3 Biotech (2021) 11:469

https://doi.org/10.1007/s13205-021-03011-3

ORIGINAL ARTICLE

Rhizosphere microbial diversity in rhizosphere of Pinellia ternata

intercropped with maize
Hamza Armghan Noushahi1,2 · Zhenxing Zhu1 · Aamir Hamid Khan3 · Umair Ahmed4 · Muhammad Haroon5 ·
Muhammad Asad2 · Mubashar Hussain6 · He Beibei1 · Maimoona Zafar1 · Mohammad Murtaza Alami1 ·
Shaohua Shu1

Received: 5 January 2021 / Accepted: 2 October 2021 / Published online: 22 October 2021
© King Abdulaziz City for Science and Technology 2021

Abstract
Dry tubers of Pinellia ternata (Thunb.) Breit are used in traditional Chinese medicine. Commonly known as “banxia” in
China, the tubers contain valuable compounds, including alkaloids and polysaccharides that are widely used in pharmaceu-
ticals. The quantity and quality of these important compounds are affected by whether P. ternata is grown as a sole crop or
as an intercrop, and P. ternata cultivation has become challenging in recent years. By intercropping P. ternata, its maximum
yield, as well as large numbers of chemical components, can be realized. Here, a large data set derived from next-generation
sequencing was used to compare changes in the bacterial communities in rhizosphere soils of P. ternata and maize grown as
sole crops and as intercrops. The overall microbial population in the rhizosphere of intercropped P. ternata was significantly
larger than that of sole-cropped P. ternata, whereas the numbers of distinct microbial genera, ranging from 552 to 559 among
treatments, were not significantly different between the two rhizospheres. The relative abundances of the genera differed.
Specifically, the numbers of Acidobacteria and Anaerolineaceae species were significantly greater, and those of Bacillus
were significantly lower, in the intercropped P. ternata rhizosphere than in the sole-cropped rhizosphere.

Keywords Pinellia ternata · Intercropping · Rhizosphere microorganisms · Microbial diversity · Alpha diversity · Beta
diversity

Introduction The herb is used as a sedative with analgesic and antiemetic

properties (Zhang et al. 2016). In addition to its antioxi-
Pinellia ternata (Thunb.) Breit, which belongs to the family dative, anti-inflammatory, immune-boosting, antibacterial,
Arceae, is a perennial herb with medicinal uses that is found anti-obesity (Ji et al. 2014; Kim et al. 2020), and bacterio-
in East Asia and commonly cultivated in China (Zhang et al. static (Ji et al. 2014) properties, it has been used for treating
2013; Moon et al. 2016). The main medicinal part is the convulsive disorders, vomiting, and coughing for decades.
dried tuber, “banxia” in Chinese, which has been widely Worthwhile uses of P. ternata in traditional Chinese medi-
used over centuries in traditional Chinese medicine (Iwasa cine are as an antitussive and anxiolytic (Gombodorj et al.
et al. 2014; Zhang et al. 2015). Major constituents of P. ter- 2017; Xu et al. 2018; Lin et al. 2019).
nata are organic acids, polysaccharides, and alkaloids (Gom- Pinellia ternata is susceptible to intense light and high
bodorj et al. 2017; Xu et al. 2018), with the latter, which temperatures during its growth period. The plant withers
have anticancer properties, being considered the principle quickly under high temperatures and strong sunlight, and
biologically active ingredients (Gombodorj et al. 2017). it develops a “sprout tumble” (Juneidi et al. 2020; Ma et al.
2020). Although P. ternata prefers warm, or hot, and humid
weather, intense sunshine, high temperatures, and waterlog-
Hamza Armghan Noushahi and Zhenxing Zhu have contributed ging are not conducive to growth. The demand for this herb
equally to this work. continues to increase but cannot be met because it is not
cultivated on a large scale. “Sprout tumbles” occur in hot
* Shaohua Shu
[email protected] weather and immediately restrict the formation of tubers,
further increasing the gap between supply and demand (Xue
Extended author information available on the last page of the article

