Effects of applying ramie fiber nonwoven films on root-zone soil nutrient and bacterial community of rice seedlings for mechanical transplanting

Raising rice seedlings in flat trays has become the main method for mechanized transplanting of rice in China. However, seedling blocks raised by this method were easily cracked in practice, and this problem can be solved by padding a thin ramie fiber nonwoven film on the bottom surface of seedling tray. This study was conducted to determine the effects of this film on root-zone environment of rice seedlings. The results showed that on the 10th day after sowing, the soil inorganic nitrogen, especially nitrate nitrogen, content in the root-zone of the film treatment were considerably higher than in the no-film treatment, in contrast, the soil organic matter content was lower in the film treatment, and by the 20th day, the gap between treatments was enlarged. After applying the film, the Chao 1 index and Shannon index values for the soil bacterial community diversity decreased, and the rice seedlings were shorter, had higher root/shoot ratios, lower nitrate contents, and higher soluble sugar contents. We conclude that application of the ramie fiber nonwoven film resulted in substantial changes in the soil nutrient and bacterial community in root-zone in a short time, which significantly impacted the growth and development of rice seedlings.

. The ramie fiber nonwoven film and its application in raising rice seedlings for mechanized transplanting: (a) ramie fiber nonwoven film; (b) the use of the film; (c) an easily broken rice seedling block (raised without the film); (d) a rice seedling block raised with the film.
Soil nutrients. Compared with the NM treatment, the M treatment had higher soil nitrogen nutrition (Table 2). On D10, mean total nitrogen, nitrate nitrogen, ammonium nitrogen, and alkali-hydrolyzed nitrogen contents in the root-zone soil were 4.9%, 25.4%, 2.3%, and 9.3% higher in the M treatment compared with the NM treatment, but all the differences were not significant except for the alkali-hydrolyzed nitrogen content (p = 0.0343). By D20, the gaps between the treatments expanded, and the increase in the M treatment compared to the NM treatment reached 4.7%, 39.3%, 14.6%, 11.5%, respectively, but the only significant difference was in the nitrate nitrogen content (p = 0.0148). Compared with the NM treatment, mean available potassium content in root-zone soils in the M treatment increased, while average organic matter and available phosphorus content decreased, but these differences were not significant (p > 0.05).  Table 1. Morphological characteristics of rice seedlings on D10 (10 th day after sowing) and D20 (20 th day after sowing). NM and M represent not padding and padding with ramie fiber nonwoven film on the bottom surface of the seedling tray, respectively.

Figure 2.
Average nitrate (a) and soluble sugar content (b) in rice seedling shoots on D10 and D20. NM and M represent not padding and padding with ramie fiber nonwoven films on the bottom surface of the seedling tray, respectively. Error bars represent SE (n = 3). ns, * and ** denote non-significance, significant differences at the 0.05 probability level, and significant differences at the 0.01 probability level between NM and M in each sampling batch (by paired T-test), respectively.
www.nature.com/scientificreports www.nature.com/scientificreports/ Soil enzyme activity. Average soil urease activity in the M treatment was 5.9 and 6.3 mg NH 4 + -N kg −1 soil 24 h −1 on D10 and D20, respectively, and was slightly higher than that under NM, which was 5.3 and 5.7 mg NH 4 + -N kg −1 soil 24 h −1 on D10 and D20, respectively (Fig. 3c). Soil neutral phosphatase activity in the M treatment was 7.6 and 9.3 mg phenol kg −1 soil 24 h −1 on D10 and D20, respectively, and was slightly lower than that under NM, which was 8.3 and 9.4 mg phenol kg −1 soil 24 h −1 on D10 and D20, respectively, but these differences were not significant (Fig. 3d).
Soil bacterial community. The Chao1 index of the root-zone soil bacterial community in the M treatment was slightly lower than that in the NM treatment on both D10 and D20 (Fig. 4a). From D10 to D20, the Chao1 index decreased, and compared with the NM group, the decline was greater in the M treatment group, indicating that the soil bacterial diversity in the seedling root-zone decreased as a function of seedling growth time, and the application of the ramie fiber nonwoven film could further reduce it. The Shannon index reflected basically the same trend as the Chao 1 index (Fig. 4b).
