Effects of straw mulching practices on soil nematode communities under walnut plantation

Agricultural management techniques such as mulching with crop straw can impact soil properties and may in turn change the structure and function of the soil food web. We investigated different straw mulching types and straw mulching coverage levels on soil nematodes community structure in walnut orchards. We set up a randomized experimental design with three straw mulch types, and three straw mulch distance treatments in a walnut plantation. The results indicated that the number of soil nematodes after straw mulching was lower than that found in the control (CK). However, the metabolic and structure footprints of the omnivore-predator nematodes showed higher values as compared to CK. The abundances of plant parasite and omnivore-predator nematodes were negatively correlated with ammonium nitrogen (NH4+–N) and dissolved organic nitrogen (DON), whereas soil moisture content (SM) had a negative correlation with the abundance of total nematodes. High structure index (SI), maturity index (MI) and low enrichment index (EI) values revealed a structured soil food web, medium soil enrichment, and fungal decomposition channel under the mix straw mulching treatments. Soil nematodes should be used as an indicator of soil functional changes resulting from straw mulching.

www.nature.com/scientificreports/ potential yield 9 . Therefore, it is an important step for the sustainable management of walnut orchards to determine suitable materials for straw mulch and location for straw mulch placement. Soil nematodes are one of the most abundant metazoans on the earth. They exist widely in various habitats and play an important role in maintaining the stability of soil ecosystems, promoting material circulation and energy flow 10 . Nematodes are simple to extract and identify, and feed on diverse nutrient resources, making them very sensitive to agricultural management measures and environmental changes; thus, they can be used as indicators of soil quality and health 11,12 . Previous experiments showed that the application of straw and other organic fertilizers could increase the number of beneficial soil nematodes and decrease the number of phytophagous soil nematodes 13,14 . However, little information is available about whether and how the microenvironment soil conditions under straw mulching affect the structure of soil nematode communities, biodiversity and function in walnut orchards.
The major objectives of this study were to explore whether and how straw mulching affects the soil nematode community. Because straw is rich in nutrients and active substances, nutrients such as C and N can be released into the soil by a degradation pathway after straw mulching a walnut orchard, which provides a rich source of food for soil nematodes, thereby increasing soil nematode numbers and improving soil nematode community structure 15 . Therefore, we hypothesized that different straw mulching treatments could increase the number of nematodes and improve the community structure of soil nematodes. We also hypothesized that mixed-straw mulching would increase nutrient availability and improve soil fertility compared with rice straw mulching and rapeseed straw mulching because it would have a more suitable C/N ratio and faster degradation rate.

Results
Soil environmental conditions. Mix straw mulching treatments significantly correlated dissolved organic carbon (DOC) (p < 0.05) and NO 3 -N content (p < 0.05) ( Table 1). In general, mix straw mulching and a cover distance of n (Mix-n) had a higher content of DOC and NO 3 -N than the other treatments (i.e., single-straw mulching). Meanwhile, the soil DOC content of the Mix-n treatment was significantly higher than that of the CK treatment. Though the content of NO 3 -N was higher in the Mix-n treatment than in the CK treatment, this difference was not significant. However, the soil pH, SMC and NH 4 + -N responses to different straw mulching treatments were not significant (p > 0.05).
