Applications of nitrate and ammonium fertilizers alter soil nematode food webs in a continuous cucumber cropping system in Southwestern Sichuan, China
Eurasian J Soil Sci
Applications of nitrate and ammonium fertilizers alter soil nematode food webs in a continuous cucumber cropping system in Southwestern Sichuan, China
Kaiwen Pan 0
Pimin Gong 0
Jinchuang Wang 0
Yanjie Wang 0
Chenggang Liu 0
Wei Li 0
Lin Zhang 0
0 Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization, Chengdu Institute of Biology , China
1 86 28 8289 0522 E-mail address:
Nitrogen fertilizer; soil nematodes; trophic groups; soil food web; cucumber cropping system
Nitrate (NO3--N) and ammonium (NH4+-N) fertilizers are the main forms of chemical inorganic
nitrogen fertilizers that are widely used in agro-ecosystem for high yield. However, the responses
of soil nematode food web to different forms and rates of inorganic nitrogen fertilizers are not
well understood. The objective of this study was to determine the responses of soil nematode
food web to the applications of inorganic nitrogen fertilizers in a continuous cucumber (Cucumis
sativus L.) cropping system. Nitrate (NaNO3) and ammonium (NH4HCO3) fertilizers were applied
to cucumber plants at the nitrogen (N) rate of 0, 67.5, 135.0 and 202.5 kg N hm-2 before planting.
It was conducted in a randomized complete block design with 4 replications at Huaizi village,
Leshan district, Sichuan province, Southwestern China. The effects were analyzed at the stages of
seedling, blooming and fruiting, respectively. The results indicated that the numbers of
nematodes were significantly higher in soils with the addition of 67.5 kg N hm-2 than the control
at the seedling and blooming stages. Nematode number strongly increased at the seedling stage
and decreased at the blooming and fruiting stages in nitrate-treated soils compared to the
ammonium-treated. The percentage of herbivores to total nematodes significantly decreased
while that of bacterivores increased with a fertilizer rate less than 135 kg N hm-2 at the seedling
and fruiting stages. Nitrate significantly reduced the percentage of herbivores, and increased that
of bacterivores to total nematodes by comparison with ammonium at the blooming and fruiting
stages. The application of nitrate significantly increased nematode diversity and evenness, and
decreased dominance at the blooming stage relative to ammonium. Nitrate significantly
decreased the values of channel index at the blooming stage and maturity index at the seedling
stage in comparison with ammonium, respectively. Enrichment index and structural index
strongly increased at the seedling stage, and decreased at the blooming and fruiting stages under
the treatment of nitrate relative to ammonium. The results suggested responses of nematode
food web dependent on the rates and forms of inorganic nitrogen fertilizers and stages of
Plant growth and yield are often dependent on nitrogen (N) supply. Therefore, applications of N fertilizers
(organic and inorganic N) have been extensively used to improve crop yields of most agroecosystems in
developing and developed countries
(Liang et al. 2009; Roosta et al. 2009)
. Since soil nematodes are
involved in important ecosystem functions, such as decomposition and nutrient mineralization by
bacterivorous and fungivorous nematodes (Djigal et al. 2010), as well as the control of plant-feeding
nematodes by predacious and omnivorous nematodes
(Yeates and Wardle, 1996)
, a large number of studies
have been recently devoted to the comparative effects between organic and inorganic N fertilizers on soil
nematodes for organic agroecosystem
(Wang et al. 2006; Liang et al. 2009; Hu and Qi, 2010)
, and toxic
effects of added mineral nitrogen on specific species of root-knot nematodes (RKN) (Meloidogyne spp.) for
biological control of plant parasitic nematodes
(Barker et al. 1971; Oka et al. 2003; Khan and Kim, 2007;
Gong et al. 2009)
. However, there is very limited information regarding soil nematode assemblage affected
by inorganic N fertilizers including different rates and forms, although its application is increasing
(Gong et al. 2009)
Nitrate (NO3--N) and ammonium (NH4+-N) are the two main forms of N available for plant uptake. They are
often used as important forms of inorganic N fertilizers in conventional agricultural practices. Ammonium
fertilizer entering into the soils may be subject to different pathways of N transformations, including
assimilation into microbial biomass, uptake by plant roots and nitrification. Nitrate fertilizer entering into
soil is normally assimilated by soil microbes, or denitrified, ammoniated and taken up by plant roots, or
rapidly leached to environment
(Pan et al. 2008)
. In such case, easily accessible nitrogen, derived from either
direct inorganic N fertilizer input or root exudate stimulated by fertilizer, might activate microbe, and then
provoke opportunistic bacteria and coloniser nematodes (r-selected, with low cp values), reduce the
proportion of K-strategist bacteria and persister nematodes (K selected taxa, with high cp values)
et al. 1997)
. In such case, the soil nematode food web might be altered by the fertilizers.