The highest relative abundance in the soil bacterial community was Proteobacteria (Fig. 5a). In the M treatment it comprised 36.28% and 38.26% of the community on D10 and D20, respectively, and in the NM treatment it comprised 35.78% and 40.08% of the community on D10 and D20, respectively. Followed by Acidobacteria, Gemmatimonadetes, and Actinobacteria, whose total relative abundance when combined with Proteobacteria was close to or over 80% in each treatment. Among different classes of Proteobacteria, α-Proteobacteria always accounted for the highest proportion (about 47%), followed by β-Proteobacteria, γ-Proteobacteria, and δ-Proteobacteria, which each accounted for about 22%, 15%, and 12%, respectively (Fig. 5b).
Compared with NM, the M treatment community showed a trend of increased relative abundance of Acidobacteria and Gemmatimonadetes, but decreased relative abundance of Actinobacteria (Fig. 5a). On D10, the relative abundance of Acidobacteria and Gemmatimonadetes was 20.38% and 14.27%, respectively in the M treatment soils, and 19.75% and 13.00%, respectively in the NM treatment soils. On D20, the relative abundance of Acidobacteria and Gemmatimonadetes was 22.05% and 11.90%, respectively in the M treatment soils, and 20.91% and 10.67%, respectively in the NM treatment soils. The relative abundance of Actinobacteria on D10 and D20 in the M treatment soils was 8.95% and 7.86%, respectively, while the NM treatment soils was 10.57% and 9.15%, respectively. www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
Our previous studies revealed that the application of the ramie fiber nonwoven film compensated for the rapid oxygen consumption in the bottom soils of seedling trays caused by soil microbial activities, effectively keeping the soil in an oxygen-rich state 10,11 . This study showed that this oxygen-enrichment effect of the ramie fiber nonwoven film further affected soil nutrients, soil microbial composition, and ultimately affected the growth and development of the rice seedlings.
The M treatment had higher inorganic nitrogen content than NM treatment soils ( Table 2). There are three possible reasons for this phenomenon. Firstly, increased oxygen supply to the root-zone soil due to the presence of the film enhanced the activity of aerobic microorganisms and consequently accelerated the decomposition of soil organic matter, which was shown by a decrease in organic matter content ( Table 2) and, according the finding that excess organic nitrogen could be mineralized into inorganic nitrogen during the process of soil organic matter decomposition when the C/N ratio is less than 25 [20][21][22] (the initial C/N ratio of the soil in this study was less than 16)), would consequently increase the inorganic nitrogen content in the soil. Secondly, improving root-zone aeration can significantly increase soil enzyme activity, especially soil urease activity 17,23 , which is consistent with the results observed in this study (Fig. 3c). Soil urease is an important regulator of the transformation of organic nitrogen to inorganic nitrogen [24][25][26] . Therefore, increases in urease activity would improve the mineralization of organic nitrogen, and consequently increase the content of soil inorganic nitrogen. Thirdly, increased oxygen content in the root-zone can also increase the activity of a series of nitrification-related microorganisms such as ammonia-oxidizing bacteria 27 , which could enhance nitrification and promote the transformation of ammonium nitrogen into nitrate nitrogen, and then promote the process of nitrogen mineralization. So, the increased activity of these microorganisms can also contribute to the increase in soil inorganic nitrogen content.
Ample evidence has accumulated in recent years that the quantity and quality of soil organic matter can significantly affect the composition of soil microbial community [28][29][30] , which is often represented by the phenomenon that soils with high organic matter content tend to have higher microbial community diversity 31,32 . In our study, both the Chao 1 index and the Shannon index in the M treatment were lower than that in the NM treatment   www.nature.com/scientificreports www.nature.com/scientificreports/ (Fig. 4a,b), indicating that application of the ramie fiber nonwoven film reduced the soil bacterial diversity in the seedling root-zone. The simultaneous decrease in soil organic matter content ( Table 2) and soil bacterial diversity with the application of the ramie fiber nonwoven film obviously accords with above finding, and the significant positive correlations between soil organic matter content and the Chao 1 as well as the Shannon indices (Fig. 6a,b) lend further support for it.
In addition, the relative abundance of Acidobacteria and Gemmatimonadetes in the root-zone soil of seedlings in the M treatment increased (Fig. 5a), which is very similar to the change in the soil bacterial community under dry-raised rice seedling 33 . However, the mechanism and its effects on the growth and development of rice seedling remains unknown.
