Cucumber
is one of the most popular greenhouse vegetables throughout the world. However,
continuous monocropping is one of the factor causing “soil sickness” which
leads to poor plant growth, increase in soil-borne pathogens and finally reduce
crop production (Zhou et al. 2017).
Soil sickness may be related to changes in soil microbial communities because
of autotoxicity (Jin et al. 2020).
Previous studies have shown that cropping systems, such as rotation,
intercropping and interplanting systems, could significantly improve soil
health for better crop production (Zhou
et al. 2017). For example, rotation of tomato-celery-cucumber-Chinese cabbage
with cucumber could overcome the soil sickness of cucumber (Zhou et al. 2017). Previous study found that
incorporation of Brassica juncea inhibit the growth of pathogenic Rhizoctonia solani and Fusarium oxysporum (Friberg et al. 2009).
Crop
rotation is the practice of rotating different crops sequentially between
seasons and years in the same field (Wibberley 1996). Previous studies have
shown that cucumber rotation with tomato, soybean, wheat and celery was
beneficial to maintain the diversity and activity of soil microbes and
inhibited the harmful microorganisms that were higher in continuously
monocropped cucumber rhizosphere (Wu et
al. 2011). For example, Jin et al.
(2019b) reported that rotation with Indian mustard could suppress cucumber
Fusarium wilt disease and increase plant-beneficial bacteria in rhizosphere.
It
has been shown that Brassica spp.
crops (i.e., Indian mustard) are commonly grown to reduce soil-borne
pathogenic fungi (Larkin and Griffin 2007) because when their tissues are
disrupted, the glucosinolate releases isothiocyanate, which is toxic to many
soil pathogenic microorganisms (Motisi et
al. 2009). It was found that Indian mustard and wild
rocket green manures increased cucumber rhizosphere bacterial diversity and
abundance of potential plant-beneficial species, decreased Fusarium wilt
disease and enhanced expression of defense-related genes in cucumber seedling
roots (Jin et al. 2019c). In this
study, we collected Indian mustard- and the fallow-treated soil samples, and further
studied the effects of rotation of Indian mustard on diversity and composition
of cucumber fungal rhizosphere using high-throughput sequencing technology.
Materials and Methods
Greenhouse experiment
Cucumber continuous cropping
soil was collected from soil upper layer (0–15 cm) of a greenhouse in the
experimental station (45°41’N, 126°37’E) of Northeast Agricultural University,
Harbin, China, where the cucumber has been cultivating since 2006. The soil
type used for pot experiments was sandy loam and the physicochemical properties
were determined by method as previously used by Zhang et al. (2018), which were as follow: EC (1:2.5, w/v) 0.43 mS cm-1;
pH 7.64 (1:2.5, w/v); organic matter 3.51%; inorganic N (NH4+-N
and NO3--N) 146.60 mg kg-1; Olsen P 284.20 mg
kg-1; and available K 341.80 mg kg-1.
A pot experiment was performed during July to September
2016 for cultivation of Indian mustard consisting of two treatments in
greenhouse (32°C day/22°C night, with a 16 h light/8 h dark and 60–80% relative humidity. Total of 30 seeds of
Indian mustard (cv. Xuelihong) were germinated in each pot (diameter 20 cm,
height 17 cm) of total 10 pots in first treatment (R). Same number of pots
without Indian mustard seeds were kept as control treatment (M), and treatments
were replicated thrice to make 30 pots in total for each treatment. Each pot
contained 2.5 ± 0.1 kg of fresh cucumber continuous monocropping soil. After germination, thinning of seedlings was done to
minimize the density of seedlings to 10 by removing bad/extra seedlings in each
pot. Each treatment was replicated thrice to make a total
of 30 pots in each treatment. Pots of both treatments
were placed randomly without any order and their place was changed after every
third day. Distilled water was added every second day to keep
soil moisture at about 65% of its water content and no fertilizer was applied.
After 40 days after sowing,
the ground portion of Indian mustard was harvested and the underground portion
was left in the soil. Each pot was wrapped in a black polyethylene plastic
film, and the soil moisture content was maintained at around 65% and incubated for 30 days. Cucumber seedlings with two cotyledons (cv. Jinyan 4) were then planted in pots, one cucumber seedling per
pot. The cultivated conditions of cucumber seedlings were same as
described above for Indian mustard.
Soil sampling and DNA extraction
After 30 days of plantation, the cucumber rhizosphere soil was collected
according to the method previously used by Zhou et al. (2017)
and sieved through 2 mm mesh. Sample of 10 plants from each replicate was mixed
to prepare a composite soil sample and stored at -80°C for DNA extraction.
