Biological Control 127 (2018) 70–77
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Biological Control
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Biocontrol potential of Paenibacillus polymyxa against Verticillium dahliae
infecting cotton plants
⁎
Fan Zhang, Xiao-Lin Li, Shui-Jin Zhu , Mohammad Reza Ojaghian, Jing-Ze Zhang
T
⁎
Ministry of Agriculture, Key Lab of Molecular Biology of Crop Pathogens and Insects, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058,
China
A R T I C LE I N FO
A B S T R A C T
Keywords:
Antagonistic activity
Cotton wilt
Biocontrol agent
Electron microscopy
Plant growth promoting rhizobacteria
Verticillium wilt of cotton is an economically important disease caused by V. dahliae throughout the world. To
control this disease, five bacterial strains from cotton-growing fields were screened for their antagonistic activity
against V. dahliae. Interestingly, they were all identified as Paenibacillus polymyxa by testing morphological and
ultrastructural characteristics as well as 16S rRNA sequences and fatty acid composition analysis. The observation of sporulation process revealed that the structure of the endospore coat was composed of three layers
including outer, middle and inner spore coats. Among five strains, ShX301 showed highest antagonistic activity
against spore germination and mycelial growth of V. dahliae. In addition, in vitro experiments showed that
ShX301 had a broad-spectrum antifungal activity against other plant pathogens. The experiments for controlling
Verticillium wilt of cotton in greenhouse demonstrated that inoculation by strain ShX301 reduced disease incidence and severity by 71.4% and 40.3%, respectively. Moreover, ShX301 significantly promoted the growth of
cotton seedling. Assessment of plant growth promotion showed that inoculation by ShX301 promoted cotton
plant growth enhancing aboveground seedling biomass by 45.5% as well as underground root length and biomass by 38.0% and 38.5%, respectively. This is the first report of P. polymyxa application to control Verticillium
wilt of cotton and promoting cotton seedling growth. This study revealed that P. polymyxa ShX301 had high
potential to be used as biocontrol agent and/or plant growth-promoting bacterium in agriculture.
1. Introduction
Cotton (Gossypium hirsutum L.) is a crop of great economic importance in many developing and some developed countries.
Verticillium wilt, caused by Verticillium dahliae Kleb., is one of the most
severe diseases of cotton in China (Cai et al., 2009; Gao et al., 2010).
Verticillium wilt disease is notoriously difficult to control as fungus has
a broad host range and produce the melanized multicellular survival
structure, known as microsclerotia in soils (Heale and Isaac, 1965;
Wilhelm, 1955).
Conventional management for Verticillium wilt of cotton is an integrated management approach including growing resistant cultivars,
cultural practices and management of water and fertilizer (Elzik, 1985).
Although using resistant cultivars is considered as the most effective
and economical mean of controlling this disease, little progress has
been made due to lacking of immune or highly resistant cotton germplasm against this pathogen (Chang et al., 2008). Crop rotation has
been effective against the disease in few studies (Xiao et al., 1998), but
it was not widely chosen due to the limitation of agricultural areas.
⁎
Although several fungicides have been reported being effective against
Verticillium diseases (Talboys,1984; Tian et al., 1998), no fungicide is
currently registered to specifically control this disease in cotton
(Merhan et al., 2009). It is not easy to control Verticillium diseases
because fungicides are not able to affect the pathogen once it enters the
xylem (Emilief and Bartphj, 2006). Moreover, extensive use of fungicides in agriculture systems has raised public concerns over the environment and human health (Dias, 2012). Many researches have focused on biocontrol as a promising alternative for controlling soil-borne
diseases in sustainable and organic agriculture. The results of these
researches revealed that a number of biocontrol fungi such as Trichoderma virens (Hanson, 1999), a number of bacteria such as Pseudomonas
spp. (Oktay and Kemal, 2010), Bacillus spp. (Mansoori et al., 2013) and
Streptomyces spp. (Xue et al., 2016a,b) and several endophytic bacteria
(Li et al., 2012) were able to significantly reduce Verticillium diseases.
More recently, studies have indicated that the plant growth promoting
rhizobacteria (PGPR) promote plant growth and suppress different
plant diseases (Remans et al., 2008; Rocheli et al., 2015). Among them,
endospore-forming PGPRs, especially gram-positive Paenibacilli, Bacilli
Corresponding author.