13
Vol.:(0123456789)
469 Page 2 of 10 3 Biotech (2021) 11:469

et al. 2019). Xue et al. (2019) showed that reducing “sprout Our limited capacity to analyse soil environments in real
tumbles” was crucial for increasing tuber yield. The rate of time is part of the challenge in examining the structures of
“sprout tumble” formation can be decreased, and the date of soil microbial communities. Research using PCR extrac-
“sprout tumble” onset advanced, by maintaining low tem- tions of genetic materials (Rastogi and Sani 2011) offers
peratures or by providing shade during the plant’s regenera- fresh insights into the immense diversity of bacterial strains
tive developmental cycle (Zhang et al. 2021). in the soil ecosystem. However, few studies have explored
Monocropping, or sole cropping, poses a challenge to the the variety and composition of bacterial communities in
cultivation of traditional medicinal plants. The production the rhizosphere of P. ternata or compared their character-
of P. ternata decreases by 60–80% within 2 to 4 years of istics between sole-cropped and intercropped P. ternata.
monocropping. Xihe County in China’s Gansu Province is Consequently, we analysed the diversity, composition, and
well known for the production of this herb. However, in that structures of bacterial communities from the rhizospheres
region, P. ternata production declined from about 1665 ha in of P. ternata and maize grown separately as monocrops and
2000 to approximately 535 ha in 2015. Recently, monocrop- together as intercrops.
ping has become a particularly severe constraint to the culti-
vation of P. ternata (He et al. 2019), with several biotic and
abiotic factors acting as barriers. The abiotic factors lower Materials and methods
the quality of the soil and hamper efficient field manage-
ment, whereas biotic factors lead to autotoxicity, pathogen The field trial design
infection, and changes in the microbial community com-
position of the rhizosphere (Vargas Gil et al. 2009), with The experiment was conducted in 2018 and 2019 in China
the latter being the main barrier to the monoculturing of at Hubei Jingheyuan Ecological Agriculture Co., Ltd., Shay-
other crops as well (González-Chávez et al. 2010; Bernard ang County, Jingmen City, Hubei Province (30° 38′ 19.55″
et al. 2012; Larkin et al. 2012). Protecting crops from intense N, 112° 34′ 15.82″ E). The average annual temperature at
sunlight by building shelters is expensive in terms of human the site was 15.6–16.3 °C, the duration of sunshine was
and material resources, and it also hampers cultural opera- 1997–2100 h a year, and the average annual precipitation
tions. However, higher yields of P. ternata can be obtained was 804–1067 mm. The soil at the experimental site was
by intercropping it with other crops, such as maize, soybean, sandy but highly fertile. Tubers of P. ternata, collected
and wheat. These crops, owing to their vigorous growth, from Jingmen City and 6–8 mm in diameter, were planted
shelter P. ternata from direct sunlight. The allelochemicals on March 20 in 2018 and 2019. The crop was sown for two
produced by P. ternata are preserved mainly in the under- consecutive years, with two harvests a year, on July 1 and in
ground tubers, which influence the photosynthetic efficiency mid-November. Maize seeds (Zea mays L., ‘Huimin 302’)
and viability of neighbouring crops through their antioxida- were purchased from the Hubei Huimin Agricultural Tech-
tive systems. Maize has been proven to be particularly suit- nology Co., Ltd., sown on April 10 and harvested on July
able as an intercrop or as a rotational crop with P. ternata. 20 in both years.
The rhizosphere, a zone of soil affected by root secre- For the experiment, a two-factorial randomized block
tions, may harbour as many as 1­ 011 units of microorganisms design was adopted. Factor 1 was designed as the maize
for every gram of roots (Hakim et al. 2021), and plants are single-line planting on the same side. Factor 2 was the dis-
closely connected to the microbes present in the soil. These tance between the rows of maize and P. ternata, which was
soil microbes represent the world’s largest source of bio- maintained at 35, 50, or 65 cm. Two controls were main-
logical diversity known to date (Gams 2007; Ho et al. 2017; tained, maize grown as a sole crop and P. ternata grown as
Jacoby et al. 2017). Beneficial rhizosphere microorganisms a sole crop. The experiment had 5 treatments, repeated over
can promote plant growth through organic mineralization, 15 blocks, each with 3 plots. Each plot was 1.1 m wide and
biological nitrogen fixation (Van Der Heijden et al. 2008; 7.5 m long, containing a 0.4-m-wide trench. The seed rate
Luo et al. 2016), host-immunity modulation (Dang et al. for P. ternata was 3,750 kg ­hm−2 of tubers. During mono-
2020), and pathogen suppression, thereby helping plants to cropping, whether maize or P. ternata, the plant-to-plant and
distinguish between beneficial and harmful entities (Zami- row-to-row distances were 35 cm and 50 cm, respectively.
oudis and Pieterse 2012). Bacterial communities are the
largest complex communities of soil microbes, account- Sampling of rhizosphere soil
ing for as many as 1­ 04 communities for every gram of soil
(Weinert et al. 2011; Xu et al. 2019). Intercropping increases The plants were gently uprooted and excess soil was removed
the number of soil microorganisms in the rhizosphere, as by shaking the roots. The soil clinging to the roots, forming
well as their diversity level (Dariush et al. 2006; Dang et al. a layer of approximately 1 mm, was retained. It was then
2020). removed using a sterilized brush, and the roots were stored

