Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Seed-borne endophytic Bacillus velezensis LHSB1 mediate

the biocontrol of peanut stem rot caused by Sclerotium
rolfsii
L. Chen , Y.D. Wu, X.Y. Chong, Q.H. Xin, D.X. Wang and K. Bian
Collaborative Innovation Center of Henan Grain Crops, Henan Collaborative Innovation Center of Grain Storage and Security, Henan University of
Technology, Zhengzhou, China

Keywords
Bacillus velezensis, biocontrol, peanut,
sclerotia, Sclerotium rolfsii, seed endophyte,
stem rot.
Correspondence
Liang Chen, Collaborative Innovation Center
of Henan Grain Crops, Henan Collaborative
Innovation Center of Grain Storage and
Security, Henan University of Technology,
Zhengzhou 450001, China.
E-mail: chen_liang.cl@163.com
2019/1679: received 20 August 2019, revised
12 October 2019 and accepted 30 October
2019
doi:10.1111/jam.14508
Abstract
Aims: This study aimed to obtain an antagonistic endophyte against
Sclerotium rolfsii from peanut seeds, evaluate the biocontrol efficacy towards
peanut stem rot and explore its antifungal mechanism against S. rolfsii.
Methods and Results: Thirty-seven endophytic bacteria were isolated from
peanut seeds, six of which exhibited stronger antagonistic activities against S.
rolfsii (inhibition rate, IR of hyphae growth ≥70%). Strain LHSB1, the
strongest antagonistic strain, was identified as Bacillus velezensis. LHSB1
showed 93
8% of radial growth inhibition of S. rolfsii hyphae and exhibited
obvious antagonistic activity against another six pathogenic fungi of peanut.
Pot experiments showed two different LHSB1 treatments both significantly
reduced the disease incidence and severity of stem rot (P < 0
05) compared to
the controls, and the biocontrol efficacy reached 62
6–70
8%, significantly
higher than that of Carbendazim control (P < 0
05). Further analyses revealed
LHSB1 culture filtrate significantly inhibited sclerotia formation and
germination, caused the abnormalities and membrane integrity damage of S.
rolfsii hyphae, which might be the possible mode of action of LHSB1 against S.
rolfsii. Three antifungal lipopeptides bacillomycin A, surfactin A and fengycin
A, were detected in LHSB1 culture extracts by UPLC-ESI-MS, which could be
responsible for the biocontrol activity of LHSB1 against S. rolfsii.
Conclusion: Our results suggested that the seed-borne endophytic B. velezensis
LHSB1 would be a tremendous potential agent for the biocontrol of peanut
stem rot caused by S. rolfsii.
Significance and Impact of the Study: This comprehensive study provides a
candidate endophytic biocontrol strain and reveals its antifungal mechanism
against S. rolfsi. To the best of our knowledge, this is the first time that seed-
borne endophytic B. velezensis was used as the biocontrol agent to control
peanut stem rot.

worldwide, and has become increasingly serious in recent

Introduction
Peanut (Arachis hypogaea L.) is one of the most impor-
tant sources of food, feed and edible oil in the world
(Sobolev et al. 2013). But it is very susceptible to many
diseases, especially soil-borne diseases caused by fungi (Le
et al. 2012a). Of these, stem rot caused by Sclerotium rolf-
sii Sacc. is the most common disease of peanut
years (Bowen et al. 1992; Le et al. 2012a). In China, stem
rot occurs in most peanut-producing areas, with the inci-
dence rate ranging from 10 to 50% and yield losses rang-
ing from 50 to 100% (Chen et al. 2018a); In central
Vietnam, 5–25% of peanut plants were infected by S. rolf-
sii (Le et al. 2012b). Disastrously, S. rolfsii is very difficult
to control, as it has more than 500 plant hosts and

