Changes of Eukaryotic Microorganism Structures in Soil during

Lettuce-spinach Rotation

Qingwen Li

, Xinyu Wang

, Jie Hong

, Yi Gao

, Zhidi Chen

, Lianquan Zhong

Xinxin Yi

1,* g

and Xiuzhi Gao

1,* h

Beijing Laboratory of Food Quality and Safety, Beijing Key Laboratory of Agricultural Product Detection and Control of

Spoilage Organisms and Pesticide Residue, Beijing Engineering Laboratory of Probiotics Key Technology Development,

College of Food Science and Engineering, Beijing University of Agriculture, Beijing, 102206, China

Beijing Changping District Plant Protection and Plant Quarantine Station, Beijing, 102200, China

zhonglianquan1@163.com,

gxz@bua.edu.cn,

yixinxin2008@163.com

Corresponding author

Keywords:

Eukaryotic Microorganism, Lettuce-spinach Rotation, High-Throughput Sequencing Technology.

Abstract:

The purpose of this study is to analyze and compare eukaryotic microorganisms in soil samples by rotation of

lettuce and spinach. High-throughput sequencing technology was used to analyze the eukaryotes present in

soil samples before and after crop planting and the effect of soil sucrase activity on eukaryotic communities,

in order to provide data that will aid in alleviating the problems resulting from the continuous cropping of

lettuce. The results showed that eukaryotic microorganism, such as Heterodera, Pseudallescheria and

Podosphaera species, were inhibited by rotation of lettuce with spinach and had a certain effect on the

recovery of soil quality. Rotation of lettuce with spinach could increase lettuce production and soil sucrase

activity by 31.4% and 9.5%, respectively. It could improve the diversity of eukaryotic microbial community

in soil.

1 INTRODUCTION

Soil microbial diversity is important for sustainable

agricultural development, because microorganisms

can participate in several biochemical processes that

promote agricultural production, including plant

nutrient recycling, soil structure maintenance and

agricultural chemical degradation (Lanzén 2013). Soil

microorganisms are mainly composed of bacteria,

fungi, actinomycetes and some algae. Among them,

soil eukaryotes play an important role in maintaining

soil nutrients and biogeochemical cycles. There are

many factors affecting soil microbial community,

including soil characteristics, environmental

https://orcid.org/0000-0003-1882-8351

https://orcid.org/0000-0002-4304-4738

https://orcid.org/0000-0002-9672-9554

https://orcid.org/0000-0002-6981-4032

https://orcid.org/0000-0003-3894-0252

https://orcid.org/0000-0001-8295-4294

https://orcid.org/0000-0002-2139-1149

https://orcid.org/0000-0002-1122-4742

conditions, crop management strategies and other

factors, among which plant species and tillage are

important factors affecting soil microbial community

(Larkin, 2003). Soil enzyme activity is also an

important indicator of microbial and biochemical

processes, usually involving the decomposition and

synthesis of soil organic matter, nutrient cycling and

availability, soil fertility and quality, and its

determination can be used to quantify and monitor

changes in microbial community structure and

activity, and soil Dynamic response index of organic

matter to human disturbance (Raiesi, 2018).

Researchers such as Lawton and Li found that the

overall composition of species and microbial

1254

Li, Q., Wang, X., Hong, J., Gao, Y., Chen, Z., Zhong, L., Yi, X. and Gao, X.

Changes of Eukaryotic Microorganism Structures in Soil during Lettuce-spinach Rotation.

DOI: 10.5220/0011393000003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 1254-1259

ISBN: 978-989-758-595-1

communities and planting methods with certain

functional characteristics could change the structure

and diversity of soil microbial communities and

affected ecosystem functions (Lawton, 1994, Li, Liu,

2017).

Lettuce is one of the most popular raw leafy

vegetables in China. So far, there have been few

reports on the interaction of eukaryotic

microorganisms in the soil during lettuce cultivation

and with different planting methods. In soil

microbiology research, soil quality indicators have

been obtained by measuring changes in microbial

activity and community structure over time. In this

paper, the soil collection of the rotation of lettuce and

spinach was used to analyze the changes in soil

eukaryotic microbial community diversity under this

planting method, in order to provide some theoretical

basis for alleviating the obstacle of continuous

cropping of lettuce.