Therefore, it will be important to investigate how the forms and rates of inorganic N fertilizers affect soil
nematode food webs, thereby using inorganic N to effectively manage nematodes. Fertilizers that contain or
release ammoniacal nitrogen, for example ammonium and urea are liable to control Meloidogyne spp. (a
group of herbivorous nematodes) for its toxicity at higher dosages
(Rodriguez-Kabana, 1986; Akhtar and
. The addition of appropriate nitrogen fertilizers both ammoniacal nitrogen and nitrate nitrogen
can improve health and defense of plants that allowed them to directly reduce the herbivorous nematodes.
Additionally, nitrate nitrogen fertilizer into soil can be transformed into ammoniacal nitrogen that has direct
toxicity for some of herbivorous nematodes. The findings from nitrogen fertilizers generally indicate that
those containing ammoniacal nitrogen are more damaging to nematodes than nitrate nitrogen
(RodriguezKabana, 1986). Although abundance of herbivorous nematodes was sometime negatively correlated with the
concentration(s) of soil ammonium
(Liang et al. 2009; Wei et al. 2012)
(Liang et al. 2009)
whether chemical nitrate fertilizers that only contain nitrate nitrogen can effectively diminish herbivores
and RKN are not well understood
(Barker et al. 1971; Collange et al. 2011)
Cucumber (Cucumis sativus L.), belongs to the Cucurbitaceae family, and is cultivated worldwide. It is the
fourth most important vegetable after tomato, cabbage and onion in Asia, and the second after tomato in
(Eifediyi and Remison, 2010)
. Cucumber monoculture often causes a decline in growth,
yield and quality in continuous cropping system because of phytotoxic effects of allelochemicals (mainly
phenolic acids) exuded by the plant
(Zhang et al. 2010)
. However, RKN increasing with year of continuous
cropping may play much more important roles in reducing cucumber production and quality when
compared to its autotoxic effects in the systems. Cultural practices such as crop rotation and mixture with
antagonistic plants are effective methods against nematode attack
(Hooks et al. 2010; Kayani et al. 2012)
However, continuous cropping of cucumber monoculture has especially attracted attention in some
countries with large population and limited farmland because it can produce higher profits than other cash
or cover crops. Interestingly, larger amount of inorganic N fertilizers (cheaper nitrate or ammonium) are
conventionally supplied into soils prior to planting and believed to be effective for reducing herbivorous
nematode infection and benefit for cucumber monoculture continuous cropping system in Leshan district,
Sichuan province, Southwestern China. One possible hypothesis given here was that the application of higher
rate of mineral nitrogen fertilizer prior to planting might not increase soil mineral concentration
(ammonium and nitrate) in the later stages of growth due to the rapid losses of mineral nitrogen, thus had
no higher inhibition for herbivores and stimulation for bacterivores than that of lower rate of mineral
nitrogen fertilizer. Previous limited reports suggested that fertilizers containing nitrate-N could better
improve root growth (Heuer, 1991), shoot growth
(Schenk and Wehrmann, 1979)
mycorrhizal (AM) colonisation of cucumber plants than those containing ammonium-N. This suggested, in
comparison with ammonium fertilizer, nitrate fertilizer might increase cucumber plant vigor, stimulate root
exudates, boosting bacteria and bacterivores as well as improving the ability of cucumber plants to resist
herbivorous nematodes. Therefore, the other possible hypothesis given here was that nitrate fertilizer might
have stronger inhibition for herbivores and stimulation for bacterivores than ammonium fertilizer in the
The objective of this study was to apply different rates of nitrate and ammonium fertilizers to field soils
prior to planting cucumber, and evaluate the response of the nematode community at the seedling, blooming
and fruiting stages of cucumber growth. We attempted to answer two questions: Is the nematode food web
structure differentially altered by the addition of different forms and rates of inorganic nitrogen? How does
the nematode food web change with different stages of cucumber growth when inorganic nitrogen fertilizer
is added to the ecosystem prior to planting?