Journal of Applied Microbiology 128, 803--813 © 2019 The Society for Applied Microbiology 803
Seed-borne endophyte biocontrol peanut stem rot
produces amounts of sclerotia which overwinter in the
soil and causes disease in the following season (Mehan
et al. 1994; Le et al. 2018). Sclerotium rolfsii causes serious
diseases in peanut and many other plants, until now the
control methods are limited or not effective (Mehan
et al. 1994; Le et al. 2018). Few resistant cultivars against
S. rolfsii are commercially available (Branch and Brene-
man 2009); applying chemical fungicides causes many
serious problems such as fungicide residue, pathogen
resistance and environmental pollution (Le et al. 2012b).
Hence, we are in urgent need of an effective and eco-
friendly control method against S. rolfsii.
In recent years, using microbial strains to control S.
rolfsii has attracted much attention, several strains were
isolated and showed great potential against S. rolfsii in
peanut or other crops by antagonism, mycoparasitism,
plant growth promotion, fungal cell wall degradation or
induced systemic resistance (ISR) (Kishore et al. 2005;
Rakh et al. 2011; Singh and Gaur 2016; Hirpara et al.
2017; Sahu et al. 2019). For example, Pseudomonas mon-
teilii 9 was reported to decrease the incidence of S. rolfsii
up to 45
5–66
7% in peanut by producing phenazine-1-
carboxylic acid, 4-diacetylphloroglucinol and pyrrolnitrin
(Rakh et al. 2011); Trichoderma virens NBAII Tvs12 was
shown to significantly inhibit the infection of S. rolfsii
towards groundnut through antibiosis and mycopara-
sitism (Hirpara et al. 2017) and Singh and Gaur (2016)
revealed that endophytic Actinomycetes reduced the mor-
tality (42–75%) of chickpea relying on the antagonistic
and plant growth-promoting activities against S. rolfsii;
Kishore et al. (2005) reported that Pseudomonas aerugi-
nosa reduced the mortality of groundnut seedlings by 54–
58% by inhibiting the plant cell wall–degrading enzymes
of S. rolfsii; endophytic Bacillus sp. 2P2 was reported for
its suppressive potential against S. rolfsii by eliciting ISR
of tomato (Sahu et al. 2019).
Plant endophytes can thrive inside plant tissues, there-
fore, they are of particular superiorities in disease sup-
pression, bacteria colonization, plant growth promotion
(Truyens et al. 2015; Afzal et al. 2019). During the past
decades, the isolation and application of biocontrol endo-
phytes from different parts of various plants was widely
reported (Shahzad et al. 2016; Afzal et al. 2019). How-
ever, the endophytic bacteria from plant seeds are less
reported (Shahzad et al. 2016). In this study, we obtained
an efficient antagonistic endophyte LHSB1 against S. rolf-
sii from peanut seeds, investigated the effects of LHSB1
on hyphal growth, sclerotial formation and germination
of S. rolfsii, and revealed the effect of LHSB1 culture fil-
trate on structure and membrane integrity of S. rolfsii
hyphae. We also evaluated the control efficiency of
LHSB1 against S. rolfsii under greenhouse conditions.
This comprehensive study provided a candidate seed-
804 Journal of Applied Microbiology 128, 803--813 © 2019 The Society for Applied Microbiology
L. Chen et al.
borne biocontrol strain and revealed its action mecha-
nisms against S. rolfsii.
Materials and Methods
Pathogens
Sclerotium rolfsii, Aspergillus niger, Diplodia gossypina and
Fusarium oxysporum used in this study were kindly pro-
vided by the Institute of Plant Protection, Henan Acad-
emy of Agricultural Sciences, other fungi were
maintained by College of Bioengineering, Henan Univer-
sity of Technology.
Isolation of endophytic bacteria from peanut seeds
Endophytic bacteria were isolated from peanut seeds by
referring to the method by Gao et al. (2017) with some
modifications. Firstly, skinless peanut seeds were surface
sterilized through immersion in 75% ethanol for 3 min,
3% sodium hypochlorite for 6 min and 75% ethanol for
