Effects of Crop Rotation and Microbial Fertilizer on Nutrient Absorption and Beneficial Bacterium Abundance in Rhizosphere of Continuous Cropped Eggplant

Sang Ping, Li Shu-min*, Meng Ling-bo, Zhang Chun-yi, Mu Yao, Li Xin-rui, Li Ying, Wu Hao-lei,Liu Xiang-jun, Ma Ze-nan, and Hou Jian

1 College of Resources and Environment, Northeast Agricultural University, Harbin 150030, China

2 School of Geography and Tourism, Harbin University, Harbin 150030, China

Abstract: Rotation is a method to effectively regulate the soil microbial community structure and alleviate the obstacles of continuous planting of eggplant in facilities. High-throughput sequencing technology was used to study the change of beneficial bacteria diversity and structure at the first fruit stage of continuous eggplant rhizosphere soil affected by rotation combined with Bacillus subtilis fertilizer.The result showed that beneficial bacteria in the rhizosphere soil samples of eggplant at the phylum classification level included Actinobacteria, Chloroflexi, Acidobacteria, Planctomycetes and Cyanobacteria. At the level of genus classification, the beneficial bacteria in the rhizosphere soil samples of eggplant were Bacillus, Arthrobacter, Sphingomonas and Streptomyces. The significantly differences were observed in the abundance of soil dominant bacteria and beneficial bacteria at phylum level and genus classification among different treatments. At the phylum level, compared with CK treatment, the relative abundances of Acidobacteria and Proteobacteria in BB treatment were increased by 34.4% and 20.2%, respectively. At the level of genus classification, the beneficial bacterial Arthrobacter abundance in BB treatment was 1.44 times than that of CK treatment and was significantly higher than that in CF treatment. Moreover,the total uptake of N, P and K by eggplants in BB treatment and BFN treatment were 1.68 and 1.35 times than those of CK treatment,respectively. The above results showed that the combined treatment of microbial fertilizer and rotation could increased the soil bacterial community diversity and the abundance of beneficial bacteria in the first fruit stage of eggplant, which indeed enhanced the absorption of N, P and K by continuous eggplant plants, thereby alleviating the obstacles of continuous cropping of eggplant.

Key words: rotation, Bacillus subtilis, beneficial bacterium, bacterial diversity, eggplant

Introduction

Eggplant (Solanum melongenaL.) is one of the main economically cultivated vegetables in China(Xinet al., 2018). Recent years, the problem of soilborne diseases has increased year by year and has became one of the important factors restricting yield and quality of eggplants (Siamak and Zheng, 2018; Heet al., 2016). Therefore, it is particularly important to explore an efficiently, applicable and environmental friendly method to controlling soil-borne diseases(Frank and Mario, 2014). Some studies have suggested that the reasonable rotation of crops not only helps to regulate soil fertility (Heet al., 2016), but also effectively controls the occurrence of crop diseases,which solve problems caused by continuous cropping obstacles in some extent (Gaoet al., 2018). Researchers found that after four years of strawberry-shelter and strawberry-rice rotation, soil pH is increased, the incidence ofVerticillium wiltdecreases and the yield and benefit of strawberry increases significantly(Chenet al., 2011). In the northeast region, two crop rotation patterns of wheat-cucumber and viciavillosa cucumber are used to find that the rotation of cucumber has significantly increased bacterial species in the rhizosphere soil, and the number of fungal communities decreases significantly (Wenet al., 2016).As a result, cucumber yield is significantly increased.Crops which containing glucosinolates are broken up and applied to the soil, the degradation of glucosinolates can effectively inhibit or kill the microorganisms in the soil (Elamin, 2012), which can effectively control crop diseases and achieve the role of soil disinfection. In cruciferous crops, the concentration of glucosinolates in brassica species is relatively higher than other vegetable crops and has a stronger anti-bacterial and anti-viral ability (Tesfayet al., 2019).