13
3 Biotech (2021) 11:469 Page 3 of 10 469

in dry ice. The intact root system was placed in a 50-mL test Statistical analyses
tube filled with sterilized phosphate-buffered saline solution,
and the root surface was removed using sterilized forceps. The treatments were compared using a one-way analysis of
The washed soil along with the phosphate-buffered saline variance (ANOVA) and Tukey’s test of honestly significant
solution was poured into a series of 50-mL sterilized test differences. All the analyses of alpha diversity relied on the
tubes and centrifuged. The supernatant was discarded, OTU clusters with a 3% dissimilarity as the cut-off point.
whereas the pellet, the rhizosphere soil, was rapidly frozen Five indexes, phylogenetic diversity (Pd), Simpson, amount
using liquid nitrogen and stored at − 80 °C for analysing the of observed species (Sobs), Chao, and Shannon, of the rich-
microbial diversity. ness and diversity of the bacterial community associated
with each treatment were computed. We compared the lev-
DNA extraction and MiSeq sequencing els of diversity of bacterial OTUs across the soil samples
from P. ternata to the rarefaction curves provided by Mothur
A PowerSoil kit (MO BIO Lab., Carlsbad, CA, USA) was and further evaluated the similarity between the community
used for extracting genomic DNA from the soil samples memberships using a principal coordinate analysis (PCoA)
(each weighing 0.5 g, dry weight). The extracted DNA with weighted and unweighted distance metrics of UniFrac
was stored at − 80 °C, and its concentration was measured (based on the phylogenetic structure). Lastly, Venn diagrams
using a spectrophotometer (Nanodrop 2000C, Thermo Sci- were constructed to examine the similarity and dissimilarity
entific, Wilmington, DE, USA). The V4–V3 region of the levels among species in the samples.
16S rRNA bacterial gene was amplified with the primers
338F (5ʹ-ACT​CCT​ACG​GGA​GGC​AGC​AG-3ʹ) and 806R
(5ʹ-GGA​CTA​CHVGGT​WTC​TAAT-3ʹ) using the following Results
PCR amplification protocol: 3 min of denaturation at 95 °C,
35 cycles of 30 s at 95 °C, 30 s at 55 °C for annealing, and Sequencing of DNA
45 s at 72 °C for elongation, followed by a final extension
at 72 °C for 10 min. The following mixture (final volume The sequenced data (OTUs, sequence numbers, base
20 μL) was used for the PCR reactions: 4 μL of 5 Fast- numbers, and mean lengths) showed the overall structure
Pfu buffer, 2 μL of 2.5-mM dNTPs, 0.8 μL of each primer of our study (Table 1). We observed that in all the trials,
(5 μM), 0.4 μL of FastPfu polymerase, 0.2 μL of bovine the OTUs were nearly equal. The maximum number of
serum albumin, and 10 ng of the DNA template. sequences (103,527 sequences) was retrieved from P. ternata
planted 65 cm away from maize as an intercrop, whereas
Sequence processing and analysis the sequence numbers in the other treatment samples did
not differ significantly. The maximum number of bases was
Purified amplicons in equimolar and paired-end sequences also recorded for sequences from intercropped P. ternata,
(2 × 300) were obtained on an Illumina MiSeq platform whereas the base numbers in the other treatment samples
(Illumina, San Diego, CA, USA) using the standard pro- were similar. The mean lengths of the sequences were equal
tocols stipulated by the company. Illumina sequencing was in all the cropping system samples.
used to generate raw gene-sequence data, and the data were
modified using Trimmonmatic and FLASH. The sequences
were grouped into operational taxonomic units (OTUs)
after 97% pair recognition by QIIME using Usearch ver. 7.1
(http:/qiime.org/). The Ribosomal Database Project classi-
fier (Release 11.1 http://​rdp.​cme.​msu.​edu/) was used for the Table 1  Sequenced data for all the cropping systems
taxonomic categorization of the representative sequences of
Sample OTUs Seq No. Base No. Mean length
bacteria against the Greengenes 16S rRNA (Release 13.5;
greengenes.secondgenome.com/) and Silva (Release 115; Ac 20427 34572.33 15164406.67 438.63
arb-silva.de) databases. Aa 24491 40401 17707564.33 438.24
Ab 22500 103527 45412416 438.64
CK 21452.67 35806.33 15701947.67 438.50
YM 22001.33 32372.67 14240639.33 439.91

Aa, Ab, and Ac indicate P. ternata intercropped at 35, 50, and 65 cm

away from maize, respectively, CK represents P. ternata monocrop-
ping and YM represents maize monocropping OTU, operational taxo-
nomic unit

13
469 Page 4 of 10 3 Biotech (2021) 11:469

Alpha diversity index of monocropped P. ternata was significantly lower

than that of intercropped P. ternata, indicating that inter-
The treatments showed no significant differences in Pd cropping increases the richness of rhizosphere soil micro-
(Table 2), whereas the Simpson index of the monocropped organisms. Similarly, Sobs and Shannon indexes of mono-
maize sample was substantially higher than that of the other cropped maize were significantly lower than those of the
treatment samples. other treatment samples, indicating that there was no sig-
The Chao index measures species richness, and the higher nificant difference in the species richness of the rhizosphere
its value, the greater the community richness (t). The Chao soils between monocropped and intercropped P. ternata.
index of monocropped maize, at 2,268.7, was significantly Thus, using P. ternata as an intercrop did not change the
lower than those of other treatment samples, and the Chao microorganism diversity level in the rhizosphere soil.