2 MATERIALS AND METHODS

2.1 Site Description

The experiments were conducted in a plastic

greenhouse in an experimental demonstration base

(east longitude 116.14°, north latitude 40.19°) in

Beijing, China. Prior to the experiments, mung beans

were grown within the greenhouse for a long period

of time. The annual average temperature in this area

was 12.6 ℃, and the annual precipitation was 680.6

mm.

2.2 Experimental Design

The experimental setup the lettuce-spinach rotation

group (S). The sample naming format was used S -

planting year – cultivation number - 1 (before

planting) / 2 (after planting) - soil depth. The adjacent

lands were protected by two furrows that were 1.2 m

wide and 6.5 m in length. The experiments were

conducted between September 2016 and June 2017.

Due to low temperatures during the winter, the

experimental field used for the continuous cropping

of lettuce was filled in, and no crops were planted

during this period. Other treatments were consistent

throughout the planting period. Randomly selected

20 × 20 cm area and the yield of lettuce in this area

was weighed by weighing method. Each field soil

sample was collected using a five-point sampling

method, during which four samples were collected at

each of four corners, and one sample was collected

from the center of the field; samples were collected

before and after crop cultivation at sampling depths

of 0-10 cm and 10-20 cm. After the removal of

residual leaves and roots, the soil samples were

placed into aseptic sampling bags. The samples from

each of the five points were combined and divided

into two parts: one part was used for the experiments,

while the other was stored at -40 ℃ for follow-up

experiments. A description of each of the soil samples

is given in Table 1.

Table 1: Description of soil samples.

Sample Collection date Depth (cm) State of crop growth Cultivation time

S.16.1.1.10 2016.09.09 0-10 Before cultivation 1st

S.16.1.1.20 2016.09.09 10-20 Before cultivation 1st

S.16.1.2.10 2016.10.20 0-10 Harvest 1st

S.16.1.2.20 2016.10.20 10-20 Harvest 1st

S.16.2.1.10 2016.10.27 0-10 Before cultivation 2nd

S.16.2.1.20 2016.10.27 10-20 Before cultivation 2nd

S.17.2.2.10 2017.03.10 0-10 Harvest 2nd

S.17.2.2.20 2017.03.10 10-20 Harvest 2nd

S.17.3.1.10 2017.03.21 0-10 Before cultivation 3rd

S.17.3.1.20 2017.03.21 10-20 Before cultivation 3rd

S.17.3.2.10 2017.04.27 0-10 Harvest 3rd

S.17.3.2.20 2017.04.27 10-20 Harvest 3rd

S.17.4.1.10 2017.05.23 0-10 Before cultivation 4th

S.17.4.1.20 2017.05.23 10-20 Before cultivation 4th

S.17.4.2.10 2017.06.20 0-10 Harvest 4th

S.17.4.2.20 2017.06.20 10-20 Harvest 4th

Changes of Eukaryotic Microorganism Structures in Soil during Lettuce-spinach Rotation

1255

2.3

DNA Extraction and PCR

Amplification

According to the manufacturer's instructions, the

Mag-Bind® Universal Metagenomics Kit was used

to extract DNA from 1.0 g of soil samples. The

extracted DNA was measured by agarose gel

electrophoresis (0.8%), and the DNA was quantified

by an ultraviolet spectrophotometer. The extracted

DNA was stored at -80°C for analysis. The V4-V5

region within the 18S rRNA gene was amplified from

each sample using general eukaryotic primers

TAReuk454F WD1 (5'-

CCAGCASCYGCGGTAATTCC-3') and TAReu

kREV3 (5'-ACTTTCGTTCTTGATYRA-3')

(Logares, R 2016) according to previously published

protocols. PCR amplification was conducted using

the Q5 high fidelity DNA polymerase (NEB, UK);

Strictly control the number of amplification cycles to

ensure that the number of cycles was as few as

possible, and the amplification conditions of each

batch of samples were consistent. The high-

throughput sequencing of the 18S rRNA gene was

conducted using the Illumina MiSeq PE300 platform

at the Shanghai Majorbio Bio-pharm Biotechnology

Co., Ltd. (Shanghai, China). The read sequences were

deposited into the NCBI Sequence Read Archive

under accession numbers SRP155301 and

SRP154689.