Except rotation, regulating soil microbial community structure is also a way to alleviate obstacles of continuous cropping of eggplants. Some studies show thatBacillus subtilishas the function of control plant soil-borne fungal diseases (Zhanget al.,2014), such asBacillus subtilisis an anti-bacterial for eggplantVerticillium wilt, which has the characteristics of fast growth, strong environmental adaptability and survival ability, and can widely survived in various soil environments and plantsinternal tissue and surface (Liet al., 2017). The application ofBacillus subtilissolution can effectively control cottonVerticillium wiltand promote the growth of cotton plants(Zhanget al., 2018). Jiet al. (2002) have successfully isolated aBacillus subtilisstrain (B67), and this strain has a significant effect on the control of watermelonFusarium wilt, with a relative prevention effect over 80%. It can be seen thatBacillus subtiliscan significantly prevent and control the plant fungal soilborne diseases.

In addition, the nutrient concentration and accumulation of plant organs can better reflect the plant's response to the environment (Tenget al., 2017).Nutrient absorption and assimilation have important effects on crop growth and development. Some studies believe that microbial fertilizer can significantly promote the utilization and transformation of the total phosphorus, potassium and other elements in the soil (Fuet al., 2019). Others consider thatPaenibacillus kribbensiscan decompose mineral potassium which cannot be absorbed by crops and produced water-soluble potassium that can be directly absorbed by crops (Liet al., 2014). Many studies have shown that microbial fertilizer has prominent advantages in promoting crop growth and changing the soil microecological environment (Piromyouet al., 2013). Rotation can change the status of nutrients in the soil, promote plant growth, improve the nutritional status of crops,and then improve the ability of crops to resist disease.

However, in continuous eggplant cropping, the effects of eggplant and cruciferous crop rotation combined withBacillus subtilison soil microorganisms have not been thoroughly studied. Some studies have shown that the rotation of mustard and eggplant has the effect on controlling eggplantVerticillium wilt,but the mechanism is not very clear. Therefore, in this study brassica vegetable mustard (napiformis var.Er DaoMei) was selected for rotation with eggplant,Bacillus subtiliswas applied to the soil before eggplant transplanting, to explore the community composition of bacterial and beneficial bacteria in the rhizosphere soil of eggplant, and analyze the nutrient absorption of different organs under different treatments in the first fruit stage of continuous cropping eggplant, which provided a basis for further revealing the mechanism of mustard rotation controlling the eggplantVerticillium wilt.

Materials and Methods

Plant materials and field experiment

The physical and chemical properties of the experi-ment soil were: organic matter 20.3 g · kg-1, available nitrogen 127.2 mg · kg-1, the total nitrogen 1.72 g · kg-1,available phosphorus 132.0 mg · kg-1, available potassium 232.5 mg · kg-1, pH 7.01.

The eggplant was "Solanum melongenaL.LongZa9";rotated vegetable with eggplant was "Brassica napiformis var.ErDaoMei" mustard; chemical disinfectant was 50% carbendazim; the microbial fertilizer was a live spore ≥5 billion · g-1Bacillus subtilis(Ke LvFeng Biochemical Technology Co., Ltd. in Baoding City of Hebei Province).

Experimental design

The experiment was carried out in a 13-year continuous cropping greenhouse which in the College of Horticulture Academy of Agricultural Sciences of Heilongjiang Province. The experiment consisted of five treatments with four replications, including continuous cropping eggplant treatment (CK), mustard rotated with continuous cropping eggplant treatment(BFN),Bacillus subtilisapplied to continuous cropping eggplant treatment (BS), mustard rotated with continuous cropping eggplant combined withBacillus subtilistreatment (BB), and chemical disinfection soil treatment (CF). CF treatment was applied with 50% carbendazim at a dosage of 0.5 g · m-2and each treatment was unified field management.

Mustard was sown in autumn and harvested in October. When the mustard reached maturity, it was evenly mixed into the soil of the plot with 4.5 kg · m-2using a rotary tiller. All the plots were fully irrigated and covered with polyethylene film for 20 days. The eggplant seedlings with the same growing state (four leaves and one heart) were transplanted and planted on April 30, 2018. Each treatment before planting was applied with a compound fertilizer of 1 200 kg · hm-2.The planting row spacing was 30 cm×50 cm and the planting density was 20 000 plants · hm-2. When planting, theBacillus subtilisfertilizerholes were applied to the corresponding plots. At the same time,rhizosphere soil samples were collected, removed 2 cm of the soil on the ground surface, and the plants were dug out from the soil. After shaking the plant to make most of the soil fall off, shook the plant vigorously. A clean brush was used to swipe the soil adhered to the surface of the root system, and then collected the detached soil as rhizosphere soil. Then,removed the plant residues and other debris and mixed them evenly and brought them back to the laboratory and stored them at -80℃ for freezing and determining of soil bacterial diversity (Liet al., 2017).

