Effects of Soil Nutrients, Enzyme Activities and Microbial Composition in Vegetable-Polygonatum Odoratum Rotation Regimes

The rotation regimes significantly alter soil properties, however, understanding the effect of crop rotation in various crops system was currently limited. The aim of this research was to reveal the variation in crop productivity, soil nutrients, enzyme activities, and bacterial community composition in vegetable-Polygonatum odoratum rotation horticulture. Thus, a 2-year field experiment was carried out under the P.odoratum-P.odoratum rotation (CC) and vegetable-P.odoratum rotation (FC) system through a randomized complete block design comprising of triplicate plots. The results showed that FC promoted most of the beneficial agronomic traits, soil nutrients, enzyme activities, and reducing root rot disease of P.odoratum. High-throughput sequencing revealed a shift in the microbial community groups with high environmental contaminants degradation ability, and a close correlation between increasing microbial diversity and major environmental factors like soil nutrients, enzyme activities under FC cropping system compared with CC. Therefore, crop rotations with vegetableP.odoratum was recommended in cultivation.


Introduction
Polygonatum belongs to the family Asparagaceae with its genus comprising of approximately 63 species and widely distributed in Europe and Asia [1]. Polygonatum odoratum, one of the species under the Asparagaceae, it has for a long time been used in traditional Chinese and Mongolian medicine for the treatment of various illnesses that range from weakness, impotence, diseases of the kidneys, gonorrhea, pains in the back and legs, coughing, tuberculosis, yellow liquorstasis, and diabetes [2]. Indeed, the extracts of P.odoratum have also been confirmed to be highly valuable in the treatment of breast cancer through its extract that have the potential of inhibiting the proliferation and induces the apoptosis of human breast cancer MDA-MB-231 cells [3]. Studies on P. odoratum have also identified higher levels of steroidal saponins, sterols, homoisoflavonoids and cholestane glycosides [2,[4][5][6][7]. Moreover, its rhizomes form a highly nutritious source of food, making it one of the most consumed members of Asparagaceae in China [1]. Despite the high economic, nutritional and aesthetic significance of P. odoratum, its production has continued to face soil quality degradation challenges [8,9].
The property of soil was an important factor in P. odoratum planting. Soil is a heterogenous environment that is modelled by several biotic and abiotic components that are involved in physical, chemical, and biological activities, abiotic factors include microorganisms, small animals, and enzymes [10]. Subsequently, due to their sensitivity to environmental stress, soil biological and biochemical properties are used to evaluate soil quality [11].

2.1.Study site and materials
The study plots were established in Lianyuan city, Hunan province (Coordinates: 27°41′31″N 111°39′50″), carried out a 2-year cropping system in a previously uncultivated bare land, the soil type was clay-loam soil. The two major crops used in rotation in this study were Polygonatum odoratum (Mill.) and cabbage-peas. Three treatments were designed, P.odoratum-P.odoratum rotation (CC) and vegetable-P.odoratum rotation (FC) and an uncultivated study plot (CK) was used as the control. The plots were organized in a randomized complete block design comprising of triplicate plots (1.5 meters by 20 meters) for each of the three cropping systems at the two different cropping entry points (3×3×2= 18 plots). Hence, each study block consisted of one crop cycles per rotation, i.e., FC: vegetable planted in the first year and P.odoratum planted in the second subsequent year. CC: P.odoratum planted in the first year and re-planted again in the second subsequent year. CK: No crop planted in the first and second subsequent year. All the study plots were amended with calcium, and fertilized with phosphorus, potassium and animal manure at the onset of the experiment, as well as routinely managed using recommended production practices to avoid overgrowth of weeds.

2.2.Soil sampling
Soil samples were collected in triplicates from each experimental plot just before planting of the crops, in crop flowering stage, in rhizome expansion phase and later during crop wilt phase, respectively. Soil sampling was conducted using 8 soil cores in every study plot and later, soil samples from each plot were mixed to form a composite sample. Each soil sample was passed through a 3.35mm sieve to eliminate rocks and large organic debris. The samples were divided into three parts and stored in sterile plastic bags that were labeled appropriately and thereafter stored at -80 °C until further processing.