2.4

Sequence Analysis

In order to integrate the original double-end

sequencing data into our analysis, the sliding window

method was used to individually screen double-end

sequences in FASTQ format. The FLASH software

(v1.2.7; http://ccb.jhu.edu/ software/FLASH/) was

used to pair double-ended sequences through primary

quality screening of overlapping bases. The

sequencing results were analyzed using the QIME

software (v1.8.0; http://qiime.org/). Sequences that

met the following criteria were filtered out: (1)

length<150 bp; (2) contained fuzzy bases; (3) number

of mismatched bases in 5'-end primers > 1; (4)

number of consecutive identical bases > 8. Chimeric

sequences were verified and removed using

USEARCH (v5.2.236;

http://www.drive5.com/usearch/). The QIIME and

UCLUST softwares were used to divide the

operational taxonomic units (OTU) at 97% similarity;

The most abundant sequence in each OTU was

selected as the representative sequence of the OTU.

Then, according to the number of sequences

corresponding to each OTU in each sample, a matrix

file containing the abundance of OTU in each sample

was constructed. For each OTU representative

sequence, used the default parameters in the QIIME

software to compare the representative sequence with

the template sequence in the Silva database to obtain

the classification information corresponding to each

OTU (Release 115; http://www.arb-silva.de).

3 RESUITS

3.1 Difference in Lettuce Yield and Soil

Sucrase Activity

Lettuce grown in rotation with spinach yields were

4.78 kg/m2 and 6.28 kg/m2. The average yield of the

two rotations is 5.53 kg/m2. The output of lettuce

increased by 31.4% after rotation, significantly. In the

process of lettuce rotation, the activity of invertase

fluctuates greatly, and the overall level remained at

15.25 U/g. Sucrose activity increased when lettuce

was harvested compared to pre-planting activity. The

soil invertase activity in rotation treatment increased

by 9.5% on average compared with that before

planting. (Fig. 1). In the crop rotation soil samples,

Ascomycota, Chlorophyta, Nematoda, Streptophyta,

Apicomplexa, Chytridiomycota, Arthropoda and

Eustigmatophyceae accounted for a large proportion

of the eukaryotes phyla present during cultivation. In

addition, Eustigmatophyceae appeared during lettuce

cultivation, and Bacillariophyta was found in all of

the 0-10 cm soil samples (Fig. 2).

Figure 1: Soil sucrase activity in rotation cropping soil

samples.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

1256

Figure 2: Relative abundance of the eukaryotes phyla

presents in the crop rotation soil samples. Relative

abundances are reported as the percentage of the total

eukaryotes sequences.

3.2 Soil Eukaryotes Diversity and

Community Structure during Crop

Rotation

In the 0-10 cm soil samples, the diversity indices

showed a rising trend during the cultivation of lettuce,

with increases observed in the Chao1

(13.46~65.04%), ACE (14.05~63.79%), Simpson

(2.90~50.35%) and Shannon (18.55~108.24%)

indices. The diversity indices, including Chao1 (-

12.14%), ACE (-12.21%), Simpson (-2.17%), and

Shannon (-13.19%), decreased during the first

cultivation of spinach. During the second spinach

planting, the diversity indices, including Chao1

(36.95%), ACE (36.85%), Simpson (9.86%) and

Shannon (32.35%), were increased. In the 10-20 cm

soil samples, the diversity indices during lettuce

cultivation were increased (Chao1, 6.48~11.89%;

ACE, 6.35~12.97%; Simpson, -3.5~ -3.11%;

Shannon, -10.70~ -9.52%. During the spinach

planting period, all of the indices, including Chao1

(17.55~24.31%), ACE (16.75~22.56%), Simpson (-

8.70~3.88%) and Shannon (-25.12~21.55%), were

increased (Table 2).

Table 2: The proportion of the shared and unique OTUs in the harvest and cultivation soil samples from each cultivation

period.