Collection and determination of eggplant plant samples

During 30 days after planting, the first fruit stage of eggplant, three eggplants in each plot were randomly selected, and separated according roots, stems, leaves and fruits. The total nitrogen, the total phosphorus, and the total potassium concentration of eggplant organs were measured, and the plant nutrient accumulation was calculated. Measure method for the determination of the total nitrogen was Semi-micro Kjeldahl method;the total phosphorus was Molybdenum Antimony Colorimetric method and the total potassium was Flame Photometry method (Liet al., 2017).

Determination of bacterial diversity in rhizosphere soil of eggplant

The total DNA was extracted with Fast DNA®Spin Kit for Soil (MP Bio medicals, U.S.A), and 27F(5'-AGAGTTTGATCCTGGCTCAG-3') and 338R(5'-TGCTGCCTCCCGTA-3') primers were used for PCR amplification. Sequencing was performed using the Illumina Miseq platform (Liet al., 2017).

Data processing

Compared with Silva 16SrDNA database (Release128 http://www.arb-silva.de), using RDP classifier(Version 2.2 Release 11.1 http://rdp.cme.msu.edu/),bayesian algorithm was similar to 97%. The horizontal OTU represented the sequence for taxonomic analysis and the composition of each sample community was calculated at each classification level (www.i-sanger.com). Data were collated and plotted using Origin 2018, and ANOVA analysis was performed using SPSS17.0.

Results

Alpha diversity of bacterial communities in rhizosphere soil

Both Ace and Chao indexes reflected the species richness of the community and could represent the abundance of the microbial community. The Simpson and Shannon indexes reflected the microbial diversity indexes of the samples. The larger the Shannon index was, the higher the species diversity and the more uniform the individual distribution, and the Simpson index was the opposite. Rotationand microbial fertilizer significantly affected the richness and diversity of soil bacteria in the rhizosphere soil of eggplant in the first fruit stage (Fig. 1). The Shannon (C) index of BB treatment was the largest, which significantly higher than that of other treatments, indicating that the combination ofBacillus subtilisand mustard rotation increased the bacterial diversity of rhizosphere soil of eggplant significantly. There was no significant difference in rhizosphere soil microbial diversity between BFN treatment and BS treatment, but both were significantly higher than those of CK and CF treatments, indicating that rotation or adding microbial fertilizer alone could significantly increase soil bacterial diversity (Fig. 1).

Fig. 1 Ace (A), Chao (B), Shannon (C) and Simpson (D) indexes of rhizosphere soil bacteria at the first fruit stage of eggplantDifferent letters indicate significant difference (p＜0.05); "-" indicates the median line; "°" indicates average value.

According to the Ace (A) and Chao (B) indexes, the soil bacterial abundance of BB treatment was significantly higher than that of other treatments, among which there was no significant difference between BFN treatment and BS treatment. This showed that the application ofBacillus subtiliscombined with mustard rotation could increase soil bacterial abundance,and more uniform distribution of bacterial species in rhizosphere soil samples. The highest bacterial diversity was BB treatment, followed by BS treatment,and CK treatment was the lowest (Fig. 1).

Rhizosphere soil beneficial bacterium community at phylum level

The total of 12 bacterial species were detected from the rhizosphere soil, showing high diversity characteristics.There wereActinobacteria,Proteobacteria,Chloroflexi,Acidobacteria,Firmicutes,Bacteroidetes,Gem-matimonadetes,Patescibacteria,Cyanobacteria,PlanctomycetesandNitrospirae(A). In all the treatments, the dominant bacteria wereActinobacteria,Proteobacteria,ChloroflexiandAcidobacteria. In each treatment, the total relative abundance of these four dominant bacteria exceeded 80%, withActinomycesandProteobacteriabeing the advantages. The abundance was particularly significant, and the total proportion of both in the rhizosphere soil samples was more than 50% (Fig. 2).