Table 2  Alpha diversity Treatments Pd Simpson Sobs Chao Shannon

index of rhizosphere soil
microorganisms under P. Aa 153.6 ± 9.8a 0.0028 ± 0.0002b 2171.0 ± 16.3a 2516.3 ± ­12b 6.72 ± 0.02a
ternata–maize intercropping
Ab 148.1 ± 8.1a 0.0027 ± 0.0005b 2185.0 ± 21.5a 2594.8 ± 15.3a 6.75 ± 0.05a
conditions
Ac 147.2 ± 7.2a 0.0024 ± 0.0003b 2204.3 ± 15.0a 2587.9 ± 11.2a 6.80 ± 0.01a
CK 142.7 ± 8.3a 0.0037 ± 0.0001b 2136.6 ± 15.7a 2497.2 ± 7.3b 6.62 ± 0.07a
YM 139.6 ± 9.2a 0.0100 ± 0.0004a 1880.0 ± 24.4b 2268.7 ± 23.0c 6.13 ± 0.17b

The variance in alpha diversity across samples is shown

Aa, Ab, and Ac indicate P. ternata intercropped at 35, 50, and 65 cm away from maize, respectively, CK
represents P. ternata monocropping and YM represents maize monocropping
a
shows significant
a
shows the non significant value (p < 5%)
Pd phylogenetic diversity; Sobs amount of observed species

Fig. 1  Principal coordinate

analysis: YM, maize as a sole
crop; ML, P. ternata as a sole
crop (control); Aa, Ab, and
Ac, P. ternata planted 35, 50,
and 65 cm away from maize,
respectively

13
3 Biotech (2021) 11:469 Page 5 of 10 469

Beta diversity

As shown by the PCoA based on UniFrac distances,

unweighted and weighted, microbial communities in the
rhizosphere of P. ternata differed, depending on whether P.
ternata was monocropped or intercropped. Microbial com-
munities in the first principal coordinate component (PC1)
(39.01% contribution) and second principal coordinate com-
ponent (PC2) (12.4% contribution) represent the two axes in
Fig. 1. The bacterial communities in intercropped P. ternata,
irrespective of its distance from maize, 35, 50, or 65 cm, and
those in monocropped P. ternata were more similar to each
other than to the bacterial communities from monocropped
maize, and they clustered along different axes.
The beta diversity, using the Bray–Curtis distance matrix,
revealed that bacterial communities in soil samples collected
from the same fields were more similar to each other than to
those in the corresponding soil samples from different fields.
For example, bacterial communities from the rhizosphere
Fig. 2  Bray–Curtis dissimilarity hierarchical cluster tree of the soil of monocropped maize clustered differently from those of
bacterial community: YM, maize as a sole crop; ML, P. ternata as monocropped and intercropped P. ternata planted at varying
a sole crop (control); Aa, Ab, and Ac, P. ternata planted 35, 50, and
65 cm away from maize, respectively distances, 35, 50, or 65 cm, from maize (Fig. 2).

Bacterial community compositions

A rarefaction curve is used to confirm that the amount of

sample sequencing data is sufficient. A flat curve indicates
that the amount of data is adequate and that more data
will add only a limited number of new species, whereas a
steeper curve indicates that adding more sequences to the
data will reveal many new species. The rarefaction curve of
the sequencing data from the present experiment is shown
in the supplementary data (Figure S1). We used a Venn dia-
gram to determine the number of unique species and their
relative proportions in the samples. A Venn diagram of the
bacterial communities, at the genus level, in the rhizospheres
of treatments is shown in Fig. 3. The numbers of genera
varied with the treatment, as follows: 559 genera were rep-
resented in the samples from P. ternata planted 35 cm or
65 cm away from maize; whereas there were 556 genera in
samples from P. ternata planted 50 cm away from maize.
As sole crops, there were 552 and 521 from P. ternata and
maize samples, respectively. Across these treatments, there
were 493 common genera (found in every treatment). Each
of the treatments in which P. ternata was planted a different
Fig. 3  Venn diagram of the composition of bacterial communities in distance from maize showed one unique genus. Addition-
rhizospheres of P. ternata intercropped with maize. Different colours ally, P. ternata as a monocrop yielded three unique genera;
represent different treatments; the numbers refer to the number of whereas maize as a monocrop yielded no unique genera.
species common to multiple treatments in overlapping and non-over-
lapping sections

13
469 Page 6 of 10 3 Biotech (2021) 11:469

Fig. 4  Hierarchical clustering tree at the genus level. The distances stacked column chart, the composition of the dominant species in
between branches represent the differences among treatments, and each sample is shown on the right
different colours represent different taxonomic categories. In the

Fig. 5  Partial least squares dis-

criminant analysis. In different
environments or circumstances,
different coloured dots or shapes
represent different treatments.
Measurements along the X- and
Y-axes are relative distances
and have no functional values.
The presumed factors control-
ling changes in the microbial
composition of the two sample
groups areComp1 and Comp2,
respectively

