Changes of Eukaryotes Microorganism Structures in Soil during

Continuous Cropping of Lettuce

Xinyu Wang

, Qingwen Li

, Jie Hong

, Zhidi Chen

, Yi Gao

, Xinxin Yi

and Xiuzhi Gao

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

zhidic416@126.com, gao15901499854@163.com, yixinxin2008@163.com

Corresponding author: gxz@bua.edu.cn

Keywords:

Eukaryotes Microorganism Structures, Continuous Cropping, Lettuce.

Abstract:

This study aimed to analyse eukaryotes in soil during the continuous cropping of lettuce. High-throughput

sequencing technology was used to analyze the eukaryotes present in soil samples before and after crop

planting, in order to provide data that will aid in alleviating the problems resulting from the continuous

cropping of lettuce. The results showed that Trichocladium, Chlorosarcinopsis, Hindakia, Zea, Diploscapter

and Tylenchorhynchus species were increased during continuous cropping. It indicates that the continuous

cropping of lettuce affected the eukaryotic microbial community.

1 INTRODUCTION

Soil microbes consist mainly of bacteria, fungi,

actinomycetes and some algae, all of which play

important roles in the ecological environment and

constitute the core of the soil ecosystem in terms of

maintaining soil quality and health (Vessey 2003).

Because of the rapid response to environmental

changes, microorganisms is regarded as an effective

biological indicator to assess soil conditions and land

management success (Chen 2012). Soil eukaryotes

play important roles in the maintenance of soil

nutrients and in biogeochemical cycles.

However, soil microbiology studies, at present,

mainly assess microbial biomass, the soil respiration

rate, and other quantitative aspects. Analysis of soil

microbial biomass may only reflect the influence of

some functionally specialized microorganisms (Tang

2007). The microbial respiration rate can be regarded

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

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

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

as an index of the total number of active soil

microorganisms. Neither of these parameters are able

to measure changes in the composition of the

microflora. To obtain complete knowledge of changes

in soil quality in terms of the microorganisms in soil at

the genus or species level, the determination of soil

microbial diversity and microbial structure should be

combined. The measurement of changes in microbial

activity and community structure over time has been

considered to be a better indicator of soil quality.

Lawton and other researchers have proposed that, in

addition to the richness of species, the presence of

species with certain functional properties and the

overall composition of the microbial community can

affect ecosystem function (Lawton 1994). During

research into the use of continuous planting and

rotation in the farming of eggplants, Li found that

continuous planting changed the structure and

diversity of the microbial community in soil (Li 2017).

484

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

Changes of Eukaryotes Microorganism Structures in Soil during Continuous Cropping of Lettuce.

DOI: 10.5220/0011217900003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 484-489

ISBN: 978-989-758-595-1

The purpose of this study was to investigate the

impact of the continuous cropping of lettuce. The

eukaryotes community structure was examined using

high-throughput sequencing technology.

2 MATERIALS AND METHODS

2.1 Site Description

The test was carried out in a plastic greenhouse at a

test demonstration base in Beijing (116.14°E

longitude, 40.19°N latitude). Before the experiment,

mung beans were grown in the greenhouse for a long

time. The annual average temperature was 12.6℃,

and the annual precipitation was 680.6 mm.

2.2 Experimental Design

The adjacent land was protected by two ditches 1.2 m

wide and 6.5 m long. These experiments were

conducted between September 2016 and June 2017.

Due to the low temperature in winter, the

experimental field for continuous lettuce cultivation

was landfilled and no crops were planted during this

period. Other treatments were consistent throughout

the planting period. An area of 20 × 20 cm was

randomly selected, and the lettuce production in this

area was weighed by a weighing method. The field

soil samples were collected by the five-point method,

four samples were collected at the four corners, and

one sample was collected at the center of the field;

samples were collected at a sampling depth of 0-10

cm and 10-20 cm before and after crop planting. After

removing the residual leaves and roots, putted the soil

sample in a sterile sampling bag. Combined the five-

point samples and divided them into two parts: one

part was used for experiments, and the other part was

stored at -40 ℃ for subsequent experiments. Table 1

gives the description of each soil sample. The lettuce

continuous cropping group (N) naming format used

was N - planting year - cultivation number - 1 (before

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

Table 1: Description of soil samples.

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

N.16.1.1.10 2016.09.09 0-10 Before cultivation 1st

N.16.1.1.20 2016.09.09 10-20 Before cultivation 1st

N.16.1.2.10 2016.10.20 0-10 Harvest 1st

N.16.1.2.20 2016.10.20 10-20 Harvest 1st

N.17.2.1.10 2017.03.10 0-10 Before cultivation 2nd

N.17.2.1.20 2017.03.10 10-20 Before cultivation 2nd

N.17.2.2.10 2017.03.21 0-10 Mid-cultivation 2nd

N.17.2.2.20 2017.03.21 10-20 Mid-cultivation 2nd

N.17.2.3.10 2017.04.27 0-10 Harvest 2nd

N.17.2.3.20 2017.04.27 10-20 Harvest 2nd

N.17.3.1.10 2017.05.23 0-10 Before cultivation 3rd

N.17.3.1.20 2017.05.23 10-20 Before cultivation 3rd

N.17.3.2.10 2017.06.20 0-10 Harvest 3rd

N.17.3.2.20 2017.06.20 10-20 Harvest 3rd

2.3 DNA Extraction and PCR

Amplification

DNA was extracted from 1.0 g of soil sample using

the Mag-Bind® Universal Metagenomics Kit

according to the manufacturer’s instructions. The

quality of the extracted DNA was determined using

agarose gel electrophoresis (0.8%), and the DNA was

quantified using a UV spectrophotometer. The

extracted DNA was stored at -80℃ prior to 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 2016) according to previously published