Soil nematode communities. The number of nematode genera in Rice-n, Rice-1.5n, Rice-all n, Rape-n, Rape-1.5n, Rape-all n, Mix-n, Mix-1.5n, Mix-all n and CK were 34, 37, 34, 31, 30, 26, 31, 30, 31 and 29, respectively (Appendix 1). Compared with those in other straw mulching treatments (rice straw, rapeseed straw, and mix straw), the total nematode genera were found to be significantly (p < 0.05) more numerous in the control treatment (CK) ( Table 2). The control treatment (CK) and mix-all n treatment had significantly (p < 0.05) higher abundances of fungivores than the Rapeseed -n and Rapeseed-1.5 n treatments. Two Rice straw treatments (those with cover distances of n and all n) and one Rapeseed straw treatment (cover distance of 1.5n) had significantly (p < 0.05) lower abundances of plant parasites than the CK treatment. However, there were no significant differences among the different treatments in the abundance of omnivore-predator nematodes ( Table 2). Table 1. Overview of main effect of straw mulching quality and distance on environmental factors based on ANOVA. Data are the means of three replicates ± SD .Within each column, the values with the same lower case letter are not significantly different. Different letters indicate statistically significant differences between treatments, according to the Duncan's multiple range test (p < 0.05). CK is no straw mulching treatment. The combination of Rice-n, Rice-1.5n and Rice-all n represents straw mulching types is rice straw and straw mulching distances is mean radius of crown width (n), one and half mean radius of crown width(1.5 n), and the whole quadrat(all n); The combination of Rape-n, Rape-1.5n and Rape-all n represents straw mulching types is rapeseed seed straw and straw mulching distances is n, 1.5 n and all n; The combination of Mix-n, Mix-1.5n and Mix-all n represents straw mulching types is mixed rice and rapeseed seed straws and straw mulching distances is n, 1.5 n and all n; similarly hereinafter. www.nature.com/scientificreports/ The Rapeseed-1.5n and Mix-1.5n treatments had significantly higher omnivore-predator footprints and structure footprints than the Rice-n and Mix-all n treatments (p < 0.05) ( Table 3). The metabolic footprints of fungivores were higher under Rapeseed-all n and Mix-all n than under the other treatments, while the metabolic footprint of bacterivores had greater values in the Rapeseed-1.5n treatment than in other treatments. There was no obvious change in the plant parasite footprint or the enrichment footprint among different treatments.
Soil nematode faunal profile. The soil food webs of the straw mulching treatments were plotted along their respective SI and EI trajectories in Fig. 1. A discernible pattern was found in the nematode faunal profile of different straw mulching treatments. The nematode fauna analysis showed that the all different coverage distance of the rice straw treatment and the coverage distances were n and 1.5n of the rapeseed straw treatment was distributed in the B quadrant (Fig. 1A,B). While, when the coverage distance was increased to cover the whole plot (all n) of the rapeseed straw treatment, it was distributed in the C quadrant. For the mix straw treatment, all coverage distances were distributed in the C quadrant. The variation in SI value increasing coverage distance is not obvious. EI value tends to decrease with increasing coverage distance, and the distribution of the straw mulching treatments gradually approaches the C quadrant level from the B quadrants ( Fig. 1).
Nematode diversity. Significant differences were observed in the basal index (BI), channel index (CI), and maturity index (MI) between the different straw mulching treatments. MI and CI were significantly (p < 0.05) higher in the mix straw mulching treatment than in the CK treatment, especially in the mixed treatment with straw mulching distance at 1.5n (Fig. 2a-c). For all straw treatments, BI and CI were higher for the whole plot mulching (all n) than for the other mulching distances (n, 1.5n). Moreover, there were no fluctuations among different treatments in terms of the Shannon-Weaver index (H ' ), Species richness index (SR), Trophic diversity index (TD) or Pielou's evenness index (J ' ) ( Fig. 2d-g). Table 2. The abundances of total soil nematodes and trophic groups (means ± standard errors, n = 3) as affected by straw mulching treatments. Different letters indicate statistically significant differences between treatments, according to the Duncan's multiple range test (p < 0.05).  (Table 4). NH 4+ -N was significantly (p < 0.05) negatively correlated with omnivore-predator nematodes. SM had a significant (p < 0.05) negative correlation with the total nematode abundance. However, DOC, NO 3 -N, and pH had no significant correlations with the soil nematode communities (Table 4).