Material and Methods
Field plot design
The field experiment was located at Huaizi village, Leshan district, Sichuan province, Southwestern China
(29?38? N, 103?45? E, elevation 364 m alt). The village is in a continental subtropical climate zone, with mean
annual temperature of 20 ? and mean annual precipitation of 1500 mm
(Gong et al. 2009)
. The soil at study
sites is silt fluvo-aquic soil. Beginning in 2005, experimental sites were planted with spring cucumber and
autumn lettuce (Lactuca sativa) in spring and autumn every year, respectively. Normally, autumn lettuce is
planted at last third of the month of August, harvested between October and November. The sits in the rest
time of each year is fallowed.
On 2 February 2007, 28 plots (1.2 m ? 5 m plot-1) were established. Plots were separated by 0.3 m wide
interval. Seven N fertilization treatments were conducted in a randomized complete block design with 4
replications: the control (no fertilizer, 0 kg N hm-2), sodium nitrate (NaNO3) at the rate of 67.5, 135.0 and
202.5 kg N hm-2, and ammonium acid carbonate (NH4HCO3) at that of 67.5, 135.0 and 202.5 kg N hm-2. On 4
February 2007, uniform seedlings of cucumber plants were randomly planted in different plots and there
were 16 seedlings per plot.
Sampling and laboratory analysis
Four soil samples were randomly collected with a 5cm diameter soil corer to a depth of 20 cm in each plot
and combined to make one composite sample per plot. Soil sampling occurred at three stages of cucumber
plant growth: (1) seedling stage (on 16 March 2007), (2) blooming stage (on 18 April 2007) and (3) fruiting
stage (on 16 May 2007). After the last sampling, all roots of cucumber plants in each plot were separately
harvested, dried and weighed. The soil samples were stored in insulated and tied plastic bags to prevent
moisture loss and transferred to the laboratory.
The organic matter content was determined by using the potassium dichromate external heating method
. Ammonium (NH4+-N) and nitrate (NO3?N) in soils were extracted with 2 M KCL and
tested by the Indolphenol-Blue method and copperized Cd reduction, respectively
(Keeney and Nelson,
. Total soil phenolics were measured by the Folin-Ciocalteau method
. Aqueous extraction
of soil by distilled water was used to determine pH by acidometer.
Nematode populations were extracted from 100 g fresh soil samples using a modified sieving and centrifugal
and identified according to a taxonomic handbook of soil nematodes
Nematode number is the total absolute abundance of individuals per 100 g dry soil. Percentages of trophic
group to total nematodes included: (a) percentage of herbivorous nematodes to total nematodes (H, %); (b)
percentage of bacterivorous nematodes to total nematodes (Ba, %); (c) percentage of fungivorous
nematodes to total nematodes (Fu, %); and (d) percentage of predator / omnivore nematodes to total
nematodes (OM, %). Nematode diversity (H'), dominance (?) and evenness (J) were calculated according to
(Yeates and Bongers, 1999)
. Maturity Index (MI) indicates the condition of an ecosystem
based on the composition of nematode community, calculated according to
. The enrichment
index (EI), based on the expected responsiveness of the opportunistic non-herbivorous guilds (Ba1 and Fu2)
to food resource enrichment, assesses food web response to available resources. The structure index (SI),
represented an aggregate of longevity, body size and disruption-sensitivity of functional guilds as captured
in the cp classification of taxa, indicates a stressed environment. Channel index (CI), an index of the nature of
decomposition channels through the soil food web, showes the predominant decomposition pathways. EI, SI
and CI were calculated to
Ferris et al. (2004)
The data of soil nutrients, pH and nematode index were analyzed by one-way analysis of variance (ANOVA)
to test differences among fertilizer rates, and between fertilizer forms. Differences at a p ? 0.05 level were
considered statistically significant using the Duncan test. Mixed model ANOVA that set fertilizer type (Type),
fertilizer rate (Rate) and growth stage (Stage) as fixed factor(s), soil nutrients, pH and nematode index as
dependent variables was used to test the effects of Type, Rate, Stage and their interactions on all dependent
variables. All statistical analyses were performed using an SPSS 16.0 software package.