Rhizosphere soil DNA was
extracted from 0.25 g soil from each sample in triplicate using PowerSoil DNA Isolation
Kit (MO BIO Laboratories, C.A., U.S.A.) following the manufacturer's
instructions. The extracted DNA (in triplicate) was then combined to make a
composite sample and stored at -80°C for further analysis.
Illumina miseq sequencing and
data processing
As previously mentioned (Zhou et al. 2018a), amplification of the ITS1 region of the fungal rRNA
gene was done using the ITS1F/ITS2 primer The forward and reverse primers also
had a unique 6 bp barcode for
each sample. The three composite sample DNA solutions were
separately subjected to PCR amplification, then the PCR product was collected and
purified and paired-end sequencing (2×300) was performed on the Illumina Miseq platform of
Majorbio Bio-Pharm
Technology Co., Ltd., Shanghai, China.
The de-multiplexing, quality filtering and processing
of the raw sequence reads were
performed by FLASH (Zhou et al.
2017). Identification and removal of chimeric sequences was done with
USEARCH 6.1 in QIIME (Caporaso
et al. 2010). Sequences
were classified by the agglomerative clustering algorithm in USEARCH (Edgar,
2010) as an Operational taxonomic units (OTUs) with 97% sequence
similarity. Each representative OTU sequence was then taxonomically classified
by BLAST in the Unite database (Koljalg
et al. 2013).
Statistical
analysis
To avoid possible deviations due to sequencing depth,
a random subsample of 30,740 sequences was performed for each sample. The defined OTUs were used to calculate the
taxon cumulative curve. The alpha diversity analysis was performed by
calculating the Shannon and inverse Simpson indices. The differences in fungal
community structures by Beta diversity analysis were assessed using the UPGMA hierarchical
clustering analysis based on Bray-Curtis distance. The shared and unique OTUs between treatments were
calculated and their distribution was shown in a Venn diagram. Differences in alpha diversity
indices and relative abundances of microbial taxa between treatments were
analyzed using Student’s t test. All of these analyses were done in 'R'
(version 3.3.1).
Results
Fungal communities alpha and
beta diversities
After reading and removing a single
OTU by basic quality control filtration, Illumina Miseq.
produced an average of 35,748 high quality fungal sequences in each sample with an average read length of 262 bp. A total of 450 OTUs were identified
at 97% sequence similarity. The OTU
rarefaction curves of all samples tended to be flat
(Fig. 1a) and the Good’s coverage was larger than 99.5% for each sample (Fig.
1b). Therefore, the number of sequences was sufficient to assess the
diversity of cucumber rhizosphere fungal communities.
Cucumber monocropping and rotation with Indian mustard
had similar fungal community richness and diversity indices (Fig. 1b). However,
cluster analysis showed that the cucumber rhizosphere fungal community
structure differed between R and M treatments (Fig. 1c).
Fig. 1: Rarefaction curves of the number of OTUs (a), The Good’s coverage, diversity and richness indices of cucumber rhizosphere
fungal communities (b),
Hierarchical clustering tree of Indian mustard (R)- and fallow (M)-treated soil
samples at the OTU level (c), and
Venn diagrams demonstrating the numbers of shared and unique observed fungal
OTUs at 97% similarity between Indian mustard (R)- and fallow (M)-treated soil
samples (d). OTUs were delineated at 97% sequence similarity.
Random subsamples of 30,740 16S rRNA gene sequences per sample were used to
generate the rarefaction curves and calculate the Good’s
coverage, diversity
and richness indices. Different letters indicate significant difference based
on Student’s t test (P≦0.05). Dendrogram
of relatedness of the soil types. Frequencies of OTUs unique to each
treatment at the fungal class level were shown
Shared and unique OTUs
For fungal communities, there were 331 OTUs in both
treatment samples, accounting for 73.56% of the total OTU observed by the fungi
(Fig. 1d). It was found that only a small fraction of OTUs were unique to
treatments. The OTUs unique in (M)-treatment samples,
fungi were mainly belonging to the classes of Sordariomycetes, Agaricomycetes
and Pezizomycetes; while the OTUs unique to (R)-treatment were belonging
to Sordariomycetes.
Fungal communities composition
A
total of 4 phyla were detected in all
the samples, among which the Ascomycota and Zygomycota were
dominant, accounting for 84.71 and 12.41% of the total
fungi, respectively (Fig.