E-mail addresses: sjzhu@zju.edu.cn (S.-J. Zhu), jzzhang@zju.edu.cn (J.-Z. Zhang).
https://doi.org/10.1016/j.biocontrol.2018.08.021
Received 11 November 2017; Received in revised form 21 August 2018; Accepted 24 August 2018
Available online 25 August 2018
1049-9644/ © 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Biological Control 127 (2018) 70–77
F. Zhang et al.
(rRNA) genes. 16S rRNA gene amplification was carried out with primers 27F and 1492R (Lane, 1991). The amplified products were submitted to the Sangon Biotech Company Limited (Shanghai, China) for
sequencing in both directions. The resulted sequence was analyzed with
the Clustalx1.83 and compared with others in the GenBank database
using BLAST in order to identify the most similar sequences. The sequences of representative strains were submitted to GenBank database
and accession numbers were obtained.
Gas chromatographic (GC) analysis of whole-cell fatty acid methyl
esters (FAME) were performed for antagonistic strains by growing the
cells at 28 °C for 24 h on TSA plates as described by (Jeon et al., 2003).
The fatty acid composition was analyzed with the Sherlock system
following the protocol of the Microbial Identification System with the
TSBA6 6.0 library. The similarity index for strains was generated based
on fatty acid data.
The endospore formation was observed using a JEM-1010 transmission electron microscope (JEOL USA Inc., Peabody, MA, USA) once
isolates were identified as endospore-forming bacteria. For this purpose, the cells were grown on specific spore-forming medium for 48 h at
30 °C (Clermont et al., 2015). The medium pieces containing cells were
fixed with glutaraldehyde (2.5%, vol/vol) in 0.1 M sodium phosphate
buffer (pH 7.0) for 18 h at 4 °C. Subsequently, the samples were postfixed in osmium tetroxide (1%, wt/vol) in the same buffer for 2 h at
20 °C. After dehydrating in a graded ethanol series, samples were embedded in Spurr’s epoxy resin. Ultrathin sections were cut with a
Reichart-Jung Ultracute E ultramicrotome with a diamond knife. Ultrathin sections collected on Formvar coated slot copper grids were
stained with uranyl acetate and lead citrate. To observe sporulation
process, representative strain was cultured under the same condition
but the samples were taken after 30–48 h.
and Streptomycetes, have showed better results than non-endosporeforming PGPRs due to being resistant to heat, drying, radiation and
toxic chemicals (Comasriu and Vivesrego, 2002; Emmert and
Handelsman, 1999).
These PGPR can produce a range of different antibiotics often associated with the ability of the bacterium to prevent proliferation of
plant pathogens. Meanwhile, a number of PGPRs produce enzymes such
as chitinases, cellulases, glucanases, proteases, and lipases which can
lyse a portion of the cell walls of many pathogenic fungi (Majeed et al.,
2015; Remans et al., 2008). However, the mechanisms by which the
PGPR suppresses different types of pathogens differ among species and
strains (Dey et al., 2004; Lucy et al., 2004; Remans et al., 2008). Although a few studies have investigated the biocontrol efficacy and
mechanism of Streptomyces (Xue et al., 2016a, Xue et al., 2016b), there
is no research involved in Paenibacillus against Verticillium wilt of
cotton.
Therefore, the objective of the present study was to screen and
identify the potential microbes that have a highly inhibitory effect
against V. dahliae from continuous cotton grown in the same field over
20 years, to characterize their antagonistic properties, and to determine
their biocontrol efficacy for controlling Verticillium wilt of cotton in
greenhouse. This will provide the potential strains for developing the
effective biocontrol agents of controlling this disease.
2. Materials and methods
2.1. Screening of antagonistic microorganisms
A highly aggressive defoliating isolate of V. dahliae (H6) was provided by Plant Pathology Department, Zhejiang University, China (Sun
et al., 2016). Twenty soil samples were collected from experimental
fields at the Sanyuan Agricultural Experiment Station of North-West
Agriculture and Forestry University, Sanyuan county, Shaanxi province,
China, on 25 November 2014. Moreover, twenty soil samples were
collected from experimental fields of the Institute of Plant Protection,
Hebei Academy of Agriculture and Forestry Sciences, Baoding city,
Hebei province, China, on 14 October 2014. In both experimental
fields, cotton plants had been continuously grown in the fields heavily
infected by V. dahliae for more than 20 years. The soil samples were
serially diluted with sterile distilled water and tested using double layer
technique (DLT). In DLT, 200 μl spore suspension of V. dahliae with
concentration of 106 conidia ml-1 was spread uniformly on the first
layer of Potato Dextrose Agar (PDA), after absorption of the spores on
the media, a second layer of Luria-Bertani Agar (LBA, yeast extract 5 g,
tryptone 10 g, NaCl 10 g, agar 15 g, pH 7.2) was added and then 200 μl
of soil dilutions were spread uniformly on the second layer. Plates were
incubated at 27 °C for 3–4 days until the fungal growth was visible on
the second layer. Antagonistic microbial colonies were chosen on the
bases of the presence of a fungal growth inhibition zone and its size.