Discussion
Soil environmental conditions. It is clear that the Mix-n treatment had higher DOC and NO 3 -N than the other treatments under all soil environmental conditions. Due to the different C/N ratios of the different straw types, N degradation and mineralization were also different. The change in soil nutrients caused by straw mulching is mainly due to the role of soil organisms. Therefore, we can explain the difference in soil nutrients by the soil biological composition of different straw mulching treatments. In general, the specific genus of soil nematode in the mix treatment can characterize the particular soil nutrient status. Previous studies have shown that some nematodes are found more often in areas with similar environmental variables and that nematode genera within the same trophic group responded differently to environmental variables 16 . We found that the higher abundances of Prismatolaimus, Cephalobus and Eucephalobus corresponded to the higher soil NO 3 -N (Appendix 1). Our results are consistent with the observations of Song et al. 17 . Moreover, the Mix-n treatment had a higher density of Mesodorylaimus, Aphelenchoides and Thonus where the DOC was higher. This result is in agreement with the findings of Olatunji et al. 18 , in which Thonus, Aporcelaimus, Mesodorylaimus, Aphelenchoides, Criconemoides, Tylenchus, and Rhabditidae were positively associated with DOC.  Table 2, it is apparent that the CK treatment had a higher total number of soil nematodes and a higher abundance of soil nematodes in different nutritional groups than any straw mulch treatment; that is, the number of soil nematodes after straw mulching was lower than that in the control. Blankinship et al. 19 used a meta-analysis method to study the response of soil nematodes to temperature increase under different ecosystem types. It was found that soil nematodes were mainly affected by annual precipitation. When annual precipitation exceeded 626 mm, the increase in temperature had a positive effect on the number of soil nematodes 19 . In this study, the annual precipitation in this area (1033.9 mm) exceeded 626 mm, and straw mulching had a cooling effect during the growth period of young walnut trees. This could be a possible reason of higher abundance of soil nematodes in the CK treatment than that in any straw mulching treatment. Moreover, this finding is also contrary to our first hypothesis that different straw mulching treatments would increase the number of soil nematodes. The reasons are as follows: on the one hand, phenolic acids enter the soil through the secretions of walnut roots and the decomposition of a large amount of straw residues, which results in an increase in phenolic acids in the soil and a decrease in the total number of soil nematodes and other nematodes 20 . On the other hand, straw mulching returns pathogenic bacteria and parasite eggs to the field directly. At the same time, the nutrients released from straw in the soil provide a favorable environment for pathogenic bacteria and parasite eggs to increase in number, which significantly inhibits soil nematodes 21 .
In addition, a key finding was that fungal nematodes were more common than bacterial nematodes in the treatments with complete mulch coverage than in the n and 1.5n coverage treatments. When rice straw, rapeseed straw and mix straw were applied at n and 1.5n distances, the decomposition pathway was a bacterial channel; when the coverage distance increased to all n, the decomposition pathway gradually changed to decomposition equally distributed between bacterial and fungal decomposition pathways. In contrast, the CK treatment was dominated by the number of bacterivorous nematodes, suggesting that the bacterial channel was the main pathway of decomposition, which was consistent with the result of the distribution map of nematode fauna in Fig. 1. At the same time, this result indicates that the coverage distance changed the dominant community of nematode trophic groups.
The footprints of different nematode trophic groups are proxies for the carbon or energy flow entering the soil food web through their respective channels 22 . In our study, we found that the footprint and the carbon biomass of the omnivore-predator nematodes and all structure metabolic footprints showed higher values under all straw mulching treatments compared with those of the other soil nematode trophic groups (Table 3). This observation may be explained by the predator-prey trophic cascade effect: straw mulching stimulates higher carbon and nutrient inputs first to microorganisms and then to microbivorous nematodes, which stimulate the metabolic activity and abundance of omnivore-predator nematodes; omnivore-predator nematodes consume more prey and thus inhibit the abundance of soil nematodes at lower trophic levels 23 .
Nematode diversity. The maturity index of nematodes is one of the key indices of soil health. In our study, the MI values for rice straw and rapeseed straw treatment alone were not significantly higher than those for the CK treatment (Fig. 2c). However, the MI values for the mix straw treatments were significantly higher than those for the CK treatment, indicating that the structure of the nematode community is stable and that the complexity of the soil food web could increase under the mix straw treatment.