2a). Compared with monocropped cucumber soil, rotation with Indian mustard had higher Ascomycota
abundance, but the abundance of Zygomycota was
relatively low (P ≦ 0.05). The top three fungal classes
(relative abundance >10%) found were Sordariomycetes, Pezizomycetes and Zygomycetes, accounting
for 92.18% of the total fungi (Fig. 2b). Rotation
with Indian mustard also increased abundance of Leotiomycetes, Ascomycota
Ineertae sedis and unclassified
fungi, and decreased abundance of Zygomycetes as compared to monocropped cucumber (P ≦ 0.05).
Hypocreales, Mortierellales, Sordariales,
Pezizales and Microascales were the dominant orders (average
relative abundance >10%) in all the samples (Fig. 2c). Futhermore, Agaricales,
Rhizophlyctidales, Thelebolales, Sordariomycetes Incertae
sedis, Agaricomycetes Incertae sedis, Eurotiales, Xylariales,
Ascomycota Incertae sedis, Onygenales, Tremellales, Pleosporales,
unclassified Sordariomycetes,
unclassified fungi and unclassified Ascomycota were
also detected at relatively higher abundance (average relative abundance >
0.1%) (Fig. 2d). Compared with monocropped cucumber, rotation with Indian
mustard had higher relative abundance of Sordariales, Eurotiales,
Agaricomycetes Incertae sedis, unclassified
Sordariomycetes and unclassified
Fungi and lower relative abundance of Mortierellales, Microascales,
Agaricales, Thelebolales (P ≦ 0.05).
At
the genus level, more than 137 fungal genera or groups were detected in both
treatment soil samples (data not shown). In (R)-treatment, the
relative abundance of Humicola, Remersonia, Myrothecium, Scedosporium and Mycothermus spp.
was higher, but that of Pseudallescheria,
Mortierella, Chaetomium, Ilyonectria, Gibellulopsis and Metacordyceps spp. was lower (Table 1).
Discussion
Table 1: Relative abundances (%) of main fungal genera in cucumber
rhizosphere soils
|
Fungal genera |
M |
R |
Fungal genera |
M |
R |
|
Pseudallescheria |
25.69 ± 1.64 |
17.46 ± 1.10 |
Aspergillus |
0.07 ± 0.00 |
0.28 ± 0.08 |
|
Mortierella |
15.74 ± 0.25 |
5.46 ± 1.13 |
Thielavia |
0.18 ± 0.01 |
0.16 ± 0.02 |
|
Humicola |
1.39 ± 0.13 |
15.04 ± 1.84 |
Trichoderma |
0.29 ± 0.17 |
0.02 ± 0.01 |
|
Chaetomium |
10.92 ± 0.06 |
5.08 ± 0.16 |
Ilyonectria |
0.19 ± 0.01 |
0.04 ± 0.01 |
|
Fusarium |
6.12 ± 1.05 |
8.68 ± 1.52 |
Gibellulopsis |
0.13 ± 0.01 |
0.04 ± 0.02 |
|
Pseudaleuria |
6.93 ± 1.81 |
7.09 ± 1.42 |
Arachnomyces |
0.09 ± 0.02 |
0.07 ± 0.03 |
|
Kernia |
2.28 ± 0.45 |
1.65 ± 0.10 |
Zygopleurage |
0.06 ± 0.01 |
0.10 ± 0.02 |
|
Acremonium |
1.54 ± 0.17 |
2.09 ± 0.25 |
Rhizophlyctis |
0.04 ± 0.01 |
0.12 ± 0.05 |
|
Preussia |
0.15 ± 0.00 |
1.39 ± 0.58 |
Penicillium |
0.08 ± 0.00 |
0.07 ± 0.02 |
|
Zopfiella |
0.45 ± 0.04 |
0.78 ± 0.17 |
Metacordyceps |
0.10 ± 0.00 |
0.05 ± 0.01 |
|
Cryptococcus |
0.60 ± 0.10 |
0.60 ± 0.08 |
Gibberella |
0.05 ± 0.01 |
0.08 ± 0.01 |
|
Remersonia |
0.40 ± 0.02 |
0.73 ± 0.04 |
Mycothermus |
0.03 ± 0.00 |
0.09 ± 0.02 |
|
Monosporascus |
0.59 ± 0.29 |
0.50 ± 0.25 |
Gymnoascus |
0.04 ± 0.01 |
0.06 ± 0.02 |
|
Chrysosporium |
0.41 ± 0.08 |
0.51 ± 0.07 |
Phialemonium |
0.04 ± 0.01 |
0.05 ± 0.01 |
|
Microascus |
0.35 ± 0.05 |
0.32 ± 0.11 |
Phialosimplex |
0.04 ± 0.01 |
0.05 ± 0.01 |
|
Myrothecium |
0.03 ± 0.01 |
0.57 ± 0.15 |
Scutellinia |
0.04 ± 0.00 |
0.04 ± 0.01 |
|
Scedosporium |
0.19 ± 0.01 |
0.35 ± 0.04 |
Papulaspora |
0.06 ± 0.03 |
0.01 ± 0.00 |
|
Myriococcum |
0.01 ± 0.01 |
0.51 ± 0.31 |
Nectria |
0.05 ± 0.02 |
0.01 ± 0.00 |
|
Cephaliophora |
0.33 ± 0.10 |
0.14 ± 0.02 |
Arthrographis |
0.03 ± 0.01 |
0.03 ± 0.01 |
|
Wardomyces |
0.21 ± 0.05 |
0.20 ± 0.00 |
Coniochaeta |