The antagonistic microbes were isolated only from the bigger zone of
inhibition for the purpose of use of biocontrol agents. They were purified three times on LBA to ensure purity and stability. The strains
purified were stored in 20% glycerol at −80 °C.
2.3. Antagonism against spore germination and mycelial growth of V.
dahliae
To obtain bacterial strains with highest antagonistic activity, all of
the candidate isolates were tested for their antagonistic effect against
spore germination and mycelial growth to V. dahliae using a disc diffusion method (Passera et al., 2017). For spore germination, 0.5 ml
fungal spore suspension (106 spores/ml) was spread onto the PDA plate
(90 mm diameter) and became dry in a fume hood. A sterile paper disk
(8 mm diameter) containing cell suspension of the strain cultured in a
ZWY-211B rotary shaker at 200 rpm at 30 °C for 12 h was placed onto
the center of a plate. The inhibition of spore germination of tested
fungus was measured as a diameter (mm) three days after incubation at
27 °C. For testing inhibitory efficacy of bacteria against mycelial
growth, the mycelial plugs (8 mm in diameter) were taken from the
edge of colony and placed onto the center of each PDA plate (90 mm
diameter). A sterile paper disk (8 mm diameter) with bacterial suspension as described above was placed 25 mm from tested fungus. The
inhibition of the mycelial growth was measured as a diameter (mm) ten
days after incubation at 27 °C. Each test was repeated three times.
2.4. In vitro antifungal activity assay against other plant pathogens
As described above, antifungal activity of the screened bacterial
stains against five isolates of plant pathogenic fungi including
Sclerotinia sclerotiorum ZH25 from cabbage, Fusarium oxysporum f. sp.
niveum ZH4 from root of watermelon, Botrytis cinerea ZH256 from leaf
of tomato, Colletotrichum truncatum ZH26 from root of soybean,
Rhizoctonia solani (AG 1-1A) HZ36 from rice sheathes and Phytophthora
capsici ZH56 (A2 mating type) from root of bell pepper were assessed
using the disc diffusion assay on PDA. The above-mentioned pathogens
were selected for this test because of their economic importance in
Zhejiang Province. The inhibition of the mycelial growth was measured
as a diameter (mm) after 2–7 days of incubation at 27 °C. Each treatment was set up in triplicates.
2.2. Identification of bacterial strains
To identify the bacterial strains with antifungal activity, they were
cultured on Lysogeny broth (LB) agar (yeast extract 5 g, tryptone 10 g,
NaCl 10 g, agar 15 g, pH 7.0–7.5) at 30 °C for 20–24 h. The bacterial
strains were characterized using the growth pattern, morphology, gram
staining and electron microscopy. The gram reaction was performed as
described by Vincent and Humphrey (1970). Morphology of the cells
was determined by a SU8010 field-emission scanning electron microscope (SEM, Hitachi, Japan) after culturing cells for 12 h in LB at 30 °C
(He et al., 2007).
The isolates were identified by the sequences of 16S ribosomal RNA
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F. Zhang et al.
2.5. Effect of bacterial strains against cotton Verticillium wilt
Table 1
Cellular fatty acid composition (%) of five strains of the genus Paenibacillus.
The seeds of a susceptible cotton (Gossypium hirsutum cv. Ejing-1)
were surface-sterilized with 0.5% NaOCl for 5 min, rinsed three times in
sterile water and sowed into the Rootrainers Seed Starter Kits (RSSKs,
21-cells per RSSK) containing the autoclaved soil inoculated with
sclerotia of V. dahliae. The mixing soil was composed of field soil, peat
and vermiculite (V/V/V = 1:1:1). To obtain abundant sclerotia, a 8-mm
plug of V. dahliae isolate H6 grown on PDA at 25 °C for 6–7 days was
placed in center of a sterile cellophane disk covering on PDA in 9-cm
plates and incubated at 22 °C for 20 days. After harvesting sclerotia, the
autoclaved soil was inoculated with sclerotia. Before sowing, the
number of sclerotia (approximately 80.5 CFU g–1) was determined using
dry sieving plating method (Butterfield, 1977). After sowing, 10 ml of
suspension containing about 108 bacterial cells grown in LB media in a
ZWY-211B rotary shaker at 200 rpm at 28 °C for 20 h was poured into
the soil around seeds. The soil inoculated with bacterial cells or sterile
water but without sclerotia was used as different controls. Each treatment (with three RSSKs) was repeated three times (Supplementary
data). The RSSKs were placed in incubators under a 16:8 h, light:dark,
28:22 °C regime for two weeks, and then moved into a greenhouse with
similar condition.