30 s, then rinsed three times with sterile water. The effect
of surface sterilization was checked by spreading the final
rinse water (200 ll) on nutrient agar (NA) plates and
culturing at 35°C for 36 h. Then five sterilized peanut
seeds were mashed aseptically with 15 ml sterile PBS buf-
fer, and the mashed sample was diluted and plated onto
NA plates. After culturing for 48 h, colonies of different
morphologies were selected to streak on NA plate to
check the purity. Then the isolate was maintained on a
NA slant at 4°C.
Screening of antagonistic endophytes against S. rolfsii
Plate confrontation method (Shan et al. 2013) was
employed to screening antagonistic strains against S. rolf-
sii from the endophytes. Briefly, S. rolfsii was precultured
on a PDA plate at 28°C for 5 days. Then a 5-mm-diame-
ter plug taken from S. rolfsii was placed at the centre of a
90-mm-diameter PDA plate, and the endophyte
was inoculated to the same plate with 25 mm away from
plate centre. For control treatment, only the S. rolfsii plug
was placed on the PDA plate. All plates were cultured at
28°C until the fungus in control plate grew to full plate.
Antagonistic activity of endophytes was evaluated by the
IR, which was calculated using the formula below:
ðD  5Þ  ðd  5Þ
IRð%Þ ¼  100
D5
where D is the diameter of S. rolfsii that grew in the PDA
plate with the absence of endophyte, d is the diameter of
S. rolfsii with the presence of endophyte, the 5 is the
diameter of the inoculated plug of S. rolfsii. The
L. Chen et al.
endophytes showing the stronger antagonistic activity
against S. rolfsii were selected for further studies.
Bacterial identification
Morphological analysis, physiological analysis and molec-
ular identification were used for endophyte identification.
Morphological and physiological analyses were conducted
according to the previous methods (Chen et al. 2019)
and included analyses of cell shape, colony characteristics,
carbon source utilization, starch hydrolysis, gelatin lignifi-
cation, glucose fermentation, oxidase reactions and
nitrate reduction; molecular identification was carried out
by 16S rDNA sequencing (Chen et al. 2019). Briefly, bac-
terial DNA was extracted using a commercial genome
DNA extraction kit (Tiangen, Beijing, China), and a pair
of universal primers for bacteria (27F, 1492R) was used
for the amplification of 16S rDNA. PCR was performed
in 25 µl volumes under the following conditions: 95°C
for 3 min, 30 cycles at 95°C for 50 s, 56
8°C for 50 s and
72°C for 2 min, followed by a final extension at 72°C for
10 min. Then the PCR product was sent to Sangon Bio-
tech Co., Ltd. (Shanghai, China) for sequencing. The
sequence obtained was compared for homology with the
reference sequences in GenBank using BLASTN 2.10.0+, and
the neighbour-joining phylogenetic tree was constructed
using MEGA X based on the 16s rDNA sequence.
Bacterial culture
The endophyte was firstly cultured for 48 h at 33°C and
175 rev min1 in a 500-ml flask containing 175 ml of
Landy medium (Chen et al. 2018b) to obtain the culture
broth. Then, bacterial cells were removed by centrifuga-
tion at 4°C and 10 000 g for 15 min, the supernatant was
collected. Through filtration with a sterile 0
22-lm mem-
brane, the culture filtrate was obtained and maintained at
4°C for further use.
Pot experiments
To evaluate the efficiency of the seed-borne endophyte to
control peanut stem rot, pot experiments were carried
out under greenhouse conditions. Peanut cultivar HY25,
one of the most widely grown cultivars in China, was
used. Four different treatments including blank control
(sterile water), fungicide control (Carbendazim), LHSB1
treatment A (irrigating seedling root with LHSB1 culture
broth) and LHSB1 treatment B (soaking peanut seed and
irrigating seedling root with LHSB1 culture broth) were
employed. Briefly, peanut seeds were surface sterilized as
described above, then the sterilized seeds were immersed