0.02 ± 0.00 |
0.03 ± 0.01 |
|
Podospora |
0.25 ± 0.05 |
0.12 ± 0.01 |
Guehomyces |
0.02 ± 0.00 |
0.03 ± 0.02 |
Note: Values
(mean ± SE) highlighted in bold are significantly different among treatments of
cucumber monocropping (M) and rotations of Indian mustard (R) at the 0.05
probability level (Student’s t test)
Fig. 2: Relative
abundances of main fungal phyla (a), classes (b), order (c,
d) in cucumber
rhizosphere. Fungal phyla and classes with average
relative abundances >10% in at least
one treatment were shown. Fungal
orders with
average relative abundances >10% (c) and
>0.1% (d) were shown
in at least one treatment. M and R represent treatments of cucumber
monocropping and rotations with Indian mustard. Values are
expressed as mean±standard error. Asterisks indicate significant difference
between treatments based on Student’s
t test (P ≦ 0.05)
Soil fungal community, acting as pathogen, decomposers,
and mutalists, play essential roles in many ecosystem processes such as energy
flow, nutrient cycling and organic matter turnover (Philippot et al. 2013). The composition of fungal
could be changed by soil environment (such as soil type, soil pH and soil
carbon content) (King and Blesh 2018). Moreover, plants are able to shape their
rhizosphere microbiome by releasing exudates containing various compounds
(Berendsen et al. 2012).
Our results indicated that the structure of Indian mustard
rotation and monocropped cucumber fungal community were distinct,
which were consistent with the previous findings (Jin et al. 2019a), demonstrating that crop rotation changes the
rhizosphere environment, thus altered the soil microbial communities
composition and structure. Miseq. sequencing showed that the main phyla
was Ascomycota across all soil
samples, and Indian mustard rotation increased the abundance of Ascomycota. Ascomycota, a group of resident soil fungi, rely on the
decomposition of soil organic matter or plant root exudates, play an important
role in maintaining soil microbial ecological balance (Wang et al. 2016). In this study, the dominant orders includes Hypocreales,
Mortierellales, Pezizales, Microascales and Sordariales
(average relative abundance >10%) and Thelebolales, Sordariomycetes
Incertae sedis, Eurotiales, Xylariales, Ascomycota
Incertae sedis, Pleosporales and unclassified Ascomycota
(average relative abundance > 0.1%) of the phylum Ascomycota, which are considered to be primary
straw residue decomposers (Hannula et al. 2012; Ma et al.
2013; Wang et al. 2017; Hu et al. 2018). Therefore, from these findings we speculate that crop rotation changed the
composition and content of root exudates, which might be the reason of higher abundances of decomposer fungi.
It has been reported that after
continuous monocropping of cucumber, phenolic compounds (such as p-coumaric acid) could accumulate in the soil, thus promoted
the growth of pathogenic fungi (Zhou et
al. 2018b). The reduction of pathogenic fungi in the soil
and the increase in beneficial fungi may be associated
with the degradation of phenolic acids. Venter et al. (2016) have shown that the
increase in soil microbial diversity and abundance can be attributed to
increase in crop diversity. These findings suggest that crop rotation can
increase the abundance of root residues, resulting in the higher diversity of
decomposers in rhizosphere. This phenomenon is caused by the
selectivity of rotation for soil fungi. Therefore, we inferred that plant residues and root
exudates could provide carbon source for soil microbes and thus changed the
composition of soil microbial communities.