The disease assessment was carried out 45 days after planting.
Disease severity was assessed for each plant on a 0 to 4 rating scale
according to the percentage of foliage affected by acropetal chlorosis,
necrosis, wilt, and/or defoliation (0 = healthy plant, 1 = 1 to 33%,
2 = 34 to 66%, 3 = 67 to 99%, 4 = dead plant)as described by
(Bejaranoalcazar et al., 1995).
Peak Name* (Fatty
acid)
b
14:0 iso
14:0
b
15:0 iso
b
15:0 anteiso
b
16:0 iso
c
16:1 w11c
a
16:0
b
7:0 iso
b
17:0 anteiso
a
Percent of fatty acid (%)
Means ± SD
ShX301
ShX302
ShX303
HB1
HB2
2.67
1.96
5.32
53.6
8.68
2.35
10.21
1.85
6.56
2.35
1.78
4.95
57.67
9.87
2.16
15.9
1.97
6.5
2.91
2.00
5.61
56.81
10.82
2.17
8.42
2.12
6.85
1.98
1.89
6.8
58.2
9.56
1.07
11.21
2.2
5.97
2.25
1.98
6.01
61.8
8.56
1.27
9.68
2.17
6.27
2.43 ± 0.37
1.92 ± 0.09
5.74 ± 0.71
57.62 ± 2.94
9.50 ± 0.93
1.80 ± 0.49
11.08 ± 2.87
2.06 ± 0.15
6.43 ± 0.33
Cells were harvested after growth on TSA medium for 3 days. Values represent
percentages of the total fatty acid compositions. Data for all strains (ShX301,
ShX302, ShX303, Hb1 and Hb6) were determined in this study under same
culture conditions.
a
Straight-chain saturated (such as 14:0, 14 carbons in the compound and 0
double bonds in the carbon chain).
b
Branched saturated (ISO: a methyl group occurs at the second to the last
carbon in the chain; anteiso: a methyl group occurs at the third to the last
carbon in the chain.).
c
Straight-chain unsaturated (14 carbons and 1 double bonds in the carbon
chain; the “w7c” refers to the 11th carbon from the “omega” end of the chain;
the carboxyl group is located at the “alpha” end).
2612, 2597 and 2584 bits for Paenibacillus polymyxa (GenBank No.
JX849658), P. jamilae (HE981797) and P. peoriae (HM209749), respectively. The three representative sequences (Accession No.
KX458008 from ShX301, KX458009 from ShX302 and KX458010 from
ShX303) were submitted to the NCBI database. Cellular fatty acid
profiles were shown in Table 1, which revealed that iso-C15:0, anteisoC15:0, C16:0, iso-C16:0, anteiso-C17:0 were predominant. All five strains
were identified as P. polymyxa, having a strong match with the MIDI
database (similarity index: 0.780–0.812).
2.6. Plant growth promotion
To evaluate role of antagonistic isolates regarding plant growth
promotion (PGP), a test for PGP was conducted. Cotton seeds (G. hirsutum cv Ejing-1) were treated with 0.5% NaOCl, sowed into the RSSKs
containing the autoclaved soil as described above, and then poured
with 10 ml of suspension with about 108 bacterial cells. They were
placed in incubators as described above and then moved into a greenhouse. The soil inoculated with sterile water was served as controls.
Each treatment (21 seeds per RSSK) was repeated three times. After
45 days of sowing, seedling lengths and their root length as well as their
aboveground and underground fresh and dry weights were determined.
3.3. Structures of the endospore and sporulation process
Transmission electron microscopy (TEM) was used to examine the
structure of endospore of P. polymyxa. The ultrastructural characteristics of the endospores were the same in five isolates grown for 48 h
(Supplementary data). A core structure was located at the center of the
spore and it was surrounded by two distinct layers (Fig. 1G). The cortex
and the spore coat that was similar to P. polymyxa as described by
Dondero and Holber (1957), and also similar to other bacterial spores
(Errington, 1993). However, in the mature endospores, a three-layered
spore coat was recognized (Fig. 1F), which was similar to P. motobuensis
as described by (Iida et al., 2007).