It is well known that the growth of crop plants, especially the growth of belowground roots, is closely related to the rhizosphere environment; changes in the ecological environment of the rhizosphere will inevitably lead to changes in crop growth factors. After application of the ramie fiber nonwoven film, increases in soil inorganic nitrogen nutrition (especially nitrate nitrogen) may promote root growth of rice seedlings, as confirmed by a large number of studies where nitrate nitrogen stimulated lateral root formation and increased root length 34,35 . This pattern is also supported by the positive correlation of soil nitrate nitrogen and alkali-hydrolyzed nitrogen content with root weight and root/shoot ratio of rice seedling found in this study (Fig. 6c-f). At the same time, the content of other forms of inorganic nitrogen and available potassium in root-zone soils had also increased in the M treatment soils, and positive correlations of the soil alkali-hydrolyzed nitrogen and available potassium with the soluble sugar content of rice seedlings was also observed in this study (Fig. 6g,h). This relationship suggests that these factors together helped to supply enough nutrients to the rice shoot, making the physiological activities of the seedlings more vigorous, and thus, contributed to the accumulation of soluble sugar.
To sum up, application of this film may cause a series of cascading effects. That is, the increasing oxygen supply in the root-zone could promote soil organic matter decomposition, increase soil urease activity as well as aerobic nitrification; and then increased soil nutrition (especially nitrate nitrogen content) and decreased soil organic matter content; consequently improved the nutrient supply of rice seedling and changed the distribution of soil bacteria; finally promoted the growth and development of rice seedling. However, the relation between the change of bacterial community and the growth and development of seedlings is still unknown.

Conclusions
In a short period of time, the thin ramie fiber nonwoven film laid under the soil in a seedling tray caused substantial changes in the root-zone environment of rice seedlings, including an increase in inorganic nitrogen content and a decrease in soil organic matter content as well as root-zone bacteria diversity, and then led a significant changes in the growth and development of rice seedlings.  . Pearson's correlations of soil organic matter with the Chao 1 index (a, n = 12) and Shannon index (b, n = 12); the soil nitrate nitrogen with root weight (c) and root/shoot ratio (d) of rice seedlings on D10 (dotted line, n = 6) and D20 (solid line, n = 6); the soil alkali-hydrolyzed nitrogen with root weight (e) and root/shoot ratio (f) of rice seedlings on D10 (dotted line, n = 6) and D20 (solid line, n = 6); and the soil alkali-hydrolyzed nitrogen (g, n = 12) and available potassium (h, n = 12) with soluble sugar content of rice seedlings. * and ** denote significant differences at the 0.05 probability level and 0.01 probability level, respectively.

Materials and treatments.
Plastic seedling trays (58 cm × 28 cm × 2.5 cm deep) were used for raising the rice seedlings. This kind of seedling tray has been widely used for raising rice seedlings for machine transplanting in southern China. The ramie fiber nonwoven film was produced by our cooperative enterprise (Haerbin Jingzhu Agricultural Science and Technology Co. Ltd, Haerbin, China). It had an approximate thickness of 0.20 mm and weight of 40 g m −2 . The experimental rice variety was Huanghua Zhan, which is widely used in one-season rice cultivation in Hunan Province.
Two treatments were used in the experiment: one with the ramie fiber nonwoven film padded on the bottom surface of the seedling tray (M), and the other without ramie fiber nonwoven film padded on the bottom surface of the seedling tray (NM). In order to ensure the consistency of water and soil conditions for the rice seedlings, paired groups consisting of M and NM were arranged in the same seedling tray, that is, half of the bottom surface of seedling tray was covered with ramie fiber nonwoven film, and the other half was not. After padding the film on half of each seedling tray, the trays were filled with seedling soil. Pre-germinated seeds were evenly sown in the seedling soil on April 20, 2018 at a sowing rate of 120 g per tray. After sowing, the seedling trays were placed on the pre-leveled seedling bed in the experimental field, and the soils were kept moist throughout the seedling stage. A total of 15 trays of seedlings were cultivated.