13
3 Biotech (2021) 11:469 Page 7 of 10 469

A Circos sample–species relationship diagram shows the Sample comparisons

correspondence between samples and species. The graph
represents not only the proportions of dominant species in A sample hierarchical cluster on the basis of beta diversity,
a sample but also the distribution ratio of each dominant at the genus level, of the rhizosphere bacteria is shown in
species across samples. The composition of the bacte- Fig. 4. Two genera, Fictibacillus and Bacillus, dominated
rial community (at the genus level) in the rhizosphere of every treatment, as did no-rank Acidobacteria. The bacterial
P. ternata intercropped with maize is shown in Figure S2. communities of the maize rhizosphere differed from those
The community was dominated by 26 major genera: Aci- of the P. ternata rhizosphere (whether as a sole crop or as
dobacteria, Bacillus, Fictibacillus, Nitrospira, Sphingo- an intercrop), which showed that the bacterial composition
monas, Anaerolineaceae, KD4-96, Knoellia, Nocardioides, of the rhizosphere was influenced by the crop.
Pseudarthrobacter, H16, Nitrosomonadaceae, JG30-KF- In Fig. 5, a PLS-DA analysis of the rhizosphere bacteria
Cm45, Actinobacteria, Rhodobiaceae, Gaiella, Microvirga, under each treatment at the genus level is shown. Here, the
Gaiellales, Streptomyces, Gemmatimonadaceae, Xanthomo- differences in the compositions of the bacterial communi-
nadales, Acidimicrobiales, Rhodospirillaceae, Roseiflexus, ties were also evident, which again indicated the influence
Rhizobium, and Pseudomonas. Among them, the relative of the crop.
abundances of three genera varied at treatment, as follows
(the proportions listed in descending order). Acidobacteria: Analysis of differences in bacterial species
25%, 24%, and 23% in P. ternata planted 65, 50, and 35 cm
away from maize, respectively, and 14% in both crops as sole The relative abundance of no-rank Acidobacteria in inter-
crops; Bacillus: 11%,12%, and 11% in P. ternata planted cropped P. ternata samples was significantly greater than
65, 50, and 35 cm away from maize, respectively, and 43% in sole-cropped P. ternata and maize samples (Fig. 6). The
and 22% in maize and P. ternata as sole crops, respectively; relative abundances of Bacillus in sole-cropped P. ternata
Fictibacillus: 7.4%, 6.5%, and 7.1% in P. ternata planted 65, and maize samples were significantly greater than in inter-
50, and 35 cm away from maize, respectively, and 68% and cropped P. ternata samples. However, Fictibacillus, was
11% in maize and P. ternata as sole crops, respectively. The more abundant in sole-cropped maize samples than in any
species composition of the rhizosphere of P. ternata as a sole other treatment, and sole-cropped maize samples also had
crop was the same as that of P. ternata as an intercrop, but lower abundances of Sphingomonas and Knoellia than any
the relative abundances of the species differed between the of the other treatments. As a monocrop, P. ternata recorded
two treatments. The composition of the rhizosphere of maize a significantly lower relative abundance of no-rank Anaero-
was also different from that of the rhizosphere of P. ternata. lineaceae than any other treatment.

Fig. 6  Variations in microbial

communities of rhizospheres
of P. ternata intercropped with
maize. At a given taxonomic
level, the Y-axis represents the
species, the X-axis represents
the average relative abundance
of species in different treat-
ments, and columns of different
colours represent different
treatments. *, **, and ***:
differences significant at the
0.05, 0.01, and 0.001 levels,
respectively