Dondero and Holber (1957) showed that the spore coat of endospores in P. polymyxa was in two layers, which was different from our
results. To illustrate and confirm the coat characteristics of the endospore, sporulation process was observed by TEM in specimens sampled from the culture of ShX301 strain. The ultrastructural micrographs
showed that the forespore was initially formed at terminal position of
the bacterial cell and it was engulfed by a bacterial membrane of the
mother cell (Fig. 1A) as observed in Bacillus spp. (Errington, 1993; Iida
et al., 2007; Ohye and Murrell, 1962; Ohye and Murrell, 1973). Hereafter, the core was surrounded by an electron-transparent layer, cortex
(CX), which was covered by a layer of lighter electron-dense granulate
materials (Fig. B and G), possibly the precursor for inner spore coat
(ISC), and then the spore wall formed beneath the cortex and the
electron-densest layer, middle spore coat (MSC), appeared around the
inner spore coat (Fig. 1C). With few spikes forming from the middle
spore coat (Fig. 1D), middle spore coat became a thick layer and was
covered by the distinctly outer spore coat (OSC) (Fig. 1E). The mature
spore in a sporangium had a three-layered spore coat (Fig. 1F) and the
3. Results
3.1. Isolation and screening of antagonistic isolates
The antagonistic microbes were isolated only from the bigger zone
of inhibition (diameter more than 2.0 cm) for the purpose of use as
biological control agents. A total of five microorganisms were picked
from 40 soil samples. After being purified, preliminary characterization
showed that all of the isolated strains were bacteria. They were designated as ShX301, ShX302 and ShX303 (from Sanyuan county, Shanxi
province) as well as Hb1 and Hb6 (from Baoding city, Hebei province).
3.2. Identification of bacterial isolates
Five strains were grown on LB agar plates and their colonies were all
round, translucent with smooth surfaces and entire margins. Vegetative
cells were rod-shaped, motile, gram-positive, with similar sizes
(0.56–0.6 × 2.8–3.0 µm) based on scanning electron microscopy micrographs (not shown).
Analysis of 16S rRNA genes with the Clustalx 1.83 showed that
sequences of four strains (ShX301, ShX302, Hb1 and Hb6) were identical but there was one base difference in strain ShX303 with the other.
The result of BLAST search showed that the query five strains had a
identities of 99.65%, 99.58%, 99.30% with variable score value of
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Biological Control 127 (2018) 70–77
F. Zhang et al.
Fig. 1. Transmission electron micrographs of
endospore-forming process of Paenibacillus
polymyxa (strain ShX-301) grown on specific
spore-forming medium at 30 °C for 30-48 h.
A. Early stage of sporulation processes. The
forespore is engulfed in the bacterial cell
membrane
(mother
cell
membrane),
Bar = 0.5 µm. B. Electron-dense granules,
possibly the precursor substances for inner
spore coat (ISC), surrounding the spore cortex
(CX). Bar = 1.0 µm. C. The spore wall (W)
forming beneath the spore cortex (CX) and
the middle spore coat (MSC, an electrondense layer) surrounding the inner spore coat
(ISC). Bar = 0.5 µm. D. The spike-like ornamentation (arrowheads) produced from the
middle spore coat (MSC). Bar = 0.5 µm. E.
The spore coat forming three layers, surrounding the pore cortex (CX). OSC: outer
spore coat. Bar = 0.5 µm. F. A mature spore
in a sporangium. Bar = 0.5 µm. G. Free
spores released from sporangia. The presence
of seven or eight characteristic projections on
the middle spore coats give the impressive
star-shaped image. Bar = 2.0 µm.
against the spore germination and mycelial growth of V. dahliae compared to other strains, it was considered to be an effective antagonist.
The strain ShX301 was also deposited at China General Microbiological
Culture Collection Center as CGMCC 11638.
outer coat was an electron-translucent layer covering the entire surface
of the star-shaped middle but the spore coats were not recognized
clearly in some case in the free spores released from sporangia
(Fig. 1G).