It is well understood that not all the fungi are plant
pathogenic but some of them can promote plant growth by decomposing plant
residues to provide nutrients to the plants (Ahmad et al. 2018). Compared with monocropped
cucumber, rotation with Indian mustard increased abundance of Humicola, Remersonia and Myrothecium spp., but decreased abundance of Mortierella, Chaetomium and Gibellulopsis spp. (P ≦ 0.05). Previously,it has been
reported that Humicola, Remersonia and Myrothecium are plant beneficial fungi, which
can promote biogeochemical cycles and the absorption of nutrients and inhibit
disease development. Humicola, a
biocontrol fungi, reduced the disease incidence of
pepper blight caused by Phytophthora capsici
and black spot on leaf of cabbage caused by Alternaria brassicicola (Ko et al. 2011; Yang
et al. 2014). Krishnan et al. (2017)
reported that due to the ability to synthesize ligninolytic and
cellulolytic enzymes, Remersonia can stimulate plant growth. Similarly,
Myrothecium could produce some secondary metabolites (such as trichothecene
macrolides) to inhibit plant pathogen
(Liu et al. 2016).
Root
exudates have been found that root exudates play important role in plant
defense against soil-borne pathogens (Park
et al. 2004). In our study, the relative
abundance of Ilyonectria, Gibellulopsis and Metacordyceps spp. (P = 0.05) were decreased by rotation of Indian mustard
which contains plant pathogens. The genera of Ilyonectria has
capable of causing black foot rot of Proteaceae
(Aiello et al. 2014). Kawaradani et al. (2013) reported
that some species of Gibellulopsis could cause the seedling rot on chrysanthemum and lettuce. Meanwhile,
some species in Metacordyceps are predominant genera in
pesticide-contaminated agricultural soils (Merlin et al. 2014). Cucumber itself could selectively recruit
microorganisms in the rhizosphere for its own benefit (Jia et al. 2019; Zhou et al. 2019). Therefore, we assume that rotation of
Indian mustard inhibited the root colonization of soil-borne pathogen as compared to monoculture.
In this study we used
Indian mustard as rotation crop with cucumber as main crop to study effects of
crop rotation system on soil fungal community. Our results indicated that
crop rotation affected the fungal composition and altered the dominant genera,
increased the abundance of fungi with potential antifungal ability and
decreased the harmful fungi.
These results show that rotation with Indian
mustard could be beneficial growth and development of cucumber which is an
important crop in many parts of the world. Overall, our findings suggest that
adopting crop rotation system with suitable crops could cure soil health by
altering its microbial communities and alleviate soil sickness.
Acknowledgement
This
work was supported by the National Natural Science Foundation of China
(31772361), 'Academic Backbone' Project of Northeast Agricultural University
(17XG05) and China Agricultural Research System (CARS-23-B-10).
References
Jia HT, JY Liu, YJ Shi, DL Li, FZ Wu, XG Zhou (2019).
Characterization of cucumber rhizosphere bacterial
community with high-throughput amplicon sequencing. Allelopath J 47:103‒112
Jin X, DD Pan, JH Zhang, DL Li, K Pan, FZ Wu, XG
Zhou (2019a). Effects of crop rotation with wild rocket on cucumber seedling
rhizosphere fungal community composition. Allelopath J 47:83‒91
Jin X, J Wang, DL Li, FZ Wu, XG Zhou (2019b). Rotations with indian mustard and wild rocket suppressed cucumber
fusarium wilt disease and changed rhizosphere bacterial communities. Microorganisms 7:1‒15
Krishnan Y, CPC Bong, NF Azman, Z Zakaria, N Othman, N Abdullah, CS Ho, CT Lee, SB Hansen, H Hara (2017). Co-composting
of palm empty fruit bunch and palm oil mill effluent: Microbial diversity and potential mitigation
of greenhouse gas emission. J Clean Prod 146:94‒100
Wang ZT, T Li, XX Wen, Y Liu, J Han, YC Liao, JM DeBruyn (2017). Fungal
communities in rhizosphere soil under conservation tillage shift in response to
plant growth. Front Microbiol 8:1‒11
Zhang JH, DD Pan, X Ge, YH Shen, PL Qiao, SY Yang,
FZ Wu, XG Zhou (2018). Effects of syringic acid on Fusarium and Trichoderma
communities in cucumber (Cucumis sativus L.) seedling rhizosphere. Allelopath J 44:181‒189
Zhou XG, J Liu, FZ Wu (2017). Soil microbial communities in cucumber
monoculture and rotation systems and their feedback effects on cucumber
seedling growth. Plant Soil 415:507‒520
Zhou XG, J Wang, X Jin, DL Li, YJ Shi, FZ Wu (2019). Effects of selected
cucumber root exudates components on soil Trichoderma spp. communities. Allelopath J 47:257‒266