Root biomass and soil chemical property
The root biomass of cucumber plants increased with rate of application of the mineral fertilizers relative to
the control (Table 1). The biomass was strongly affected by fertilizer rates of ammonium nitrogen or nitrate
nitrogen (p ? 0.05) but not by different forms (p > 0.05).
The pH values of soils greatly decreased with the rate of the ammonium application (p ? 0.05) at the seedling
stage (Table 2). Significant difference in pH values was only observed between the two forms of fertilizers at
the seedling stage (p ? 0.01). The rate and form of fertilizer application had no effect on the concentration of
soil organic matter except for the rate of ammonium application at fruiting stage (p > 0.05). However, there
was significantly higher concentration of organic matter in soils with the addition of ammonium in
comparison with the control at the fruiting stage (p ? 0.05 except for application rate of 135.0 kg N hm-2).
The concentrations of soil NO3--N strongly increased with increasing application rates of nitrate and
ammonium fertilizers at the stage of seedling (p ? 0.05 and p ? 0.001 at rates of nitrate and ammonium
fertilizers, respectively). Of all stages, ammonium addition greatly increased concentration of NO3--N in soils
at the stage of blooming relative to nitrate addition (p ? 0.05). Soil NH4+-N strongly increased with rates of
fertilizer additions at the seedling stage (p ? 0.001). There was significantly higher concentration of NH4+-N
in soils treated with ammonium in comparison with nitrate at the seedling stage (p ? 0.05). The application
of nitrate strongly enhanced soil NH4+-N at the blooming stage (p ? 0.05). Total phenolic acids in soils
gradually decreased with the rates of fertilizer application at the stage of seedling (p > 0.05) (Table 2).
Nematode composition and number
Herbivorous nematodes comprised 10 genera, in which Tylenchus dominated. Bacterivorous nematodes
included 13 genera (Table 3), in which Rhabditis, Mesorhabditis, Caenorhabditis and Cephalobus were
predominant, and Rhabditophanes, Cervidellus and Acrobeles were co-dominant. Fungivorous nematodes
comprised of dominated Aphelenchus and co-dominant Paurodontus and Nothotylenchus.
Predaceous/omnivorous nematodes contained 7 genera, in which Dorylaimus was dominated. The number
of nematodes significantly increased and reached the highest values after application of 67.5 kg N hm-2 (p ?
0.05, Fig. 1), and then weakly decreased with the fertilizer rate at seedling and blooming stages. Moreover,
nematode number in nitrate-treated soils was strongly higher than that in ammonium-treated soils at
seedling, but the contrary results were found at the other two stages (p ? 0.05, Fig. 2).
K. Pan et al. / Eurasian J Soil Sci 2015, 4 (4) 287 - 300
Proportional contributions of trophic groups
The rate of nitrogen fertilizer significantly affected the percentages of herbivores and bacterivores at the
seedling and fruiting stages (p ? 0.01 at seedling and p ? 0.001 at fruiting) (Fig. 3). The percentage of
herbivores to total nematodes strongly decreased with fertilizer rate when not exceeding 135 kg N hm-2 (p ?
0.05), but weakly increased when application dose was 202.5 kg N hm-2 at the seedling and fruiting stages
(Fig. 3). On the contrary, the percentage of bacterivores greatly increased with fertilizer rate when not
exceeding 135 kg N hm-2 and then decreased with the rate at the seedling and fruiting stages (p ? 0.05).
Furthermore, the percentage of fungivores was greatly decreased with the rate of nitrogen application at the
blooming and fruiting stages (p ? 0.001), and that of predators / omnivores to total nematodes was
significantly impacted by the rate at all the stages (p ? 0.001 except for p ? 0.05 at the seedling stage).
There were significantly higher percentages of bacterivores and lower that of herbivores in nitrate-treated
soils than ammonium-treated soils at blooming and fruiting stages (p ? 0.001, Fig. 4). However, decrease of
herbivores and increase of bacterivores by NO3--N relative to NH4+-N were highest at fruiting stage, and
lowest at blooming stage. Significant difference in the percentage of predators / omnivores between two
treated soils was not observed at all the stages (p > 0.05, Fig. 4).