3.5. Inhibitory activity of strain ShX301 against other plant pathogens
3.4. Antagonism against spore germination and mycelial growth of V.
dahliae
The antagonistic activity of P. polymyxa ShX301 against five plant
pathogens was assessed using the disc diffusion assay. P. polymyxa
ShX301 showed in vitro antagonistic activity against five tested pathogens (Fig. 2). The test evaluating PIRG values showed that antagonistic
activity of ShX301 against five pathogens was different. The highest
PIRG value (75.0%) was observed against C. truncatum ZH26 (Fig. 2C)
and the least recorded (59.52%) was observed against S. sclerotiorum
ZH25 (Fig. 2E), respectively. The PIRG values was found to be 73.02,
72.22, 70.63 to 69.84% against B. cinerea ZH256 (Fig. 2D), P. capsici
ZH56 (Fig. 2A), R. solani HZ36 (Fig. 2F) and F. oxysporum f. sp. niveum
ZH4 (Fig. 2B), respectively, with no significant differences among them.
Five isolates screened were determined further for their inhibitory
activity against V. dahliae using the disc diffusion assay. All five isolates
showed inhibitory effect against spore germination and hyphal growth
with different efficacy (Table. 2). In the test evaluating inhibition of
spore germination, both ShX301 and Hb6 strains produced the largest
zone diameter with the same size (4.2 ± 0.21 mm) under in vitro
conditions (P < 0.05). However, in the test assessing growth inhibition
of fungal mycelia, isolate ShX301 showed the highest percentage inhibition of radial growth (PIRG) (87% ± 2%) 10 days after inoculation
(P < 0.05). Because ShX301 showed the highest antagonistic activity
3.6. Inhibitory efficacy against cotton Verticillium wilt
Table 2
Antifungal activity of five strains of Paenibacillus polymyxa against V. dahliae.
Bacterial strains
PIRG (%)*
Inhibition zone diameters (mm)
Hb1
Hb6
ShX302
ShX301
ShX303
72
81
56
87
59
35
42
18
42
21
±
±
±
±
±
4
2
3
2
2
c
b
d
a
d
±
±
±
±
±
0.18
0.21
0.31
0.21
0.34
Inoculation tests showed that P. polymyxa ShX301 was able to delay
foliage symptoms and significantly reduce disease incidence and severity. After 22 d, typical foliage symptoms with acropetal chlorosis
were observed on plants inoculated with V. dahliae. In addition, similar
symptoms appeared on plants coinoculated with V. dahliae and P.
polymyxa ShX301 29 days after inoculation. With development of disease, severe Verticillium symptoms with necrosis, wilt, and defoliation
gradually appeared in plants inoculated with V. dahliae (Fig. 3A). By
comparison, symptoms only with chlorosis and necrosis occurred on
plants coinoculated with V. dahliae and P. polymyxa ShX301 (Fig. 2).
b
a
c
a
c
* Percentage Inhibition of radial growth of V. dahliae hyphae 10 days after
inoculation. Means in the columns followed by the same letter(s) are not different significantly at P < 0.05 according to LSD.
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F. Zhang et al.
Fig. 2. In vitro antagonistic activity of P. polymyxa ShX301 against six fungal plant pathogens using disc diffusion assay. A. Phytophthora capsici ZH56; B. Fusarium
oxysporum f. sp. niveum ZH4; C.Colletotrichum truncatum ZH26; D. Botrytis cinerea ZH256; E. Sclerotinia sclerotiorum ZH25; F. Rhizoctonia solani (AG 1-1A) HZ36. Tests
were done in triplicate and representative plates are shown. CK: control.
positive, rod-shaped and endospore-forming bacterium (Ash et al.,
1993). The coat structure of its endospores was previously considered
to have two layers, an electron-dense outermost layer (middle spore
coat) and a closely applied, lighter, inner layer (inner spore coat) in the
cross sections (Dondero and Holber, 1957). However, we found that
coat structure of P. polymyxa endospores from five strains with a distinct geographical and possibly genetic background had three layers
based on observation of sporulation process (Supplementary data),
being similar to that described in P. motobuensis by Iida et al. (2007). In
addition, we re-examined the ultrastructural photographs in previous
literature (Dondero and Holber, 1957) and found that the blurry outer
spore coat (except obvious middle and inner outer spore coat), in fact,
also could be distinguished in Fig. 3 in P. polymyxa. The reason for the
earlier observations of a two-layered spore coat recognized by Dondero
and Holber (1957) might be because of technical limitations of studies
(such as lower magnification and resolution) at that time. These evidences indicate that the spore-forming pattern is the same in P. polymyxa from different original strains. This also hints that the threelayered spore coat is possibly a common feature in genus Paenibacillus
but this observation needs to be confirmed further.