13
469 Page 8 of 10 3 Biotech (2021) 11:469

Discussion key factor affecting the diversity and distribution of bacterial

communities (Zi et al. 2020). Thus, the P. ternata–maize
Pinellia ternata, an important medicinal herb distributed intercropping system was more efficient in recycling nutri-
over most of China, cannot be cultivated continuously in ents than P. ternata monocropping.
north-western China, especially in Gansu Province; whereas, Understanding the structure and diversity of bacterial
in southern China, the plant has been cultivated continually communities in the rhizosphere provides novel insights
for more than 10 years. Wild P. ternata grows as a weed of into microorganisms that are potentially hostile to plant
dryland crops, such as soybean, maize, and cotton. Reports pathogens (Berendsen et al. 2012). The present study
have indicated that diseases are more severe when a crop is observed a significant decline in bacterial richness (Chao)
grown continually than when it is rotated with other crops, under sole-cropped P. ternata conditions, but there were
and the soil microbial community is believed to contribute no significant effects on diversity (Pd) (Table 1). This
greatly to countering plant diseases. In the present study, result is consistent with those of several earlier studies. For
although the P. ternata yield was greater when it was inter- example, in black pepper, the bacterial diversity decreases
cropped with maize, the species composition of its rhizos- significantly after 55 years of monocropping (Xiong et al.
phere remained the same whether it was grown as a sole crop 2015), and similar results have been observed for cucum-
or as an intercrop. However, the bacterial species were more ber (Chen et al. 2012) and potato (Liu et al. 2014). The
abundant in P. ternata’s rhizosphere than in that of maize. richness and diversity of soil bacteria play essential roles
The difference was traced to carbon sources. Decomposing in soil quality and health, as well as in the sustainability of
plants release sugars and amino acids into the rhizosphere, soil ecosystems (Kennedy and Smith 1995), whereas con-
which act as carbon sources for soil microorganisms. How- tinued monocropping is detrimental to both richness and
ever, monocropping relies more on chemical fertilizers and, diversity. A decrease in the diversity level of soil microbes
therefore, releases smaller amounts of carbon sources than aids the development of soil borne plant diseases (Maz-
intercropping. As a result, the microbial diversity and activ- zola 2004). Overall, any reduction in the richness and
ity levels may be lower under monocrop conditions (Wel- diversity levels of a soil microbial community as a result
baum et al. 2004). The growth rates of the microorganisms of continual cropping may impair plant performance and
at each point in the soil were assumed to be controlled by the make plants vulnerable to diseases and pests, as shown in
soluble organic substrate concentrations. The relative com- Camellia sinensis. Moreover, the UniFrac-weighted PCoA
position of rhizosphere bacteria also differed, as reported in (Fig. 1) showed marked variations in the structures of
another study (Zi et al. 2020), in which Acidobacteria was bacterial communities across the treatments, and this was
the more abundant genus under intercropping conditions, consistent with the findings of Xiong et al. (2015), in that
with a relative abundance of 21.61%. This finding was con- the long-term continual cropping of black pepper results
sistent with that of the present study, in which the relative in noticeable variations in the structure of the bacterial
abundance of no-rank Acidobacteria was significantly higher community. Li et al. (2014), using a 454-pyrosequencing
under intercropping conditions. Members of Acidobacteria analysis, also noted significant differences in the composi-
degrade cellulose and other polysaccharides and are, there- tions and structures of soil microbial communities of three
fore, important to the ecosystem’s carbon cycle (Eichorst peanut fields, each having a different monoculture history,
et al. 2011). Growing the same crop year after year lowers and the number of unique bacterial OTUs declined over
the soil pH (Lauber et al. 2009), which is probably less con- time during vanilla monoculturing (Zhao et al. 2014).
ducive to microbial activity; therefore, the carbon cycle in To conclude, intercropping P. ternata with maize
the P. ternata–maize intercropping system may be more effi- affected the relative abundances of the different genera
cient. Bacillus is a facultative anaerobic autogenous nitro- in the microbial community of the rhizosphere more than
gen-fixing bacterium (McSpadden Gardener 2004), and its the composition, and the former was greatly increased as
relative abundance under sole-cropped P. ternata conditions a result of intercropping: no-rank Acidobacteria and no-
was greater than under intercropped P. ternata conditions, rank Anaerolineaceae were significantly more abundant.
which are, therefore, more conducive to nitrogen fixation. Therefore, we believe that P. ternata–maize intercropping
The added nitrogen may be exploited by Anaerobacteria will use soil nutrients more efficiently. More generally, the
which, under anaerobic conditions, degrade carbohydrates diversity, function, and application of rhizosphere micro-
and participate simultaneously in C, N, Fe, and S cycles, organisms to medicinal plants deserves greater attention,
thereby interacting with nitrogen-fixing bacteria to make and associated research will provide valuable insights
the soil more fertile (Zhang et al. 2019). Several soil factors into the roles of microbes in promoting plant growth and
are associated with unique bacterial communities, and the increasing the accumulation of active compounds in plant
ammonium nitrogen ­(NH4-N) content of the soil may be the tissues.