The rate of fertilizer significantly affected soil nematode diversity (H') at the fruiting stage (p ? 0.01), and
dominance (?) and evenness (J) at the seedling (p ? 0.05) and fruiting stages (p ? 0.01 for ? and p ? 0.01 for J,
Table 4). H' significantly decreased at the fruiting stage, and J strongly increased with the rate at the seedling
and fruiting stages (Table 4). The forms of fertilizers strongly altered H' and ? at the blooming (p ? 0.01), and
J at the seedling and blooming stages (p ? 0.01, Table 5). H' at the seedling stage was weakly lower but at the
blooming stage was significantly higher in soils with application of NO3--N relative to NH4+-N (Table 5).
Nitrate application strongly incurred a decrease of ? at the blooming stage and increase of J at the seedling
and blooming stages in comparison with ammonium application (p ? 0.01, Table 5).
The rates of fertilizer significantly impacted on CI at the blooming (p ? 0.001) and fruiting stages (p ? 0.01,
Table 4), and on MI at all stages (p ? 0.05 except for p ? 0.01 at the seedling stage, Table 4). EI was strong
altered by the rate at the blooming stage (p ? 0.01), and SI at the blooming and fruiting stages (p ? 0.01 and p
? 0.05, respectively). The values of CI and MI greatly decreased but that of EI and SI increased at some of
stages and rates (p ? 0.05, Table 6). Nitrate addition resulted in significant decrease of CI at the blooming
stage (p ? 0.01) and MI at the seedling stage (p ? 0.05) but incurred slight increase of that at the fruiting
stage compared to ammonium addition. However, the values of EI and SI in soils with nitrate fertilizer
significantly, by comparison with ammonium application, increased at the seedling stage (p ? 0.01 for EI and
p ? 0.05 for SI) and decreased at the blooming and fruiting stages (p ? 0.01 except for p ? 0.05 for EI at
blooming, Table 5).
The results by Mixed model ANOVA (MOVA) indicated no significant effects of the fertilizer rate and type on
soil organic matter (SOM) (Table 6). The addition of the fertilizers weakly increased the concentrations of
soil organic matter (SOM) at almost of the stages and rates, and ammonium application significantly
stimulated SOM at the fruiting stage (Table 2). This was in consistent with previous findings of higher
organic carbon in a semiarid soil after nitrogen fertilizer
(Rasmussen and Rohde, 1988)
. The type, rate, stage
and its interaction significantly affected soil NH4+-N (Table 6). Although the type and rate had no direct
effects on soil NO3--N, but they significantly indirectly affected soil NO3--N via its interactions with the stages
(Table 6). The concentrations of NO3--N and NH4+-N were significantly enhanced both in nitrate-added or
ammonium-added soils relative to the control at almost rates and the seedling stage (Table 2). However,
concentration of NO3--N increased by 59.36 ? 238.41 mg kg-1 N via nitrification in ammonium-treated soils,
while that of NH4+-N by 0.15 ? 40.05 mg kg-1 N via ammonification in nitrate-treated soils. This indicated that
soils were undergoing ammonification although nitrification dominated in the ecosystem
(Pan et al. 2008)
The percentages of herbivores, accounting for 60-92% of total soil nematodes in the whole experiment (Fig.
3), showed that herbivorous nematodes were the dominant trophic groups of the cucumber cropping
system. This was in accordance with previous studies in a wheat field
(Li et al. 2007)
and a Chinese maize
(Hu and Qi, 2010)
. The percentage of omnivores/predators remained still lower after nitrogen
application (Fig. 3). Higher percentage of herbivores with little predator/omnivore suggested fragility and
degradation in soil nematode food web of the cucumber cropping ecosystem. Therefore, restored damaged
omnivores and predators could be required for soil health because they could stimulate soil nutrient
elements mineralization, as well as preying on other nematodes such as plant-feeding nematodes
The addition of inorganic nitrogen increased total nematode density during the whole period of cucumber
growth, especially the rate of 67.5 kg N hm-2 (Fig. 1). This was largely due to the increase of the enriched
bacterivores and general opportunistic bacterivores and fungivores.