in sterile water for 4 h at 28°C. For LHSB1 treatment B,
Journal of Applied Microbiology 128, 803--813 © 2019 The Society for Applied Microbiology
Seed-borne endophyte biocontrol peanut stem rot
the seeds were subsequently soaked in LHSB1 culture
broth (5 9 108 CFU per ml) for 30 min, while for other
treatments, the seeds were soaked in sterile water for
30 min instead. Then the seeds of four treatments were
transferred to Petri dishes for germinating 12 h, subse-
quently sown in pots containing 150 g of sterilized sandy
soil. After sowing for 14 days, peanut seedlings with con-
sistent growth were chosen for pot experiments. Two bar-
ley seeds infected by S. rolfsii were inoculated to the base
of peanut seedlings stem. Then each seedling root was
irrigated with 50 ml of respective treatment solutions
(sterile water for blank control, 1000-fold dilution of
50% Carbendazim for fungicide control, LHSB1 culture
broth (5 9 108 CFU per ml) for LHSB1 treatment A and
LHSB1 culture broth (5 9 108 CFU per ml) for LHSB1
treatment B) respectively. Each treatment had 10 pots
and repeated in triplicates. All pots were kept at 28°C in
a sunlight greenhouse and watered regularly. After
28 days, all plants were uprooted, washed in running tap
water to remove soils and investigated for disease inci-
dence.
The disease severity was graded according to the fol-
lowing severity levels, 0 for no stem lesion, 1 for small
stem lesion (<25% of the stem circumference girdled), 2
for moderate stem lesion (26–50% of stem circumference
girdled), 4 for large stem lesion (>51% of stem circum-
ference girdled) and 5 for dead plant (stem completely
girdled) (Fery and Dukes 2002). The disease incidence
rate (DIR), disease index (DI) and control efficacy (CE)
were calculated as follows (Li et al. 2019):
n
DIR ð%Þ ¼  100;
N
P5
i¼1 ðNi  iÞ
DI ¼  100;
N 5
DIck  DIt
CEð%Þ ¼  100
DIck
where N is the total number of investigated plants, n the
number of infected plants, Ni the number of infected
plants at a certain severity level, i the certain severity
level, 5 the highest severity level, DIck is the disease index
in control, DIt = disease index in treatment.
Sclerotia formation and germination inhibition tests
To determine the effect of bacterial culture filtrate on
sclerotia formation of S. rolfsii, bacterial culture filtrate
was mixed with PDA media in the proportion 1 : 4 (v/v)
before pouring into 90-mm-diameter plates. Equivalent
sterile water mixed with PDA media served as control.
After solidification, a 5-mm-diameter plug of S. rolfsii
was inoculated at the centre of the plate and cultured at
28°C. After 30 days, mature sclerotia were picked,
805
Seed-borne endophyte biocontrol peanut stem rot
counted, dried to constant weight under 60°C and
weighed. Each treatment was repeated three times, each
replicate contained 10 plates.
To evaluate the effect of bacterial culture filtrate on
sclerotia germination of S. rolfsii, bacterial culture filtrate
was mixed with PDA media in the proportion 1 : 4 (v/v)
before pouring into 90-mm-diameter plates. Equivalent
sterile water mixed with PDA media served as control.
The sclerotia were surface sterilized through immersion
in 75% ethanol for 3 min, 3% sodium hypochlorite for
6 min, and 75% ethanol for 30 s, then rinsed three times
with sterile water. Then 10 surface-sterilized sclerotia
were transferred to the prepared plate, the number of
germinated sclerotia was recorded and the IR was calcu-
lated after 24, 48 and 72 h. Each treatment was repeated
three times, each replicate contained five plates.
Effect of bacterial culture filtrate on cell membrane
integrity of S. rolfsii
Propidium iodide (PI) staining was employed to assess the
effect of bacterial culture filtrate to cell membrane integrity
of S. rolfsii (Xu et al. 2019). PI, a red fluorescent nuclear
and chromosome dye, is not membrane permeable, mak-
ing it useful to assess cell membrane integrity. Briefly, five