protocols. PCR amplification was conducted using

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

number of amplification cycles was strictly controlled

to ensure that the least number of cycles were used as

Changes of Eukaryotes Microorganism Structures in Soil during Continuous Cropping of Lettuce

485

possible and the amplification conditions used for

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

To integrate the original double-ended sequencing

data into our analysis, a sliding window method was

used to individually screen the double-end sequences

in FASTQ format. The FLASH software (v1.2.7;

http://ccb.jhu.edu/ software/FLASH/) was used to

pair the double-ended sequences via a primary quality

screen of the 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, the

matrix file containing the OTU abundances in each

sample was constructed. For each OTU representative

sequence, the default parameters were used in the

QIIME software to obtain the taxonomic information

corresponding to each OTU by comparing the

representative sequence to the template sequence in

the Silva database (Release 115; http://www.arb-

silva.de).

3 RESULTS

3.1 Lettuce Yield

The yields of continuous cropping lettuce were 4.88

kg/m

, 5.54 kg/m

and

5.29 kg/m

respectively.

3.2 Soil Eukaryotes Diversity and

Community Structure during

Continuous Cropping

After the DNA sequences obtained from the soil

samples were trimmed and filtered for quality and

chimeric reads, pyrosequencing was conducted. These

experiments resulted in a total of 2 995 049 sequence

reads that were obtained from eukaryotes 18S rRNA

in 30 soil samples. The sequences that had a similarity

of greater than 97 % were classified as belonging to

the same OTU. The description of the indices that

were used, including ACE, Chao1, Shannon, Simpson,

goods-coverage and Simpson- evenness, are shown in

Table 2. During the first cultivation period, the Chao1

(78%), ACE (83%), Simpson (2%), and Shannon (2%)

indices for the 0-10 cm harvest soil samples were

increased compared with those for the samples

obtained before cultivation, and the same trend was

observed for the 10-20 cm soil samples, for which the

Chao1, ACE, Simpson and Shannon indices were

increased by 4%, 2%, 2% and 4%, respectively.

During the second cultivation period, the Chao1 (-1%)

and ACE (-2%) indices were decreased for the 0-10

cm soil samples, in contrast with the Simpson (1%),

and Shannon (1%) indices, which were increased. For

the 10-20 cm soil samples, all of the diversity indices,

including Chao1 (-34%), ACE (-32%), Simpson (-

23%), and Shannon (-40%), were decreased. The same

phenomenon was observed during the third cultivation

period; however, compared to the first period, all of

the diversity indices were increased. For the 0-10 cm

samples, the increases were as follows: Chao1

(113%), ACE (115%), Simpson (13%) and Shannon

(63%); for the 10-20 cm samples, the increases were:

Chao1 (14%), ACE (17%), Simpson (17%) and

Shannon (62%). The good-coverage index was

between 99.7%-100%, indicating that the sequencing

depth was sufficient to cover all of the species present

in the sample (Table 2).

In contrast, there were 414 shared OTUs (7.21％)

found in all of the soil samples, and there were 436

unique OTUs (45+37+48+92+214; 7.59％) (Fig.1),

accounting for 3.6%, 2.4%, 3.1%, 6.0%, and 16.6%

of the total number of OTUs in each sample,

respectively. The overall trend in the proportion of

unique OTUs in the soil samples was observed to be

one of gradual increase, which indicates that the

planting of lettuce affected the eukaryotic microbial

community. The proportion of unique OTUs

increased during the first cultivation period but

declined during the second and third cultivation

periods. During each cultivation period, the

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

486

proportion of shared OTUs in the harvest and

cultivation soil samples decreased as the cultivation

frequency increased (Table 3).

At the genus level, there were significant

differences in the species present in soil at different

depths. In the 0-10 cm soil samples, Mortierella and

Lactuca species decreased over time during the three

periods of lettuce planting. In contrast,

Trichocladium, Chlorosarcinopsis, Diploscapter,

Hindakia, Tylenchorhynchus and Zea species

increased over the same period of time. In addition,

some eukaryotes were affected by the idle

period; Chlamydomonas, Copromyxa, Pterygota,

Desmochloris, Heterococcus and Rubus species

showed a rise-fall-rise trend, while Plasmodiophora

and Alogomyces species presented a fall-rise-fall

trend. In the 10-20 cm soil samples, Fusarium,

Trichophaeopsis, Pseudallescheria and Orbicula

species increased during continuous cropping, while

Mortierella, Eocercomonas, Pythium,

Chlorosarcinopsis, Tetracystis, Macrobiotus, and

Gallus species demonstrated a rise-fall-rise trend.