The abundance of those nematodes in soils is documented to growth swiftly when new resources are input
(Liang et al. 2009; Hu and Qi, 2010)
. The percentage of bacterivores greatly increased at seedling by
applications of 135 kg N hm-2 and at fruiting stages by applications of 67.5 and 135 kg N hm-2, while that of
fungivores only remained weak increase at seedling stage (Fig. 3). Those additions of nitrogen fertilizer did
not strongly increase soil organic carbon that had a increase tendency along with the rate, but significantly
improved soil mineral nitrogen (Table 2, 6) which could motivate microbe growth
(Gong et al. 2009a)
turn, provided rich food resources for opportunists, especially for enrichment opportunists and thereby
increased the percentage of bacterivorous nematodes.
Most of the previous reports revealed that chemical nitrogen fertilizer increased percentages of soil
herbivores relative to the control, for example,
Wang et al. (2006)
found that ammonium nitrate addition
enhanced the percentages of herbivorous nematodes in the soils from a squash (Cucurbita pepo)
agroecosystem. The similar results were also observed in the soils of maize after the application of urea and
ammonia sulfate respectively
(Liang et al. 2009; Hu and Qi 2010)
. However, the percentage of herbivores
was greatly decreased by the applications of 67.5 and 135 kg N hm-2 at the seedling and fruiting stages (Fig.
3). Relative abundances of Meloidogyne spp. were reduced 0.8% and 4.5% by 67.5 kg N hm-2, as well as 1.3%
and 3.3% by 135 kg N hm-2 compared with the control. The additions of NH4+-N and NO3--N significantly
increased NH4+ content of soils directly, or indirectly through ammonification with the conversion of organic
nitrogen or NO3--N into NH4+-N at the seedling stage (Table 2). The type, rate and stage can also indirectly
alter soil NH4+-N via its interactions (Table 6). These results to a certain extent, explained why percentage of
herbivores was lowered at the seedling stage since higher dose of ammoniacal nitrogen can reduce
(Rodriguez-Kabana, 1986; Akhtar and Malik, 2000)
. However, negative effect of
ammoniacal nitrogen on herbivores could not explain significantly lower percentage of herbivores than the
control at the fruiting stage as soil NH4+-N was not strongly enhanced at that time (Table 2). Underground
biomass of the cucumber plant was obviously enhanced by the addition of inorganic nitrogen (Table 1). It
could suggest that cucumber plant might have higher biological activity after the application of inorganic
nitrogen, in turn, secrete lots of root exudates into soils to activate antagonistic microorganisms that
reduced herbivorous nematodes by parasitization or prey at the fruiting stage (Fig. 3). Moreover, the
defense responses of cucumber plant motivated by the addition of mineral nitrogen could produce new food
sources and environments that are harmful for herbivores or herbivores abominated, and thereby reduced
the percentage of herbivorous nematodes
(Gong et al. 2009)
N fertilizer strongly reduced H' and ? at the fruiting stage (Table 4). J was greatly stimulated at almost of
rates in the periods of the seedling and fruiting stages (Table 4). Those results were not in agreement with
previous findings of
Wang et al. (2006)
that the addition of 100 kg N hm-2 ammonium nitrate hardly had
impacts on H' and ? relative to the control in a squash agroecosystem. This could be attributed to the
differences of plant species and growth stages between the both studies.
The values of EI could reflect the availability of resources to the soil food web and the response of primary
decomposers to the resources
(Ferris et al. 2001)
. The value of CI may assess soil fertility levels and nutrient
(Ferris et al. 2001)
. Inorganic nitrogen strongly reduced values of CI at the blooming stage (Table
4), which was consistent with previous findings that the value of CI in soils treated by urea was significantly
lower than the control
(Liang et al. 2009)
. Higher EI and lower CI suggested more enriched conditions of soil
food web and greater bacterial activity as a result of nitrogen treatment
(Gong et al. 2009a)
. Therefore, soils
with added fertilizers were undergoing a bacteria-dominated decomposition pathway in the cucumber
cropping system (Fig. 3).