Combined with the ecological indices BI, which is related to soil properties and decomposition pathways 24 , we found that higher CI value for the three straw mulching treatments appeared in the whole-plot coverage treatments (all n). Our results contrast with those of other studies, which found that bacterial-dominated decomposition pathways were the most common pathways 20 . This discrepancy could be explained mainly by the observed variations in the abundances of bacterivores and fungivores among the different coverage distances. Specifically, bacterivore nematodes predominate in different soil nematode trophic groups when the coverage distance is n, while bacterivore nematodes and fungivore nematodes predominate in different soil nematode trophic groups when the coverage distance is increased to all n ( Table 2). In addition, soil nematode decomposition pathway changed with the increase in coverage distance in the three straw mulching treatments, which may have been caused by the increase in contact area between straw and soil. The specific mechanism needs to be further studied in our next work. www.nature.com/scientificreports/ Soil nematode faunal profile. The SI is considered to indicate the structure of the soil food web response to disturbance and during remediation, while the EI reflects soil food web responses to available resources and the resource response to the primary decomposers 17,25 .
In the present study, the rice straw mulching treatments and rapeseed straw mulching treatments with high EI and SI values at different straw mulching distances were in quadrant B, indicating that the structure of the food web was fairly mature, the N concentration was high, the C:N ratio was low, the decomposition pathways of fungi and bacteria was balanced, and the disturbance level of the soil environment was low to moderate. These conditions occurred is mainly because of the large amounts of dissolved organic carbon and dissolved organic nitrogen in the soil due to straw degradation and the straw mulching water retention effect making the soil moisture content higher than that found in the CK treatment (Table 1).
However, the mix straw mulching treatments with high SI and low EI values at different straw mulching distances were in quadrant C, which indicates a structured food web, medium soil enrichment, a moderately high C/N ratio, fungal decomposition channels, and no disturbance. Our previous research suggested that the mix straw mulching treatment had a moderate carbon nitrogen ratio (C:N) and that mix straw degrades more quickly than rice straw or rapeseed straw 9 . In addition, the mix straw may have provided stable moisture content and higher dissolved organic carbon and dissolved organic nitrogen than rice straw or rapeseed straw (Table 1), thus increasing nutrient availability and soil fertility levels. This result is supported by other agricultural management practices 20,26,27 . This evidence supported our hypothesis that the mix straw mulching treatment led to a more stable soil food web and higher soil fertility levels.
Environmental factors affecting soil nematode community variability. Straw mulching directly increases the mineral nitrogen and DON contents in the soil through decomposition, which significantly increases the content of nitrogen in the soil, thus increasing the amount of soil nutrients and soil organisms. Plant parasite and omnivore-predator nematode abundances were negatively correlated with NH 4+ -N and DON contents, but there was no significant correlation between the nematode community and soil DOC content. This finding indicates that nitrogen in the soil of the agroforestry ecosystem had a more significant impact on the nematode community than carbon. This result is also consistent with previous results 28,29 . Another possible explanation was that ammonium toxicity may occur when soil nematodes feed on root fluid, resulting in a negative correlation between omnivore-predator nematodes and NH 4+ -N 30 . Compared with the control condition, straw mulching significantly increased soil moisture content and soil anoxia, while soil total nematodes were negatively correlated with SM value. The results showed that the increase in soil moisture changed the soil environment, inhibited the growth of soil microorganisms, and inhibited the growth of total nematodes through changes in nutrient levels and the environment in the food chain.
In terms of straw coverage distance, our results showed that the decomposition pathway gradually changed from the bacterial decomposition channel to the bacterial/fungal decomposition channel when the coverage distance increased from a narrow coverage distance (n) to a wide coverage distance (all n) in the three straw mulching treatment groups. In terms of straw mulch types, the mix straw mulching treatment had a higher maturity index, a more stable soil food web and higher soil fertility levels than the rice straw or rapeseed straw mulching treatments. There was a significant negative correlation between plant parasite and omnivore-predator nematodes and NH 4+ -N and DON, but there was no significant correlation between the nematode community and the soil DOC content. This finding was unexpected and suggests that nitrogen in the soil of agroforestry ecosystems had a more significant impact than soil carbon on the nematode community. Recommendations for sustainable walnut orchard management based on the complexity and stability of nematode food webs should advocate the use of mix straw mulching (mix) covering the whole plot (all n) and thus promote the accumulation of soil dissolved organic nitrogen and carbon nutrients.