13
3 Biotech (2021) 11:469 Page 9 of 10 469

Supplementary Information The online version contains supplemen- PINELLIA TUBER (dried rhizome of Pinellia ternata) as crude
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 13205-0​ 21-0​ 3011-3. drug. J Oleo Sci 63:127–135
Jacoby R, Peukert M, Succurro A et al (2017) The role of soil microor-
Author contributions HAN, ZZ, and SS designed the study; HAN ganisms in plant mineral nutrition—current knowledge and future
wrote the MS; HAN, AHK, UA, MH, MA, and MMA helped create directions. Front Plant Sci 8:1617
diagrams; HAN, MZ, UA, HB, AHK, MH, MMA, MH, and SS revised Ji X, Huang B, Wang G, Zhang C (2014) The ethnobotanical, phy-
the MS; SS supervised All the authors have read and agreed to the tochemical and pharmacological profile of the genus Pinellia.
published version of the manuscript. Fitoterapia 93:1–17
Juneidi S, Gao Z, Yin H et al (2020) Breaking the summer dormancy
of Pinellia ternata by introducing a heat tolerance receptor-like
Funding This work was supported by the National Key Research and
kinase ERECTA gene. Front Plant Sci 11:780
Development Project of China [No.2017YFC1700700].
Kennedy AC, Smith KL (1995) Soil microbial diversity and the sus-
tainability of agricultural soils. Plant Soil 170:75–86
Declarations Kim Y-G, Komakech R, Choi JE et al (2020) Mass production of Pinel-
lia ternata multiple egg-shaped micro-tubers (MESMT) through
Conflict of interest On behalf of all authors, the corresponding author optimized growth conditions for use in ethnomedicine. Plant Cell,
states that there is no conflict of interest. Tissue Organ Cult 140:173–184
Larkin RP, Honeycutt CW, Olanya OM et al (2012) Impacts of crop
rotation and irrigation on soilborne diseases and soil microbial
communities. Sustainable Potato Production: Global Case Stud-
References ies. Springer, Dordrecht, pp 23–41
Lauber CL, Hamady M, Knight R, Fierer N (2009) Pyrosequencing-
Berendsen RL, Pieterse CMJ, Bakker PAHM (2012) The rhizosphere Based assessment of soil pH as a predictor of soil bacterial
microbiome and plant health. Trends Plant Sci 17:478–486 community structure at the continental scale. Appl Environ
Bernard E, Larkin RP, Tavantzis S et al (2012) Compost, rapeseed Microbiol 75:5111–5120
rotation, and biocontrol agents significantly impact soil micro- Li X, Ding C, Zhang T, Wang X (2014) Fungal pathogen accumula-
bial communities in organic and conventional potato production tion at the expense of plant-beneficial fungi as a consequence of
systems. Appl Soil Ecol 52:29–41 consecutive peanut monoculturing. Soil Biol Biochem 72:11–18
Chen L-H, Huang X-Q, Zhang F-G et al (2012) Application of Lin S, Nie B, Yao G et al (2019) Pinellia ternata (Thunb.) makino
Trichoderma harzianum SQR-T037 bio-organic fertiliser sig- preparation promotes sleep by increasing REM sleep. Nat Prod
nificantly controls Fusarium wilt and affects the microbial com- Res 33:3326–3329
munities of continuously cropped soil of cucumber. J Sci Food Liu X, Zhang J, Gu T et al (2014) Microbial community diversities
Agric 92:2465–2470 and taxa abundances in soils along a seven-year gradient of potato
Dang K, Gong X, Zhao G et al (2020) Intercropping alters the soil monoculture using high throughput pyrosequencing approach.
microbial diversity and community to facilitate nitrogen assimi- PLoS One 9:e86610
lation: a potential mechanism for increasing proso millet grain Luo S, Yu L, Liu Y et al (2016) Effects of reduced nitrogen input on
yield. Front Microbiol 11:2975 productivity and ­N2O emissions in a sugarcane/soybean intercrop-
Dariush M, Ahad M, Meysam O (2006) Assessing the land equiva- ping system. Eur J Agron 81:78–85
lent ratio (LER) of two corn [Zea mays L.] varieties intercrop- Ma G, Zhang M, Xu J et al (2020) Transcriptomic analysis of short-
ping at various nitrogen levels in Karaj. Iran J Cen Europ Agri term heat stress response in Pinellia ternata provided novel
7:359–364 insights into the improved thermotolerance by spermidine and
del González-Chávez MCA, Aitkenhead-Peterson JA, Gentry TJ melatonin. Ecotoxicol Environ Saf 202:110877
et al (2010) Soil microbial community, C, N, and P responses Mazzola M (2004) Assessment and management of soil microbial com-
to long-term tillage and crop rotation. Soil Tillage Res munity structure for disease suppression. Annu Rev Phytopathol
106:285–293 42:35–59
Eichorst SA, Kuske CR, Schmidt TM (2011) Influence of plant poly- McSpadden Gardener BB (2004) Ecology of Bacillus and Paenibacil-
mers on the distribution and cultivation of bacteria in the phy- lus spp. in agricultural systems. Phytopathology 94:1252–1258
lum acidobacteria. Appl Environ Microbiol 77:586–596 Moon BC, Kim WJ, Ji Y et al (2016) Molecular identification of the
Gams W (2007) Biodiversity of soil-inhabiting fungi. Biodivers traditional herbal medicines, Arisaematis Rhizoma and Pinel-
Conserv 16:69–72 liae Tuber, and common adulterants via universal DNA barcode
Gombodorj S, Yang MH, Shang ZC et al (2017) New phenalenone sequences. Genet Mol Res 15:1–14
derivatives from Pinellia ternata tubers derived Aspergillus sp. Rastogi G, Sani RK (2011) Molecular techniques to assess microbial
Fitoterapia 120:72–78 community structure, function, and dynamics in the environ-
Hakim S, Naqqash T, Nawaz MS et al (2021) rhizosphere engineer- ment. Microbes and Microbial Technology. Springer, New York,
ing with plant growth-promoting microorganisms for agriculture pp 29–57
and ecological sustainability. Front Sustain Food Syst 5:16 Van Der Heijden MGA, Bardgett RD, Van Straalen NM (2008) The
He Z, Mao R, Dong JE et al (2019) Remediation of deterioration in unseen majority: soil microbes as drivers of plant diversity and
microbial structure in continuous Pinellia ternata cropping soil productivity in terrestrial ecosystems. Ecol Lett 11:296–310
by crop rotation. Can J Microbiol 65:282–295 Vargas Gil S, Meriles J, Conforto C et al (2009) Field assessment of
Ho Y-N, Mathew DC, Huang C-C (2017) Plant-microbe ecology: soil biological and chemical quality in response to crop manage-
interactions of plants and symbiotic microbial communi- ment practices. World J Microbiol Biotechnol 25:439–448
ties. Plant Ecology-Traditional Approaches to Recent Trends. Weinert N, Piceno Y, Ding GC et al (2011) PhyloChip hybridization
InTech, pp 93–119 uncovered an enormous bacterial diversity in the rhizosphere of
Iwasa M, Iwasaki T, Ono T, Miyazawa M (2014) Chemical compo- different potato cultivars: Many common and few cultivar-depend-
sition and major odor-active compounds of essential oil from ent taxa. FEMS Microbiol Ecol 75:497–506