2.4.Measurement of soil chemical properties
Soil chemical properties including pH, N, P and K content were analyzed as described by Liu et al. [13]. Briefly, soil pH was determined in suspensions with deionized water (1:5 soil/water, w/v). Total N (TN) was determined by Kjeldahl methodology. Available N (AN) was estimated by the alkaline hydrolysis diffusion method. Total P (TP) and K (TK) was measured by sodium hydroxide fusion, followed by colorimetric analysis; the available P (AP) and K (AK) was extracted with 0.5M NaHCO3 (pH 8.5) and quantified as described above.

2.5.Measurement of agronomic traits
Leaf area, leaf index, number of leaves, plant height, plant productivity and polysaccharide content were measured on ten randomly P. odoratum plots in FC and CC cropping treatments. Plant productivity were determined at 3 points in each plot (1.5 m 2 per point), and the newborn rhizome was cut out and weighed. The polysaccharide percentage content was determined through Phenol-sulfuric acid spectrophotometric method.

2.6.Measurement of root rot disease parameters
The root rot disease parameters were measured for all treatments (FC and CC) during three stages (Seedling stage (SS), Flowering stage (FS), and Rhizome extension stage (RE)). The disease cases, rate, index, and strains were assessed. Disease rate was calculated as a percentage of the number of affected plants against the total plant population. The disease index (DI) was calculated as DI = [(Root rot disease grades × number of infected plants)/ (total checked plants ×7)] ×100.

2.7.Statistical analysis
All data were analyzed using Microsoft Office Excel 2007. A variance analysis (ANOVA) was performed on all data using SPSS 19.0 (IBM) software, and differences between the treatments were calculated using the LSD at the 0.05 probability level. Figures were generated using Origin 9.0 (Origin Lab) software [22].

2.8.PCR Amplification of 16S And18s Rrna, illumina sequencing and data processing
This protocol was conducted as provided in detail by Han [22]. In brief, DNA was extracted from soil samples using the E.Z.N.A.® Soil DNA kit (Omega Bio-Tec, Inc., USA) following the manufacturer's guidelines. The PCR was performed for the bacterial 16S ribosomal RNA gene with primer sets F: 5′-GCC TTG CCA GCC CGC TCA GTC AGA GTT TGA TCCTGG CTC AG-3′; R: 5′-GCC TCC CTC GCG CCA TCA GNN NNN NNN NNC ATG CTG CCT CCC GTA GGA GT-3′ and for the fungus identification with the primer sets F: 5′-GCC TTG CCA GCC CGC TCA GAC GGT ATC T (AG)A TC(AG) TCT TCG-3′; R: 5′-GCC TCC CTC GCG CCA TCA GNN NNN NNN NNN GGC AAG TCT GGT GCC AG-3′. The sequencing of the barcoded amplicons was done using the paired-end method with an Illumina MiSeq (Illumina, San Diego, CA, USA) system through a 6cycle index. Orthogonal Taxonomic Units (OTU) status and identification was achieved by using QIIME software that compared the OTU representative sequence to the template sequence of the corresponding database to get the taxonomic information corresponding to each OUT based on the procedures by DeSantis et al. [23] and Huson et al. [24]. Community diversity was calculated through Simpson indices using QIIME software. The specific composition of each sample at genus level was calculated based on the results on OTU classification and taxonomic status identification.

2.9.Spearman correlation analysis of microbial diversity and environmental factors
To further understanding the correlation of microbial diversity and environmental factors such as the content of N, P, K, and the activities of PPO, SC, UE and CAT, the Spearman correlation analysis was carried out as reported previously [25]. https://doi.org/10.37358/Rev. Chim.1949

3.1Agronomic Traits in Different Crop Rotations
The agronomic traits markedly increased under the FC condition compared with the CC (Figure 1). The values of leaf area (LA), number of leaves, leaf area index (LAI), plant height, crop productivity, and polysaccharide content under FC treatment significantly higher than CC treatment. Additionally, the values for Chlorophyll-a, Chlorophyll-b, and total chlorophyll content were significantly increased in FC cropping system compared with the CC (Figure 2).