The values of SI in soils with nitrogen additions were greatly higher than the control at the blooming stage
(Table 4), which was consistent with previous reports that the SI was increased by the addition of the cover
crops (Poaceae or Fabaceae species) in banana agro-ecosystems
(Djigal et al. 2012)
Wang et al. (2006)
reported the abundance of bacterivores, fungivores, predatory nematodes, and total
nematode abundance increased with rates of organic fertilizer. However, soil nematode density at seedling
and blooming stages was only strongly enhanced by the application of 67.5 kg N hm-2 in comparison with the
control (Fig. 1). The percentages of herbivores were significantly reduced by 135 kg N hm-2 at seedling and
by 67.5 and 135 kg N hm-2 at fruiting (Fig. 3). It suggested that optimal nitrogen application for reducing
percentages of herbivores covered Meloidogyne spp was 67.5 ~ 135 kg N hm-2 per year in cucumber
cropping system. This was inconsistent with previous findings of the excess of 300 kg N kg-1 was required for
satisfactory control in herbivores
. The fertilizers in the study using inorganic not
organic nitrogen (like urea) could result in different effects dependent on doses in decrease the percentages
of herbivorous nematodes. Most importantly, excess nitrate nitrogen into soils could be transformed into N2
or N2O through the process of denitrification, and escape into air, or directly through nitrate leaching to the
(Pan et al. 2008)
. Excess ammonium nitrogen into soils is rapidly converted into nitrate
nitrogen by nitrification, and thereupon easily loses through the processes of denitrification or leaching.
These processes could conceal true effects of fertilizer rates on herbivorous nematodes and thus present a
decrease of the percentage with rates of fertilizers. Similar function and consequences could also exist in the
effects of fertilizer rates on the percentages of bacterivores and fungivores (Fig. 3). All indexes fluctuated
with fertilizer rates at most stages of cucumber growth (Table 4). This phenomenon was also reported by
Sarathchandra et al. (2001)
who found MI was highest at the rate of added 200 kg N hm-2, while lower at the
rate of added 400 kg N hm-2 in comparison with the control. This was attributed to direct and indirect effects
including fertilizer rate, growth stage and its interactions.
Nematode number was significantly higher in soils with treated nitrate nitrogen than ammonium nitrogen at
the seedling stage, but opposite result was observed at the blooming and fruiting stages (Fig. 2). This was
ascribed to the explosive increase of nitrate nitrogen in soils at the seedling because of added nitrate
nitrogen (Table 2), which easily stimulated growth of soil microorganism and nematode because nitrate
nitrogen was preferred to be resource for immobilization. Nitrate nitrogen is easily absorbed by plant root,
immobilized by microorganisms, or lost through denitrification and nitrate leaching
(Pan et al. 2008)
Through this process, nitrate nitrogen of soils after the application of nitrate prior to planting reduced at the
blooming and fruiting stages. However, to a certain extent, the addition of ammonium relative to nitrate
supplemented nitrate nitrogen in soils through nitrification (Table 2), which strongly enhanced nematode
number at the blooming and fruiting stages (Fig. 2).
The percentages of herbivores were significantly lower in the nitrate-treated soils than the ammonia-treated
soils at the blooming and fruiting stages, suggesting that the addition of nitrate nitrogen had strong decrease
in comparison with ammonia nitrogen (Fig. 4). This was contrary to expectation as those containing
ammoniacal nitrogen are more damaging to nematodes than those containing nitrate nitrogen, on the basis
of the comparison of decrease in nematode between ammonia nitrogen and nitrate nitrogen
Barker et al. (1971)
found inhibitory effects of the application of sodium nitrate or
ammonium nitrate were positively correlated with concentration of N. This could explain significantly lower
percentages of herbivores in the nitrate-treated relative to the ammonia-treated because there was higher
concentration of mineral N in the nitrate-treated soils at the blooming stage (Table 2, Fig. 4). Previous
reports found that nitrogen addition reduced herbivores through inhibition of higher ammonium
concentration and pH value in soils
(Oka et al. 2003; Wei et al. 2012)
. However, there were no significant
differences in pH value, concentrations of mineral N and ammonia nitrogen in the soils between the nitrate
and ammonia treatments at the fruiting stage (Table 2). Perhaps the explanation for strongly decrease
effects of the application of nitrate nitrogen at the blooming stage, especially fruiting stage, lay in activation
of the plant resistance to herbivores or antagonists of herbivorous nematodes as cucumber plant and soil
organisms prefer to utilize nitrate nitrogen in soils. Preferred nitrate nitrogen for uptake also strongly
incurred higher percentages of bacterivores at the blooming and fruiting stages relative to soils with the
addition of ammonia nitrogen (Fig. 4).