13
469 Page 10 of 10 3 Biotech (2021) 11:469

Welbaum GE, Sturz AV, Dong Z, Nowak J (2004) Managing soil Zhang GH, Jiang NH, Song WL et al (2016) De novo sequencing and
microorganisms to improve productivity of agro-ecosystems. CRC transcriptome analysis of Pinellia ternata identify the candidate
Crit Rev Plant Sci 23:175–193 genes involved in the biosynthesis of benzoic acid and ephedrine.
Xiong W, Li Z, Liu H et al (2015) The effect of long-term continuous Front Plant Sci 7:1–14
cropping of black pepper on soil bacterial communities as deter- Zhang Y, Li Q, Chen Y et al (2019) Dynamic change in enzyme activ-
mined by 454 pyrosequencing. PLoS One 10:e0136946 ity and bacterial community with long-term rice cultivation in
Xu JY, Dai C, Shan JJ et al (2018) Determination of the effect of Pinel- mudflats. Curr Microbiol 76:361–369
lia ternata (Thunb.) Breit. on nervous system development by Zhang H, Zhang Z, Xiong Y et al (2021) Stearic acid desaturase gene
proteomics. J Ethnopharmacol 213:221–22 negatively regulates the thermotolerance of Pinellia ternata by
Xu Y, Wang T, Li H et al (2019) Variations of soil nitrogen-fixing modifying the saturated levels of fatty acids. Ind Crops Prod
microorganism communities and nitrogen fractions in a Robinia 166:113490
pseudoacacia chronosequence on the Loess Plateau of China. Zhao J, Ni T, Li Y et al (2014) Responses of bacterial communities in
CATENA 174:316–323 arable soils in a rice-wheat cropping system to different fertilizer
Xue T, Zhang H, Zhang Y et al (2019) Full-length transcriptome analy- regimes and sampling times. PLoS One 9:e85301
sis of shade-induced promotion of tuber production in Pinellia Zi H, Jiang Y, Cheng X et al (2020) Change of rhizospheric bacterial
ternata. BMC Plant Biol 19:565 community of the ancient wild tea along elevational gradients in
Zamioudis C, Pieterse CMJ (2012) Modulation of host immunity by Ailao mountain, China. Sci Rep 10:1–11
beneficial microbes. Mol Plant-Microbe Interact 25:139–150
Zhang JY, Guo QS, Zheng DS (2013) Genetic diversity analysis of
Pinellia ternata based on SRAP and TRAP markers. Biochem
Syst Ecol 50:258–265
Zhang X, Cai Y, Wang L et al (2015) Optimization of processing
technology of Rhizoma Pinelliae Praeparatum and its anti-tumor
effect. Afr Health Sci 15:101–106

Authors and Affiliations

Hamza Armghan Noushahi1,2 · Zhenxing Zhu1 · Aamir Hamid Khan3 · Umair Ahmed4 · Muhammad Haroon5 ·
Muhammad Asad2 · Mubashar Hussain6 · He Beibei1 · Maimoona Zafar1 · Mohammad Murtaza Alami1 ·
Shaohua Shu1

Hamza Armghan Noushahi Mohammad Murtaza Alami

[email protected] [email protected]
Zhenxing Zhu 1
College of Plant Science and Technology, Huazhong
[email protected]
Agricultural University, Wuhan 430070, China
Aamir Hamid Khan 2
Department of Agronomy, University of Agriculture
[email protected]
Faisalabad, Faisalabad 38000, Pakistan
Umair Ahmed 3
National Key Laboratory of Crop Genetics Improvement,
[email protected]
College of Plant Science and Technology, Huazhong
Muhammad Haroon Agricultural University, Wuhan 430070, China
[email protected] 4
Key Laboratory of Horticultural Plant Biology, Ministry
Muhammad Asad of Education, College of Horticulture and Forestry Sciences,
[email protected] Huazhong Agricultural University, Wuhan 430070, China
5
Mubashar Hussain National Key Laboratory of Crop Genetic Improvement,
[email protected] Huazhong Agricultural University, Wuhan 4300070, China
6
He Beibei Institute of Applied Mycology, College of Plant Science
[email protected] and Technology, Huazhong Agricultural University,
Wuhan 430070, China
Maimoona Zafar
[email protected]

13