plugs of S. rolfsii were inoculated into a 250-ml flask con-
taining 50 ml of Czapek’s medium. After culturing for
12 h at 28°C, 10 ml fungal broth was centrifuged for
10 min under 4°C and 7500 g, the hyphae were collected,
washed twice with sterile water and resuspended with 1 ml
normal saline. Two hundred microlitres of hyphae suspen-
sion was mixed with 800 µl of enzyme solution containing
10% snailase and 10% cellulose. After exposure for 1 h at
37°C, the hyphae were collected through centrifugation,
washed and resuspended with 1
8 ml bacterial culture fil-
trate. 1
8 ml normal saline served as the control. After
exposure for 6 h at 28°C, the hyphae were collected,
washed and resuspended with 1 ml normal saline. For PI
staining, 20 µl PI solution (1 mg ml1) was added to 1 ml
hyphae suspension to staining in the dark for 30 min.
After that, the hyphae were centrifugated, rinsed and
observed using a confocal laser scanning microscope
(FV3000, Olympus, Tokyo, Japan).
Detection and identification of antifungal metabolites
To explore the antifungal basis against S. rolfsii in the
seed-borne endophyte, an ultra-high liquid chromatogra-
phy (UPLC, Ultimate 3000; Thermo, Bremen, Germany)
coupled with high-resolution electrospray ionization
(ESI) mass spectrometer (MS, Q Exactive Orbitrap,
Thermo) was employed for the detection and identifica-
tion of antifungal metabolites from the seed-borne
806 Journal of Applied Microbiology 128, 803--813 © 2019 The Society for Applied Microbiology
L. Chen et al.
endophyte (Chen et al. 2018b). Briefly, bacterial culture
filtrate was adjusted to pH 2 by the addition of 6 mol l1
HCl. After precipitation overnight at 4°C, the precipitate
was collected by centrifugation (4°C, 12 000 g, 15 min)
and extracted with methanol three times. Methanol
extracts were collected, filtered and evaporated to one-
tenth under vacuum. Then 5 ll of the above aliquot was
injected to UPLC system (C18 column, 100 9 2
1 mm,
1
7 µm) for separation, monitoring at 205 nm with a
flow rate of 300 ll min1. Water (A) and acetonitrile
(B), both containing 0
1% Formic acid (v/v), were used
as mobile phases, elution gradient was 95%A/5%B to 5%
A/95%B within 60 min. The following MS operational
parameters referred to the previous study (Chen et al.
2018b).
Statistical analysis
Each experiment was carried out at least three times. All
data were expressed as mean  SD and analysed by one-
way analysis of variance at the 5% level. Statistical differ-
ences between treatments were analysed by Duncan’s
multiple-range test at 5% significance level.
Results
Selection of antagonistic endophytes against S. rolfsii
A total of 37 endophytic bacteria were isolated from pea-
nut seeds, six of those showed stronger antagonistic activ-
ity in vitro against S. rolfsii (IR ≥70%) (Fig. 1). Among
the six, strain LHSB1 was most effective in suppressing
the radial growth of S. rolfsii hyphae with a 93
8% IR,
and exhibited a 20-mm-width inhibition zone against S.
rolfsii (Fig. 1). Additionally, as shown in Table 1, LHSB1
also exhibited significant antagonistic activity against
another six pathogenic fungi which also cause the
destructive diseases of peanut (Pal et al. 2014), indicating
LHSB1 has the great potential in the control of peanut
fungal diseases.
Identification of the seed-borne endophyte LHSB1
The cells of strain LHSB1 are rods, Gram-positive, pre-
sent singly or in pairs, with central spore. On NA plate,
LHSB1 forms creamy white and surface rough colonies
with irregular edge. Strain LHSB1 is chemo-organ-
otrophic, produce oxidase and catalase, hydrolyse starch,
gelatin, casein and blood, and produce acids with fruc-
tose, glucose, maltose, mannitol, mannose, ribose,
sucrose. The 16S rDNA sequence of strain LHSB1
(1449 bp) was obtained and deposited in GenBank (ac-
cession number MN044879), showing 99
72% homology
L. Chen et al. Seed-borne endophyte biocontrol peanut stem rot

