dionex AMMS-ICE
suppressor, and conductivity detector. The eluent was 1.0 mM heptafluorobutyric acid in HPLC-grade water
at a flow rate of 2 mlmin at a column pressure of ap- proximately 4800 kPa 700 psi. The column was re-
generated with 2.75 tetrabutylammonium hydroxide TBAH at a flow rate of 1 mlmin. Quantification of
the acids was by comparison of the sample peak area with the peak area of a standard amount of the respec-
tive acids. The leachate concentrations were converted to the concentration of organic acids in soil solution
at 80 of water-holding capacity by taking into ac- count the dilution occurring during the leaching pro-
cess and the amount of solution recovered from each pot.
The experiment was conducted using a randomized complete block design with blocks corresponding to
the incubator’s six shelves top two, middle two, and bottom two shelves being utilized as threee separate
blocksreps. There were a total of 15 experimen- tal units pots per block. The treatments rates of
rye and sample times 0, 12, 36, 84, and 180 h af- ter rye application were randomly assigned to the
pots within each block. Each pot was leached only once. The overall statistical model corresponds to a
randomized complete block design with rates of rye and sample dates as treatments, following the form
Y
ij k
= µ+R
i
+ T
j
+ S
k
+ TS
j k
+ e
ij k
, where R
i
repre- sents reps, T
j
represents rye rates, and S
k
represents sampling dates. A statistical analysis was not con-
ducted on the organic acid data because insufficient data above the detection limit precluded a meaning-
ful analysis. Differences between treatments at each sample date for the pH data were determined by cal-
culating the least significant difference LSD at the 0.05 probability level.
2.6. Nematode population assay Fourteen days after the rye and root-knot nematode
eggs were incorporated into the pots, the experiment was moved from the incubator to a greenhouse, with
246 R.G. McBride et al. Applied Soil Ecology 15 2000 243–251
the blocking and randomization maintained. At this time, one 10 cm tall tomato seedling Rutgers variety
was transplanted into the center of each pot. It was necessary to wait 2 weeks after the initial incorpora-
tion of the rye in order to complete the organic acid sampling and to allow some initial decomposition
of the rye to occur. Planting the tomato seedlings at the time of the rye incorporation may have re-
sulted in phytotoxicity with such high rye application rates. The tomatoes were watered daily and fertil-
ized weekly with a solution containing N, P, and K. The plants were watered carefully to avoid leaching
the pots and to maintain aerobic conditions for the nematodes.
Five weeks after transplanting, the entire tomato plant was removed from each pot. The leaves and
petioles were removed from the top two complete branches for nutrient analyses. The entire shoot was
weighed after 24 h of oven drying at 60
◦
C. The leaves and petioles were analyzed for N with a Perkin-Elmer
2400 CHN elemental analyzer, while P, K, and mi- cronutrients were measured by means of inductively
coupled plasma spectroscopy following acid digestion. The roots were removed intact by submerging the soil
and root ball in water and gently rinsing away the soil. The roots were rinsed, weighed, given a visual rating
for percent root infection, and the root-knot nematode induced galls were counted.
The plant response data was analyzed as a factorial arrangement in a randomized complete block design.
The three rye rates and the five sample dates were used in the model Y
ij k
= µ+R
i
+ T
j
+ S
k
+ TS
j k
+ e
ij k
, where R
i
represents reps, T
j
represents rye rates, and S
k
represents sampling dates. Although not a factor in this experiment, sample dates were retained in the
model due to the possibility of a relic effect from the organic acid extraction experiment. The dry shoot
weight, fresh root weight, and root gall count data were transformed by taking the square root of the
data to obtain homogeneity of variance. There was insufficient leaf tissue from each plant to do individ-
ual analyses for each plant. For this reason, three leaf tissue samples, one from each block with the same
sample date, were combined. Data were analyzed following a randomized complete block design with
sampling dates as the blocking factor and rye rates as treatments following the model Y
ij
= µ+S
i
+ T
j
+ e
ij
, where S
i
represents sampling dates replications, and T
j
represents rye treatments. The differences between rye treatments were assessed for all the
tomato plant data by performing the least signifi- cant difference LSD test at the 0.05 probability
level.
2.7. Experiment 2: fate of added organic acids The soil collected for Experiment 1 was utilized.
The soil was maintained in a field-moist condition to sustain the pre-existing microbial population. Analyt-
ical grade formic 95–97, acetic 99.7, butyric 99, propionic 99.5, and valeric 99 acids
were combined and mixed with HPLC-grade water to form a working solution. Clay pots 7.6 cm diame-
ter were filled with the equivalent of 100 g oven-dry soil. The drain hole was covered with a piece of
woven fiberglass cloth to contain the soil and allow free drainage. The organic acid solution was added
to the soil with each pot receiving 1500 mmol of each acid.
The soil-filled pots were organized in a randomized complete block design in a temperature-controlled
incubator maintained at 26.5
◦
C. A pan of water was placed in the incubator to raise the humidity and
reduce desiccation of the soil. Samples of the soil solution were taken by flooding the top of the pots
with 35.6 ml of water and collecting the leachate. This amount of water theoretically diluted the
final acid concentration to 500 mmoll. The leach- ing took place in a refrigerator at 1
◦
C to inhibit further microbial decomposition of the added acids.
Samples were taken by leaching the soil imme- diately after application of the acids time zero
and new pots were leached every 2 h for 10 h. The leachate samples were adjusted to pH 5 by adding
0.1 mol NaOHl and then analyzed by ion exclusion chromatography.
Three shelves were used to separate the samples into three blocks. The experimental design consisted
of one soil treatment and six sampling periods. Six pots were randomly assigned within the shelves blocks.
At each sample period one pot was selected from each block with a pot being sampled only once. At each
sampling period, organic acid analysis was performed on the leachate. The model Y
ij
= µ+R
i
+ S
j
+ e
ij
, was utilized, where R
i
represents reps, and S
j
represents sampling periods.
R.G. McBride et al. Applied Soil Ecology 15 2000 243–251 247
3. Results and discussion