3.2.Root Rot Disease Index in Different Cropping Systems
The root rot disease index was affected by different rotation (Figure 3). In this study, there were 89 strains of root rot diseases that were identified across all the cropping systems (CC and FC) at three stages (SS, FS, RE stage). The number of disease cases, disease rate and disease index were significantly lower under FC compared with CC system. During RE stage, the number of disease cases, disease rate and disease index were lower than SS and FS under the CC.

3.3.Soil Properties in different cropping systems
Soil chemical properties were significant influenced by crop rotations at different grow stages of plant ( Table 1). In rhizome enlargement stage, TN content under FC was significantly higher compared with the content reported under CC. The value of AN content showed no significant difference between the FC and CC systems in flowering, and wilting stages. The value of TP and AP under the FC was lower than CC in all stages. However, the TK and AK under CC rotation were significantly lower than FC rotation in all stages, except the before planting one. The value of pH was significantly decreased under the FC compared with CC during all stages.

3.4.Soil enzyme activities in different cropping systems
The activities of soil enzymes were affected by crop rotations systems, the value of enzyme activities was significantly different between FC and CC in all three stages ( Table 2).The activities of UE, PPO under the FC rotation system were significantly higher than under the CC system but the CAT and SC activities under CC rotation system were higher compared with those reported under FC rotation system in all stages.

3.5.Soil Microbial composition and diversity in different cropping systems
According to 16S analysis, orthogonal taxonomic units (OTU) results were analyzed in three crop rotation systems ( Figure 4). More than 40,000 sequences that represent about 3000 to 4000 OTU's were generated in each crop rotation system. Upon quality filtering and trimming of all the generated OTU's, high quality sequences were obtained from soil samples representing the three rotation systems. The top ten abundant phyla were Proteobacteria, Acidobacteria, Actininobacteria, Chloroflexi, Gemmatimonadetes, Verrucomicrobia, Planctomycetes, Bacteroidetes, Firmicutes and Latescibacteria under all rotation systems ( Figure 5). The phylum Proteobacteria was the most dominant in all crop rotation systems with the average relative abundance of 47.31%. On the other hand, the top ten dominant bacterial genera were Sphingomonas, unidentified Burkholderiaceae, Haliangium, Gemmatimonas, Staphylococcus, Streptomyces, Kitasatospora, Candidatus, Udaeobacter, unidentified Gammaproteo bacteria and Candidatus Solibacter under all cropping systems. Sphingomonas had the highest relative abundance followed by Haliangium under the FC rotation system. However, Haliangium had the highest relative abundance then followed by Sphingomonas under CC and CK.
The top ten fungal phyla are Ascomycota, Basidiomycota, Mortierellomycota, Glomeromycota, Mucoromycota, Chytridiomycota, Rozellomycota, Kickxellomycota, Blastocladiomycota and Olpidiomycota ( Figure 6). The top three were consisted of Ascomycota, Basidiomycota and Mortierellomycota with relative abundance of 33.76, 13.31, and 6.25%. https://doi.org /10.37358/Rev. Chim.1949 Rev. Chim., 71 (6) The OTU richness, Simpson index, and Shannon index values were significantly different under different cropping systems (Figure 7). The FC rotation system significantly increased OTU richness compared with CC rotation system ( Figure 7A). Simpson index indicated a higher microbial diversity in FC rotation systems than in the CC rotation system ( Figure 7B). Moreover, the FC cropping system significantly increased Shannon diversity index compared with CK and CC ( Figure 7C).