Samples Chao1 ACE Simpson Shannon

Goods_coverag

Evenness

S.16.1.1.10 900.4000 880.5166 0.9421 5.5672 0.9978 0.5788

S.16.1.2.10 1021.5543 1004.2072 0.9694 6.6001 0.9980 0.6694

S.16.2.1.10 1068.1728 1085.3210 0.9722 6.8789 0.9986 0.6860

S.17.2.2.10 938.4615 952.7641 0.9511 5.9713 0.9991 0.6055

S.17.3.1.10 670.1648 681.4558 0.6239 2.8731 0.9981 0.3126

S.17.3.2.10 1106.0067 1116.1486 0.9381 5.9829 0.9977 0.5978

S.17.4.1.10 820.0132 821.0723 0.8845 5.2434 1.0000 0.5417

S.17.4.2.10 1123.0038 1123.6392 0.9718 6.9342 1.0000 0.6843

S.16.1.1.20 998.0000 998.0000 0.9645 6.9054 1.0000 0.6931

S.16.1.2.20 1116.6429 1127.4693 0.9307 6.2483 0.9994 0.6173

S.16.2.1.20 831.0000 843.8775 0.9412 5.9878 0.9983 0.6239

S.17.2.2.20 1033.0339 1034.2674 0.9777 7.2781 0.9999 0.7269

S.17.3.1.20 939.9874 958.8076 0.9654 6.6445 0.9987 0.6752

S.17.3.2.20 1000.9198 1019.7156 0.9354 5.9335 0.9991 0.5961

S.17.4.1.20 878.6081 882.2762 0.9696 7.0500 0.9993 0.7227

S.17.4.2.20 1032.8430 1030.0534 0.8852 5.2790 0.9977 0.5347

Changes of Eukaryotic Microorganism Structures in Soil during Lettuce-spinach Rotation

1257

Figure 3: Relative abundance of different eukaryotic genera in the crop rotation soil samples

During crop rotation, Ecumenicus,

Bracteacoccus, Pythium, Chlorosarcinopsis,

Tetracystis and Macrobiotus species showed an

upward trend in the 0-10 cm soil samples obtained

during the cultivation of lettuce and spinach. In the

10-20 cm soil samples, Trichocladium,

Plasmodiophora and Filamoeba species showed an

increasing trend during the rotation process, while

Copromyxa and Echinamoeba species showed a

downward trend. There were also a number of

eukaryotic genera that demonstrated opposing trends

during the cultivation of lettuce and spinach, such as

Lactuca, whose species increased in number during

lettuce cultivation but decreased during the spinach

planting period in the 10-20 cm soil samples (Fig. 3).

4 DISCUSSION

Sucrase is an important enzyme to characterize the

biological activity intensity of soil, which is

positively correlated with the content of humus,

organic matter and clay, the content of N and P, the

number of microorganisms and the respiration

intensity (

Wang, 2005

). In this experiment, the activity

of sucrase and the yield of lettuce both increased

9.5% and 31.4%, respectively, after rotation.

Nematoda were the most dominant faunal group in

this experiment, which is highly diverse, ranked third

in terms of richness during these experiments. In the

soil samples obtained during crop rotation, the

changes in Nematoda were different due to the

different crops that were planted. Among them,

Heterodera species showed an opposing trend during

the process of lettuce and spinach cultivation.

In this experiment, changes in the genus of some

fungi were also different, these differences in

eukaryotic community compositions might be the

result of the different crops. Some studies have found

that members of Ascomycota are the main soil fungal

decomposers (

Ma, 2013

). Interestingly, in the crop

rotation soil samples, this phylum was increased

during lettuce cultivation and decreased during

spinach cultivation, due to different planting

methods. After the lettuce-spinach rotation the

relative abundance of genus such as Bacillus,

Pseudomonas, Sphingomonas, Nitrospira and Zephyr

in soil is higher than the crop rotation. But the the

relative abundance of Acidocaldarius was going low.

Research findings after rotation, the proportion of

bacteria in the soil, such as Acidbacteria, Firmicutes

and Fusarium, is less than that of crop rotation soil.

And Proteobacteria and Bacillus constitute the

dominant flora in the soil in rotation (

Pieterse, 2014

Pseudomonas and its secondary metabolites,

including the antibiotic 2,4-diacetylphloroglucinol

(DAPG), protect plants directly by inhibiting plant

pathogens by inducing plant systemic resistance

(

Zhang, 2018

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

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5 CONCLUSIONS

The results showed that crop rotation of spinach and

lettuce could effectively control increased in

Heterodera, Pseudallescheria and Podosphaera

species. However, some eukaryotes species, such as

those of Fusarium and Plasmodiophora, did not

decrease during crop rotation. Further experiments

were needed to identify the specific components of

spinach root exudates that have inhibitory effects on

eukaryotes. In addition, rotation with spinach

improved soil sucrase activity and lettuce yield.

ACKNOWLEDGEMENTS

This work was supported by the Beijing Leafy

Vegetables Innovation Team of Modern Agro-

industry Technology Research System (BAIC07-

2021) and fund for Academic Degree & Graduate

Education of Beijing University of Agriculture

(2021YJS029).

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