Fig. 2 Relative abundance of bacteria (A) and beneficial bacteria (B) at phylum level in eggplant rhizosphere soil* means p≤0.05, **means p≤0.01.

At the level of phylum classification, beneficial bacterial populations in rhizosphere soil samples in the first fruit stage of eggplants includedActinobacteria,Chloroflexi,Acidobacteria,PlanctomycetesandCyanobacteria(B). The results showed that the difference inCyanobacteriaabundance between treatments reached a significant level (p＜0.05), and the abundance ofCyanobacteriain BFN treatment and BB treatment was significantly higher than that in CK treatment.Chloroflexiabundance was significantly different among treatments, but did not reach a significant level.Chloroflexiabundance was significantly higher in BB,BFN and BS treatments than that in CK treatment and CF treatment. BS treatment had the highest abundance values.Chloroflexiin BS treatment was 3% and 2.5%higher than those in CK treatment and CF treatment,respectively. At the phylum level, compared with CK treatment, the relative abundances ofAcidobacteriaandProteobacteriain BB treatment were increased by 34.4% and 20.2%, respectively.Acidobacteriaabundance of BFN treatment, BB treatment and BS treatment was higher than that of CK treatment, and the abundance of BB treatment was nearly 3.5% higher than that of CK treatment. The relative abundance ofActinobacteriain BFN treatment and BB treatment was significantly higher than that in CF treatment (Fig. 2).

Rhizosphere soil beneficial bacteria community at genus level

Based on the genus classification level, community composition analysis was performed on the rhizosphere soil samples of eggplant in the first fruit stage with different treatments. The results showed that there was a large number of bacteria in the experiment soil samples and unknown genus was classified. In the samples, the distribution of bacteria was significantly different among the samples. There were 29 dominant bacterial genera in the experiment soil samples.

The main bacterial genera includedArthrobacter,Sphingomonas,Streptomyces,norank_f_Gemmatimonas,norank_c_Subgroup_6, andnorank_o_Gaiellales, etc. (A). The relative abundance of the above genus classification level was selected as 1%as the critical range, and the total number of various genus types below 1% was displayed as others. It could be shown that the highest relative abundance of soil bacterial population wasnorank_c_Subgroup_6,which showed a higher abundance value in all the treatments, and BS treatment and BB treatment were significantly higher than that in CK treatment.Gainellalesabundance in BFN treatment was significantly higher than that in other treatments, about twice as much as that in CK treatment.Arthrobacterabundance was significantly higher in BS treatment than that in CF treatment and CK treatment andBacilluswas significantly higher in BB treatment than in other treatments. It could be seen that the combination ofBacillus subtilisand mustard was beneficial to the enrichment of beneficial bacteria (Fig. 3). The beneficial bacteria of the eggplant rhizosphere soil samples in each treatment wereGaiellales,Bacillus,Arthrobacter,SphingomonasandStreptomyces(B). TheArthrobacterabundance was significantly different among treatments. Among them,Arthrobacterabundance in BB treatment was significantly higher than that in CK treatment and CF treatment and the abundance in BS treatment was 2.83 times that in CF treatment, which was a very obvious promotion onArthrobacter. The differences in the abundance ofSphingomonasbetween treatments were significantly.At the level of genus classification, the beneficial bacterial Arthrobacter abundance in BB treatment was 1.44 times that of CK treatment and was significantly higher than that in CF treatment.Sphingomonasabundance values of BFN, BB and BS treatments were higher than those of CK treatment and CF treatment.The abundance of BFN treatment was 0.71% higher than that of CK treatment.Streptomyceswas a common beneficial bacterium in soil at the genus level. From the analysis results, it could be seen that the abundance ofStreptomycesin BFN treatment was significantly higher than other treatments. It indicated that mustard rotation could significantly increase the abundance ofStreptomycesin rhizosphere soil of eggplant (Fig. 3).

Fig. 3 Relative abundance of bacteria (A) and beneficial bacteria (B) at genus level in eggplant rhizosphere soil* means p≤0.05.