To obtain effective biocontrol strains, we collected the soil samples
from two experimental fields heavily infested by V. dahliae for more
than twenty years in which cotton varieties, with excellent agronomic
traits but poor resistance to the pathogen, have been cultivated continuously for more than twenty years, for screening new resistant
varieties. To our surprise, five strains screened with highly antagonistic
activity all belong to P. polymyxas species. The reasons for this may
reside in the screening criterion and bacterial properties. On the one
hand, it is possible that, although other potential antagonistic microbes
were present in isolated soil, they were neglected due to restrictive
screening criteria. On the other hand, the long-term P. polymyxa-V.
dahliae interaction in the same field may induce differences in the
However, the disease incidence and severity increased in both treatments over time but control plants inoculated only with P polymyxa
ShX301 (Fig. 3C) and sterile water (Fig. 3D) were symptomless. After
45 days of inoculation, the disease incidence and severity on plants
coinoculated with V. dahliae and P. polymyxa ShX301 were 21.4% and
13.5%, respectively. The disease incidence and severity on plants inoculated with only V. dahliae were measured as (92.8%) and (53.8%),
respectively. In addition, a phenomenon that promoted root growth
was observed in the treatment inoculated with ShX301. Inoculation
increased the length of cotton roots (Fig. 3F, left) comparing with
controls (Fig. 3F, right).
3.7. Effect of P. polymyxa on plant growth promotion
In order to confirm and evaluate effectiveness of plant growth
promotion (PGP), inoculation tests were carried out with P. polymyxa
ShX301. The results showed that the phenomenon of root growth
promotion was similar with that observed above in Fig. 3F. The root
lengths and biomass of cotton significantly increased due to inoculation
with ShX301 compared with control plants in both aboveground and
underground parts (Table. 3). In the aboveground part, inoculation
increased seedling dry weight (biomass) by 45.5% (or fresh weight by
60.5%) as compared to the controls. Similarly, inoculation also increased seedling lengths by 12.7% but their differences were not statistically significant (Table. 3). In the underground part, inoculation
with ShX301 led to the enhancement of underground root lengths by
38% and root dry weight (biomass) by 38.5% (or fresh weight by
47.8%).
4. Discussion
P. polymyxa, formerly known as Bacillus polymyxa, is a gram
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Biological Control 127 (2018) 70–77
F. Zhang et al.
Fig. 3. Effect of inoculation with Paenibacillus polymyxa (ShX301 strain) on the disease incidence and severity of Verticillium wilt and growth on Gossypium hirsutum
cv Ejing-1 45 days after inoculation. A. Inoculation with sclerotia of V. dahliae. B. Co-inoculation with sclerotia of V. dahliae and bacterial cells of P. polymyxa ShX301.
C. Inoculation with bacterial cells of P. polymyxa ShX301. D. Inoculation with sterile water. E. Growth promotion of cotton roots comparing left roots from Fig. C with
right roots from Fig. D. Arrowhead: indicating the root origins.
gain a competitive advantage in soil under this cultivation condition, as
showed by strain ShX301, which exhibited a broad-spectrum antagonistic activity against the oomycetes, ascomycetes and basidiomycetes.
P. polymyxa strains have the broad-spectrum antagonistic activity to
be related to the component of antimicrobial compounds secreted by
themselves, which effectively prevented various plant diseases caused
by fungi, bacteria, and nematodes (Timmusk et al., 2009; Weselowski
et al., 2016). Previous studies had shown that P. polymyxa strains were
able to produce two types of peptide antibiotics, such as polymyxins,
polypeptins, gavaserin, saltavalin, and jolipeptin against bacteria and a
series of LI-F antibiotics, gatavalin, and fusaricidins against fungi,
Gram-positive bacteria and actinomycetes (Deng et al., 2011; Lal and
Tabacchioni, 2009). More recently, it was found that P. polymyxa Sb3-1
was able to reduce the growth of V. longisporum directly, and via its
volatiles (Rybakova et al., 2016). Some of these antimicrobial volatiles
have been identified, such as 2-nonanone and 3-hydroxy-2-butan
(Rybakova et al., 2017). However, the variations in the composition of
antagonistic compounds might be primarily due to differences among P.
polymyxa strains (Lal and Tabacchioni, 2009), leading to diverse antagonistic activity against different pathogens. Furthermore, P. polymyxa HKA-15 metabolites effectively prevented the citrus canker
(Mageshwaran et al., 2011), and P. polymyxa GBR-1 was able to
Table 3
Percent differences of various growth parameters between treatment and control due to interaction between Paenibacillus polymyxa (strain ShX301) and
cotton*.