3.6.Correlation of microbial and environmental factors
The Spearman correlation analysis applied for the microbial diversity across different cropping systems and environmental factors uncovered that there are 6 bacteria including Spirochaetes, Elusimicrobia, Nitrospirae, Oxyphotobacteria, Bacteroidetes and Proteobacteria exhibiting positive correlation with the most environmental factors such as AN, AP, TN and TP, UE, CAT and ACP, and other 7 bacteria including Berkelbacteria, Euryarchaeota, Rokubacteria, Latescibacteria, Firmicutes, Chloroflexi and Actinobacteria showed negative correlation with the environmental factors ( Figure 8). In the same time, the fungi as Aspergillus, Rhizophagus, Colletotrichum, Paraglomus, Ceratotrichum, Zasmidium, Neocosmospora, Scutellinia, Podospora, Volutella, Fusarium and Polythrincium were positive correlated with environmental factors, while other fungi such as Agaricus, Striaticonidium, Talaromyces, Humicola and Cladorrhinum were negative correlated with environmental factors (Figure 8). In addition, the dominant bacteria and fungi showed opposite effect on soil pH and other factors.

3.7.The Increased productivity and agronomic traits under crop rotation system
Many perennial and annual horticultural crops growth and yield have been restricted when repeatedly planted in the same site over a long period of time [26]. This phenomenon has been referred as replant problem. The mechanisms of replant problem are very complex, and have been considered as including multiple cultural practices, soil conditions and other environmental factors. Previous studies have found the link of long-term continuous cropping systems to many agricultural traits [27]. Leaf area used to measure plant photosynthesis, which was a key factor for agronomic traits. Increased leave area of plants could capture more light in photosynthesis and improved leaf area index, thereby increased crop yield [28]. In this study, leave area and chlorophyll contents significantly increased by FC system (Figures 1&2). Similarly, the higher plant productivity and percentage of the polysaccharide content in FC system compared with CC system could also be based on the same fact as reported by Xia et al. [29]. The putative mechanism may explain the increase of the productivity reported in P.odoratum under FC system.

3.8.Reduced crop diseases under FC system
Previous studies have reported that the continuous cropping system may lead to the accumulated populations of harmful microbes, such pathogenic bacteria, fungi and viruses [16]. Therefore, crop rotation was occasionally applied in control and management of crop diseases. In this study, the number of disease cases, disease rate and disease index were significantly increased under the CC system compared with FC system (Figure 3). The results indicated that crop rotation not only improved the soil quality but also has a significant contribution in control of crop diseases in agriculture. In addition, recently developed novel biocontrol techniques on plant pathogens, like plantgrowth-promoting rhizobacteria (PGPR), can be used in combination with crop rotation practice to control and manage crop diseases. PGPR entails the use of naturally occurring and beneficial soil bacteria that are able to colonize rhizosphere and plant roots, promoting plant growth and defense against certain crop pathogens. This method has been successfully applied in control of plant pathogens in various crops [30], which may explain the mechanism how the rotation cropping system reduce crop disease through improving microbial diversity in soil. https://doi.org /10.37358/Rev. Chim.1949 Rev. Chim., 71 (6)

3.9.Soil Nutrients improved under FC system
Soil fertility and agronomic traits were influenced by the soil physicochemical characteristics such as pH, salinity, and the property of cropping system. In the present study, the soil pH corresponding to FC was higher compared with the soil pH corresponding to CC rotation system (Table 1). Studies have affirmed that soil pH decreased in continuously cropped fields due to the release of protons by root organic anions, as malate, citrate and oxalate. The significant variation in the soil chemical properties across all the cropping systems indicated the effects of agricultural practice on the quality of soil (Table 1). Some studies highlighted the impact of tillage practices on the soil chemical properties [29]. Moreover, the lowest total N and P contents were reported in all CK plots in different stages, while in FC and CC systems they increased. The physicochemical properties of soil fertility in cropped fields were improved in cultivated lands compared with barren land. This result may be explained by the assumption that crop stimulate microbial activities by shading-off harsh environmental conditions such as solar radiation, thereby increased decomposing plant organic content. Studies show that continuous cropping has a negative contribution to soil chemical traits and nutrient quality compared to rotational cropping system. The present results showed that FC significantly increased the content of TN, AN, TK, AK in soil, compared with CC system (Table 1), which suggests that FC system may improve soil physicochemical characteristics, and enhance nutrient content [31][32][33][34][35][36][37][38].