Sampling. Sampling was carried out on the 10 th and 20 th days after sowing. At each sampling, three trays of seedlings were randomly selected, and six, 4 × 4 cm seedling blocks were cut from both treatment areas of each tray. Three of the six seedling blocks for each treatment were randomly selected for measurements of rice seedling traits. These were carefully washed to remove the soil. Thirty seedlings with complete root systems were selected from the three seedling blocks, and their plant height was determined. These seedlings were divided into shoots and roots; any surface moisture was removed with paper, and their fresh weight was determined. We measured dry weights after drying at 80 °C to a constant weight, and the dry shoot samples were kept for the determination of soluble sugar and nitrate content. A layer of soil about 1 cm thick at the bottom of the other three seedling blocks was separated and retained as root-zone soil. The visible roots and stones were carefully removed, and the visible film was also removed in the samples taken from the M treatment area. This root-zone soil was mixed and divided into three parts: one part was immediately used for the determination of soil nitrate and ammonium nitrogen content. The second part was air-dried for the determination of soil physiochemical properties, and the third part was kept at −80 °C to determine the composition of the soil bacterial community.
Soil physiochemical properties. The soluble sugar and nitrate contents were determined by the phenol and salicylic acid methods according to Li 36 . Soil pH and electrical conductivity (EC) were measured in a soil:water mixture (1:5 w/v) using a pH meter (FE20, Mettler-Toledo, Switzerland) and a conductivity meter (DDS-12A, Shanghai Hongyi Instrumentation CO., LTD, China), respectively. We used Bao's method 37 for the determination of soil nutrients. Organic matter content (OM) and alkali-hydrolyzed nitrogen (AN) were determined using an outside heating method and an alkaline hydrolysis diffusion method, respectively. Total nitrogen (TN) was determined with an automatic kjeldahl nitrogen determiner (Kjeltec8400, FOSS, Denmark). Soil nitrate nitrogen (NO 3 -N) was extracted with a 0.01 mol L −1 CaCl 2 solution and determined by ultraviolet spectrophotometry. Ammonium nitrogen (NH 4 + -N) was extracted with a 2 mol·L −1 KCl solution and determined using an indigo blue colorimetric method. Total phosphorous (TP) and available phosphorous (AP) were extracted using an HClO 4 -H 2 SO 4 method and an HCl-H 2 SO 4 method, respectively, and both were measured using the Vanadium molybdate blue colorimetric method. Total potassium (TK) and available potassium (AK) were extracted by a NaOH molten method and an NH 4 Ac digestion method, respectively, and determined by a flame photometric method. Soil enzyme activity was assayed according to the methods described by Guan 38 . Urease activity was measured by determining the amount of NH 4 + released from a hydrolysis reaction after incubating the samples with urea (10% w/v) for 6 h at 37 °C. Neutral phosphatase activity was estimated by determining the amount of phenol released after incubating the samples with phenyl disodium phosphate (0.5% w/v) for 6 h at 37 °C.
Soil bacterial community composition. The diversity and composition of the bacterial communities in each sample of root-zone soil were assessed by 16 S rDNA, as described next. Total microbial community DNA was isolated from approximately 0.25 g soil per sample using the E.Z.N.A. ® Soil DNA Kit (D5625, Omega, Inc., USA) according to the manufacturer's instructions. The reagent was designed to uncover DNA from trace amounts of the sample and has been shown to be effective for the DNA preparation of most bacteria. The V3-V4 region of the 16 S rDNA gene was amplified with slightly modified versions of primers 338 F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806 R (5'-GGACTACHVGGGTWTCTAAT-3′) 39 . The 5′-ends of the primers were tagged with sample-specific barcodes and universal sequencing primers. PCR amplification was performed in a total volume of 25 µL reaction mixture containing 25 ng of template DNA, 12.5 µL PCR Premix, 2.5 µL of each primer, and PCR-grade water to adjust the volume. The PCR conditions to amplify the prokaryotic 16 S fragments consisted of an initial denaturation at 98 °C for 30 seconds, 35 cycles of denaturation at 98 °C for 10 seconds, annealing at 54 °C for 30 seconds, a primary extension at 72 °C for 45 seconds, and then a final extension at 72 °C for 10 minutes. The PCR products were confirmed with 2% agarose gel electrophoresis, purified by AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA), and quantified by Qubit (Invitrogen, USA). The amplicon pools were prepared for sequencing, and the size and quantity of the amplicon library were assessed on an Agilent 2100 Bioanalyzer (Agilent, USA), and with the Library Quantification Kit