Nitrate nitrogen significantly increased H' and J, decreased ? at the blooming stage when comparison with
ammonia nitrogen (Table 5). This could be attributed lower concentration of mineral nitrogen in the nitrate
treated soils (Table 5), which reduced abundance of enrichment opportunists. Significant decrease of EI and
SI at the blooming and fruiting stages in soils added with nitrate nitrogen relative to ammonia nitrogen
indicated that the addition of ammonia nitrogen could provide much more rich food resources for the soil
food web, and then enhanced the length of food chains of soil nematode than nitrate nitrogen. The result
suggested that ammonium instead of nitrate could be a desirable source of N under certain conditions in
order to reduce N leaching and persist providing available mineral nitrogen for plant and soil organisms
during the whole growth period
(Roosta et al. 2009)
. EI and SI significantly increased at the seedling and
decreased at the blooming and fruiting stages (Table 4), which suggested that response of EI and SI to
inorganic mineral fertilizer also depended on growth stages of cucumber (Table 6).
The rate strongly altered soil phenolic acids (Table 6). Additionally, Plant can rapidly produce lots of
phenolic acids into soils when plant exposed to biotic and abiotic stress environment
(Li et al. 2009)
. Of all
stages of the plant growth, the percentage of herbivores reached a peak at the blooming stage, and was
strongly altered by the additions of different rates of mineral nitrogen at the seedling and fruiting stages
(Fig. 3). Higher percentages of herbivores stimulated exudation and release of phenolic acids produced by
the cucumber plant for improving its resistance to herbivorous nematodes and thereby resulted in highest
accumulations of phenolic acids in soils at the blooming stage (Table 2). Increase of phenolic acids as
available carbon source for soil microorganisms and enhanced defense function of the plant significantly
increased the percentages of bacterivores, and strongly decreased that of herbivores at fruiting stage (Fig.
3). The results suggested that plant growth could alter effects of fertilizer rates and forms on soil nematode
food web (Table 6), thus incurred fluctuations of soil nematode with growth stages (Table 4 and 5).
From an applied perspective, the amount of the application of inorganic nitrogen better for the decrease of
the percentage of herbivorous nematodes and for the increase of the percentage of bacterivores was 67.5 ~
135 kg N hm-2 a-1 in the cucumber cropping system. Excess nitrogen addition did not provide better decrease
for herbivores and increase for bacterivores and fungivores. Nitrate addition had higher decrease for the
percentages of herbivores and fungivores and increase for that of bacterivores relative to ammonium.
However, application of inorganic nitrogen did not increase higher trophic levels of the soil nematode food
web. Therefore, inorganic nitrogen mixed with organic fertilizers such as manure and compost should be
advocated to restore lost higher trophic levels (for example, omnivores and predators) of the nematode food
web in soils of the ecosystem.
The paper revealed that rates and forms of inorganic nitrogen fertilizers, which contain either NO3--N or
NH4+-N, strongly altered the percentages of herbivores and bacterivores, and also affected nematode
functional indexes included H', J, ?, CI, MI, EI and SI at one or more growth stage(s) of the cucumber plant.
However, the comparisons of effects among nitrate nitrogen, ammonia nitrogen and ammonium nitrate that
contains both NO3--N and NH4+-N, on soil nematode food webs need to be further studied for well
understanding of responses of soil nematodes to NO3--N and NH4+-N fertilizers.
The application of mineral nitrogen into soils in the cucumber cropping system greatly altered the nematode
assemblage, and produced obviously effects on soil nematode food web functioning. Effects of the
applications of mineral nitrogen fertilizers prior to planting on soil nematodes were dependent on rates and
forms of mineral nitrogen fertilizer, and also on growth stages of the cucumber plant.
We thank the Leshan Municipal Science and Technology Bureau who allowed our work in their fields, and
provided us great help during the field work. This research was supported by National Natural Science
Foundation of China (#31370632), Ministry of Science and Technology of the People?s Republic of China
, and cooperative project by the Chinese Academy of Sciences and the people's
Government of Leshan Municipality.
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