Nutrient absorption of different organs of eggplant

The root potassium concentration of eggplant in the first fruit stage was greatly affected by rotation. In the experiment treatments, the potassium concentration of eggplant roots was the highest in BB treatment, and followed by BFN treatment, which was significantly higher than those in CK treatment and CF treatment.The potassium concentration of eggplant stem was significantly different among treatments. The potassium concentration of eggplant stem in BFN, BB and BS treatments was significantly higher than those in CK treatment and CF treatment. The potassium concentration of leaves and fruits in BB treatment was the highest between treatments, and was significantly higher than that of other treatments. The potassium concentration of BB treatment was 23.5% higher than that of CK treatment. It showed that the concentration and accumulation of potassium in each organ of BB treatments was higher than that in other treatments.Among them, the potassium accumulation in the leaves of BB treatment was 28% higher than that in CK treatment and the potassium accumulation in the fruits of BB treatment was significantly higher than that in CK treatment, with an increase of 43.7% points (Table 1).

Table 1 Effects of rotation and microbial fertilizer on N, P and K concentration and absorption of eggplant organs

There were significant differences in the phosphorus concentration and phosphorus accumulation in di-fferent organs of eggplant, during the first fruit stage.Among them, the roots and fruits phosphorus concentrations of BB treatment were the highest in the soil sample, which were 1.43 and 1.69 times of CK treatment, respectively. The phosphorus accumulation of BS treatment was 61.4% and 41.2% higher than those of CK and CF treatments, respectively. The total uptake of N, P and K by eggplants in BB and BFN treatments were 1.68 and 1.35 times than those of CK treatment, respectively. It showed that microbial fertilizer had a significant promotion effect on the accumulation of phosphorus in the root system. BFN treatment had the highest phosphorus concentration,which was 22% and 22.8% higher than those of CK treatment and CF treatment, respectively. Phosphorus accumulation in leaves of BB treatment was 1.6 times that of CK treatment. Phosphorus accumulation in the stems of BS, BB and BFN treatments was not significantly different among the treatments, but was higher than those of CK treatment and CF treatment.It could be indicated that mustard rotation promoted the accumulation and utilization of phosphorus in the stems and leaves of eggplant (Table 1).

The nitrogen concentration of different treatments in different organs of eggplant in the first fruit stage was significantly different. The accumulation of nitrogen in each organ of BB treatment was significantly higher than other treatments. Compared with CK treatment, nitrogen accumulation in stems increased by nearly 57.52%. The nitrogen accumulation in leaves in BB treatment was 2.3 times than that of CK treatment. The nitrogen concentration in roots and stems of BFN treatment and BS treatment was significantly higher than that of CK treatment. It could be seen that both rotation and microbial fertilizer contributed to the accumulation of nitrogen in the stems of eggplant (Table 1).

Discussion

Soil microbial community structure and composition were one of the important indicators to measure soil quality (Liet al., 2015). The present study showed that mustard rotated with eggplant and applied to microbial fertilizers increased soil microbial diversity and richness. Plants of ginseng appliedBacillus subtiliscould effectively adjust the soil microbial system,balance the diversity and stability of the microbial community (Donget al., 2018), improve the soil micro-ecological environment, and reduced continuous cropping obstacles. Through inter-specific competition and antagonism,Bacillus subtiliscould inhibited the reproduction of pathogenic bacteria (Liet al., 2017),and had its own bacteriolytic effect to promote plant growth (Huang and Yuan, 2014).

The bacterial diversity of rhizosphere soil was analyzed. It was found that the bacterial diversity index and abundance of rhizosphere soil of eggplant in different treatments were higher than that of CK treatment. BB treatment significantly increased Ace, Chao and Shannon indexes, followed by BFN treatment, and CK treatment was the lowest. The bacterial diversity of the soil under single rotation and microbial fertilizer treatment was higher than that of CF treatment. This showed thatBacillus subtilisand rotation could effectively increase soil bacterial diversity and significantly change the structure of bacterial communities. The reason might be that after the plant residues were incorporated into the soil,the physical and chemical properties of the soil were changed, which led to an increase of the microbial diversity in the soil.