3.10.Enzyme activities promoted under FC system
Soil enzymes usually derived from soil microorganisms, plants and animals play a key role in various soil biochemical processes and decomposition of soil organic matter [21]. Enzyme activities influence the cycling of nitrogen, phosphor usually utilized to evaluate the health of soil. Soil enzymes are the best indicators for microbial activities because they are sensitive to any disruption or alteration in the soil structure or microbial community [36]. Thus, the enzyme activities with significant changes at different stages of crop may be driven by the microbial activities because the bacterial functions were related to various enzymatic activities ( Table 2). This is consistent with some studies showing that crop rotation significantly regulated soil microbial activities, hence affected soil enzymatic https://doi.org /10.37358/Rev. Chim.1949 Rev. Chim., 71 (6) activities [37]. In this study, the activities of UE, CAT, ACP and PPO significantly increased under FC system compared with CK. These results may be due to the changes in the soil organic matter, and the adaptation and proliferation of soil microbes in FC systems. However, the enzyme activities at flowering stage were significantly higher than other growth stages among FC and CC systems, which suggested the highest microbial activities at the FS. The activities of UE, PPO and ACP dominantly decreased under CC system compared to FC system. These results suggested that FC promotes microbial activities that mediate N and P cycle in the soil, which may drive nutrient (e.g., N and P) cycle, thus increasing nutrient availability to crops.

3.11.Benefit of plants as result of changes in soil microbial community composition under FC
Increased plant diversity has been shown to improve bacterial abundance of soil ecosystem and further reduce negative feedback. Planting of perennial crops has similarly been shown to have a lesser diversity compared to growth of seasonal crops in a rotational systems. In this study, lower OTUs were observed in the CC compared with FC system (Figure 4). Further, the relative bacterial abundance and diversity were also lower in CC system than in FC system (Figure 7). The results suggested that soil microbial diversity was higher under the FC system than under the CC system. This is consistent with the result we reported concerning the enzyme activities and soil nutrients under the CC system lower than the FC cropping system. The studies illustrated that highly diverse cropping systems were less affected by soil-borne pathogens than the plant mono-fields or low-diversity fields. This is consistent with the results of crop diseases (Figure 3), and highlighted that FC system not only increased soil bacterial relative abundance but also decreased soil borne pathogens [25][26][27][28].
Sphingomonas have been reported to be capable of metabolical versatility, and to play a role in the degradation of various compounds, including some environmental contaminants, which harms the plants. The relative abundance of Sphingomonas increased under FC cropping system, and this indicated that FC system showed potential for degrading soil contaminants, improving soil environment and promoting plant growth. Haliangium was obligate halophile, and requires a saline https://doi.org /10.37358/Rev. Chim.1949   environment. The relative abundance of Haliangium decreased when CC system was practiced, suggesting that CC system affected the abundance of some bacteria because of the decreased soil pH (Table 1).This is consistent with some studies that uncovered the relatively high acidity conditions within the soils under a rotational system [30]. Previous studies demonstrated that some fungi such as Podospora, Furasium, Neocosmospora and Ceratobasidium, and some bacteria such as Proteobacteria, Bacteroidetes and Nitrospirae may lead to the higher occurrence of plant diseases and replant problem. In this study, the Spearman correlation analysis showed the close and positive relationship between these fungi together with bacteria and the environmental factors including AN, AP, TN and TP, UE, CAT and ACP (Figure 8). These factors may be associated with replant problem under CC system (Table 2), which suggests the possible negative effects of microbial diversity on the environmental factors under CC system [39][40][41][42]. P; AK, available K; TN, total N; TP, total P; TK, total K; UE, Urease; PPO, Polyphenol Oxidase; CAT, Catalase; SC, Sucrase; ACP, Acid Phosphatase. * represents significant difference at p< 0.05, ** for highly significant difference at P< 0.01

4.Conclusions
In summary, the present results suggested that the FC system improved productivity and other agronomic traits of P.odoratum compared with CC system, and revealed the underlying mechanisms