Materials and methods
Experimental site. The study was conducted in a large walnut orchard field in Langzhong (31° 57′ 82″ N, 105° 96′ 65″ E; 712.5 m above sea level), which is the hilly area of the central Sichuan Basin, southwestern China. The area has a humid mid-subtropical monsoon climate, with an average annual precipitation of 1033.9 mm and an annual temperature of 18.7 °C. This site has purple soil, classified as Pup-Orthic Entisol in the Chinese Soil Taxonomy (CST) and Entisol in the USDA Soil Taxonomy 31 . The specific soil in this study was a loam soil with the following nutrient profile (0-15 cm depth): total nitrogen (2.4 g kg −1 ), available phosphorus (0.96 g kg −1 ), available potassium (86.57 mg kg −1 ) and total carbon (5.95 g kg −1 ) 32 . In July 2016, we established a straw mulching experiment with a randomized block design in a walnut plantation with 3 m × 3 m spacing to investigate the potential effect of straw mulching on nematode community abundance and diversity and the associated agroecosystem function. We selected three different straw mulch types, rice straw, rapeseed straw, and mix straw (of equal quality, mixed 1:1), as the main plot. Then, under different main plots, we set up three different straw mulching distances (covering the mean radius of the crown width (n), covering 1.5 times the mean radius of the crown width (1.5 n) and covering the whole experimental plot (all n)) as sub-plots. Plots with no straw mulching were used as the CK plots. The quantity of straw mulch in each treatment was 3 kg/m 2 , selected based on previous research results 33,34 . There were a total of 10 treatments, based on the three straw mulching types, the three different straw mulching distances and CK, and each treatment had three replicates. All treatments were subjected to random permutations. Analysis of soil physicochemical properties. Soil moisture content was estimated gravimetrically by oven drying 20 g of each field composite soil sample at 105 °C for 24 h. Soil pH was determined with deionized water and an air-dried and fine-ground sample at a ratio of 1:2.5 (weight to volume, w/v) with an electronic pH meter. Soils were extracted with 2 M KCl, and the filtrate was analyzed for NH 4 + -N and NO 3 -N content (with an Acquity Ultra-Performance Liquid Chromatograph, AA3, Bran + Luebbe, Germany). Dissolved organic carbon (DOC) and nitrogen (DON) were estimated using a TOC/TN analyzer (Multi N/C 2100(S), Analytik Jena AG, Germany).
Nematode extraction and identification. Soil nematodes were extracted from 50 g of fresh soil using a modified cotton-wool filter method 35 . The extractions were used for identification (at least 100 nematodes) at the genus level using a microscope (OLYMPUS BX51) at 100 × magnification (resolution: 0.25 μm) according to Ahmad et al. 36 . If fewer than 100 nematodes were observed in one sample, all specimens were identified. Nematode abundance was adjusted according to soil moisture and was expressed as the number of nematodes per 100 g dry soil. After identification (within one week), based on their feeding habits, nematodes were classified into four trophic groups: (1) bacterivores (Ba), (2) fungivores (Fu), (3) omnivores-predators (OP) and (4) plant parasites (PP) 12 .
The metabolic footprint approach uses existing data on nematode biovolumes and growth rates, and the weightings used in the enrichment index (EI), structure index (SI), and channel index (CI) calculations 41 to estimate the C metabolism of the nematode community 22 . The nematode metabolic footprints (NMF) was also divided into the enrichment footprint (efootprint), representing lower trophic levels (c-p 1-2), and the structure footprint (sfootprint), representing higher trophic levels (c-p 3-5). The above data were calculated using the online Nematode Indicator Joint Analysis (NINJA) tool 42 . Data analysis. The nematode abundances were ln (x + 1) transformed prior to statistical analysis for the normality of data. One-way ANOVA was used to test the effect of straw mulching on soil properties, nematode abundance, and nematode ecological index under each treatment. Correlation analyses between abiotic and biotic drivers, including pH, SMC, NH 4 + -N, NO 3 -N, DON, DOC and nematode community data, were conducted. Statistical significance was determined at p < 0.05. Differences between data means were analyzed with t-tests using SPSS v. 17.0 (SPSS Inc., Chicago, IL) statistical software. Least significant difference (LSD) was used to test for differences among treatment means.