Growth parameters
Control
Seedling length (cm)
Root length(cm)
Seedling fresh weight (g)
Seedling dry weight (g)
Root fresh weight (g)
Root dry weight (g)
30.8
15.2
8.29
1.12
1.36
0.13
±
±
±
±
±
±
1.71
2.18
1.42
0.08
0.39
0.01
Treatment
Percent difference %
34.7 ± 2.27
20.98 ± 2.4 a
13.31 ± 0.86 a
1.63 ± 0.06 a
2.01 ± 0.48 a
0.18 ± 0.01 a
12.7
38.0
60.5
45.5
47.8
38.5
*
Root fresh and dry weight as well as seedling fresh and dry weight (means and
standard deviation; n = 21 seedlings per treatment) of cotton seedlings inoculated with Paenibacillus polymyxa ShX301, harvested 45 days after inoculation. Control: seedlings inoculated with sterile water; Treatment: seedlings inoculated
with
Paenibacillus
polymyxa
ShX301.
Percent
difference = [(treatment − control)/control] × 100%. Means between control
and treatment in the rows with the letter “a” are different significantly at
P < 0.05 according to LSD.
physiological characteristics of natural isolates of the same species,
which may reflect in the selection of subpopulations of P. polymyxa
with different antagonistic activity. Otherwise, P. polymyxa can possibly
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Biological Control 127 (2018) 70–77
F. Zhang et al.
effectively suppressed the tomato root knot formation (Khan et al.,
2008). In this study, the strains of different or same origins (Table. 2)
indicated different antagonistic activity against the same pathogen,
possibly involving in difference of antimicrobial compounds. Although
the structure of the antifungal compounds produced by P. polymyxa
strains examined in this study is still unknown, our study provides a
basis for further research aimed at revealing differences in antagonistic
activity among strains.
In addition, we also found strain ShX301 was also able to markedly
promote cotton seedling growth, being similar to other strains, such as
P. polymyxa Sb3-1 with plant growth promoting and biocontrol properties on the oilseed rape seedlings (Rybakova et al., 2016). Some
studies showed that the PGP mechanisms in P. polymyxa were correlated with its nitrogen fixation, soil phosphorus solubilisation and
producing plant growth regulating hormones such as indole-acetic acid
and cytokinins (Lal and Tabacchioni, 2009; Spaepen et al., 2007) but
exact mechanisms in ShX301 still need to be analyzed.
Our data indicates that strain ShX301 has great potential using as
biocontrol and/or plant growth-promoting bacterium. However, it is
possible that this strain is not in fact the best of the five isolates, since
comparison was only carried out during in vitro assays, which are not
directly comparable to in vivo performance. A few studies indicated that
some isolates against pathogens were ineffective in vitro on media but
they were effectively in an in vivo assay using plants (Essghaier et al.,
2009). Furthermore, although biosafety of P. polymyxa was analyzed
and discussed (Raza et al., 2008) and it had also been used in microbial
pesticide products in the United States (Hydroguard) and Korea (Topseed and NH) (Kabaluk and Gazdik, 2007), there were safety concerns
related to its applications mainly because of the possible hazards connected with its use in biocontrol (Raza et al., 2008). Therefore, further
studies will focus on the in vivo assays, analysis of antifungal component, PGP mechanisms and safety evaluation to gain a greater understanding of the mode of action for development of biocontrol agent.
This will facilitate the long term efforts toward weaning off dependence
on agricultural chemicals.
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CRediT author statement
Fan Zhang: Conceptualization, Methodology, Software, Validation,
Formal Analysis, Investigation, Resources, Data Curation, WritingOriginal Draft, Writing-Review & Editing, Visualization.
Xiao-lin Li: Conceptualization, Methodology, Software, Validation,
Formal Analysis, Investigation, Resources, Data Curation, WritingOriginal Draft, Visualization.
Mohammad Reza Ojaghian: Writing-Review & Editing,
Jing-Ze Zhang*: Conceptualization, Software, Data Curation,
Writing-Original Draft, Writing-Review & Editing, Supervision, Project
Administration, Funding Acquisition.
Shui-Jin Zhu*: Writing-Review & Editing, Supervision, Project
Administration, Funding Acquisition
Acknowledgments
The research was funded by the Special Fund for Agro-scientific
Research in the Public Interest of China (No. 201503109) and the Key
Science and Technology Project of Zhejiang Province (No.
2015C02023).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.biocontrol.2018.08.021.
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