Based on the classification level of the phylum,Actinobacteria,ProteobacteriaandChloroflexiwere found. Among them,Actinobacteria,Proteobacteria,ChloroflexiandAcidobacteriawere the main dominant bacteria in the rhizosphere of eggplant, and their total relative abundances had exceeded 80%.Compared with the CK treatment, the abundance ofAcidobacteriaincreased by 34.4% in BB treatment.The abundance ofFirmicutesin BFN, BB and BS treatments was significantly higher than that in the CF treatment. It showed that rotation and microbial fertilizer could significantly increase the abundance of firmicutes in the soil. Wanget al(2009)had found that the main groups of soil bacteria in the black soil farmland of the Northeast Plain wereAcidobacteria,ActinobacteriaandProteobacteria.Javidet al.(2016) and Chenet al.(2016) also found thatProteobacteriaandAcidobacteriawere also the dominant flora in the vegetation restoration soil and the plantation soil.

The results also found that at the phylum classification level, the beneficial bacteria, in the rhizosphere soil of eggplant indudedActinobateria,Chloroflexi,Acidobacteria,PlanctomycetesandCyanobacteria.The abundance ofActinobacteriain BB, BFN and BS treatments was significant higher than that in CF treatment (p＜0.05).Actinobacteriain the soil could be symbiotic with plants and fix nitrogen in the atmosphere (Liet al., 2018).Acidobacteriahad an important relationship with the metabolism and activate of phosphorus in the soil. The abundance ofPlanctomycetesin BFN treatment was significantly higher than that in CK treatment.Planctomyceteswas one of barren trophic bacteria and participated in soil carbon and nitrogen cycles and it played an important role in decomposition of soil organic matter.

On the genus classification, a total of 29 bacteria species were detected and the abundance between treatments showed differences. For example, the low abundance value ofnorank_c_Subgroup_6 was detect-ed in CK treatment, but it was the dominant bacteria in BB treatment. The research found that in BS treatment the abundance ofSphingomonaswas significantly higher than that in CF treatment, which might be due to the diversity of available substrates, providing a good source of microorganisms, and promoting the degradation of organic matter bySphingomonas, and BB treatment had the highest the nutrient absorption of potassium concentration in eggplant roots, followed by BFN treatment, which was significantly higher than that of CK treatment. Some studies indicated thatSphingomonascould use a variety of substrates,such as polycyclic aromatic hydrocarbons, polyvinyl alcohols, inorganic nitrogen, etc (Shardenduet al.,2018). It could be indicated that the combined withBacillus subtiliscould help to increase the abundance ofSphingomonas. Furthermore, it promoted the absorption of nutrient by crops and changed the soil microbial community structure. The result showed that the abundance ofArthrobacterin BS treatment was significantly higher than that in CF treatment and CK treatment, which showed that mustard rotated with eggplant was beneficial to increase theStreptomycesabundance.Arthrobacterhad the strong environmental adaptability and stress resistance, and it widely distributed in soil, with had the ability to efficiently degrade organic pollutants and help soil organic matter improvement (Shobhitet al., 2017).Streptomyceswas widely distributed in terrestrial soil and other ecological environments and had been an important source of antibiotic production for a long time (Niuet al., 2017), which could alleviate the incidence of soil-borne diseases in continuous cropping soil and the cropping obstacles. The results showed that the nutrient accumulation of eggplant in BB treatment was significantly higher than that in CK treatment and CF treatment. This might be due to the application ofBacillus subtilisand the rotation methods to promote the increase of beneficial bacteria in the soil of eggplant, improve the absorption and utilization of nutrients by the crops, and the potassium and phosphorus in the soil could improve disease resistance of the crops, thereby alleviating the obstacles on continuous cropping.

Conclusions

Bacillus subtilisand rotation methods could not only promote the growth of eggplant plants, but also change the soil bacterial community structure. The abundance ofChloroflexi,AcidobacteriaandCyanobacteriaat the phylum level was significantly increased and the abundance of some beneficial bacteria (ArthrobacterandSphingomonas) at the gene level was significantly enhanced. Therefore, this application of microbial fertilizer and rotation could improve soil bacterial diversity, regulate soil bacterial community structure,which promoted eggplants to absorb nutrients and alleviated continuous cropping obstacles.