Additionally, Stachyamoeba species showed a fall-

rise-fall trend (Fig. 2).

Table 2. Eukaryotes diversity within the continuous cropping soil samples.

Samples

Chao1

ACE

Simpson

Shannon

Goods_coverage

Evenness

N.16.1.1.10 517.0000 517.0000 0.8408 4.4452 1.0000 0.4931

N.16.1.2.10 919.5301 945.0977 0.8612 4.5395 0.9977 0.4662

N.17.2.1.10 1202.7557 1220.3624 0.9064 5.6456 0.9974 0.5571

N.17.2.2.10 1103.1321 1115.4176 0.8984 5.4381 0.9975 0.5444

N.17.2.3.10 1188.3860 1199.2055 0.9190 5.6780 0.9976 0.5610

N.17.3.1.10 621.0000 621.3391 0.8673 4.4233 1.0000 0.4767

N.17.3.2.10 1320.7807 1337.8595 0.9766 7.2261 0.9978 0.7010

N.16.1.1.20 1031.8201 1043.0437 0.8846 5.5050 0.9982 0.5538

N.16.1.2.20 1068.5283 1063.3434 0.9066 5.7253 0.9983 0.5734

N.17.2.1.20 1207.3285 1204.0233 0.9023 5.7121 0.9978 0.5633

N.17.2.2.20 1239.6391 1229.8505 0.8946 5.6729 0.9976 0.5584

N.17.2.3.20 798.2095 823.3581 0.6946 3.4453 0.9979 0.3616

N.17.3.1.20 1070.6346 1035.2957 0.8375 4.5212 0.9972 0.4597

N.17.3.2.20 1220.2435 1213.2584 0.9769 7.3027 0.9981 0.7178

Figure 1: Venn diagram showing the shared eukaryotes OTUs (at a distance of 0.03) in the continuous cropping soil samples.

SN11, before the first planting; SN12, after the first planting; SN21, before the second planting; SN22, after the second

planting; SN32, after the third planting.

Changes of Eukaryotes Microorganism Structures in Soil during Continuous Cropping of Lettuce

487

Table 3: The proportion of shared and unique OTUs in the harvest and cultivation soil samples from each cultivation period.

Unique OTUs in

cultivation soil samples (%)

Unique OTUs in

harvest soil samples (%)

Shared OTUs (%)

1st 15.7 32.0 52.3

2nd 22.9 22.3 54.8

3rd 39.6 28.1 32.3

Figure 2: Relative abundance of different eukaryotic genera in the continuous cropping soil samples.

4 DISCUSSION

The main purpose of this study was to analyze the

changes in eukaryotes communities in soil during the

continuous cropping of lettuce via the high-

throughput sequencing of microbes in soil samples.

Nematoda are the most dominant faunal group in

this experiment, which is highly diverse, ranked third

in terms of richness during these experiments. There

are approximately 100,000 - 1 million nematodes

worldwide, which account for 80% of the total

number of animals (Parkinson 2004). Nematoda are

divided into three types of species, namely, plant-

feeding nematodes, bacterial-feeding nematodes, and

fungal-feeding nematodes; some of these species

cause economic losses during plant cultivation

(Mcsorley 2016). During the continuous cropping

process, the abundance of Nematoda in the soil was

gradually increased as the planting times were

increased. Prismatolais and Ceratoplectus were two

bacterial nematode genera that appeared during the

third planting. Studies have shown that nematodes

that feed on bacteria have a great potential to function

as predators of soil bacteria (Xiao 2014). When

nematodes reach a certain abundance, their feeding

reduces the number and activity of bacteria, which

results in a reduction in nitrogen mineralization due

to the consumption of fixed and nutritive components

(Mao 2005). Compared with the early stages of

planting, the number of nematodes that feed on fungi

in the soil was increased; Aphelenchoides species

were increased in the 0-10 cm soil, samples, while the

percentage of Aphelenchus and Tylencholaimus

species were increased in the 10-20 cm soil samples.

This may be related to the increase in the amount of

eukaryotes in the soil that resulted from the increase

in planting times.

5 CONCLUSION

Using Illumina MiSeq to sequence eukaryotes 18S

rRNA, changes in eukaryotes community structures

occurring during the continuous cropping of lettuce

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

Relative abundance

Sample name

Pterocystis

Stachyamoeba

Heterococcus

Tylenchorhynchus

Desmochloris

Orbicula

Macrobiotus

Pseudallescheria

Hindakia

Diploscapter

Tetracystis

Alogomyces

Copromyxa

Pythium

Eocercomonas

Fusarium

Lactuca

Mortierella

Plasmodiophora

Trichocladium

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

488

were observed. The results showed that

Trichocladium, Chlorosarcin- opsis, Hindakia, Zea,

Diploscapter and Tylenchorhynchus species were

increased during continuous cropping.

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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