(a) (b)

100
(c)

80
Inhibition rate (%)

60

40

20

0
LHSB1 LHSB4 LHSY2 LHSP3 LHSH7 LHSK2

Figure 1 Selection of antagonistic endophytes from peanut seeds against Sclerotium rolfsii. (a) Without the inoculation of LHSB1, Sclerotium rolf-
sii overgrew the PDA plate. (b) In the presence of LHSB1 inoculation, the radial growth of S. rolfsii was significantly inhibited, showing an obvious
inhibition zone between fungal colony and bacterial colony. (c) Six peanut endophytes, showing stronger antagonistic activity in vitro against S.
rolfsii ( ) inhibition rate ≥70%), were obtained. Error bar represents the standard error of means of replicates. [Colour figure can be viewed at
wileyonlinelibrary.com]

to Bacillus velezensis strain Y9 (GenBank accession num-
ber MH394318) and 99
93% homology to B. velezensis
strain CBv_BE1 (GenBank accession number
MH788975). Based on the 16s rDNA sequence, a neigh-
bour-joining phylogenetic tree was constructed, clearly
showing strain LHSB1 was closely related to B. velezensis
strains (Fig. 2). Collectively, the seed-borne endophyte
LHSB1 should be identified as a member of B. velezensis.
Biocontrol of peanut stem rot under greenhouse
conditions
As shown in Table 2, compared to sterile water control
and fungicide control, the two LHSB1 treatments signifi-
cantly reduced both the disease incidence and severity of
peanut stem rot (P < 0
05). The biocontrol efficacy of
LHSB1 treatment A and B was significantly higher than
that of the fungicide control, reaching 62
6 and 70
8%
respectively (P < 0
05). The results indicated that strain
LHSB1 has great potential to be used as a biocontrol
agent to control peanut stem rot caused by S. rolfsii.
Incidentally, the biocontrol efficacy of LHSB1 treat-
ment B significantly increased by 20
8% compared with
LHSB1 treatment A. It can be inferred that soaking seed
provided an opportunity for the seed-borne endophyte
LHSB1 into the peanut in advance, so as to form early
protection against the diseases. The DIR of LHSB1 treat-
ment B significantly decreased from (60
8  2
4)% to
(41
2  2
2)% compared to LHSB1 treatment A, thereby
verifying the above inference. The result suggested that
soaking seed with LHSB1 culture broth was useful for the
control of peanut stem rot, providing references for the
practical application of the seed-borne endophyte LHSB1
and disease control.

Journal of Applied Microbiology 128, 803--813 © 2019 The Society for Applied Microbiology 807
Seed-borne endophyte biocontrol peanut stem rot L. Chen et al.

Table 1 Inhibition of peanut endophytic bacterium LHSB1 against Table 2 Biocontrol efficacy of the seed-borne endophytic Bacillus
peanut pathogens velezensis LHSB1 on peanut stem rot

Width of Inhibition Disease

inhibition ratio of radial incidence Control
Pathogen (peanut disease) zone (mm) growth (%) Treatments rate (%) Disease index efficacy (%)

Aspergillus flavus (Aspergillus, 11  2
Damping-off)
Aspergillus niger (Crown rot) 13  1
Diplodia gossypina (Collar rot) 10  1
Fusarium oxysporum (Fusarium wilt) 11  2
Fusarium moniliforme (Root rot) 10  1
Rhizopus sp. (Damping-off, Rhizopus) 8  1 65
9
The width of inhibition zone was measured from the edge of fungal
colony to the edge of bacterial colony.
LHSB1 culture filtrate inhibited sclerotia formation and
germination of S. rolfsii
The LHSB1 culture filtrate significantly inhibited the for-
mation of S. rolfsii sclerotia (P < 0
05) (Fig. 3). Com-
pared with the control, the number, dry weight and the
size of sclerotia in the treated group significantly reduced
(P < 0
05), with the reduction rate of 44
9, 51
1, and
30
7% respectively. LHSB1 culture filtrate also effectively
suppressed the germination of S. rolfsii sclerotia
(P < 0
05), the IR of sclerotia germination at 24, 48 and
72 h was 98
4, 97
2 and 96
7% respectively. The IR from
24 to 72 h showed a slight decrease, while there was no
significant difference (P > 0
05). To sum, LHSB1 culture
filtrate could significantly inhibit sclerotia formation and
germination of S. rolfsii.
LHSB1 culture filtrate destroyed membrane integrity of
S. rolfsii hyphae
Confocal laser scanning microscope observation showed
that the untreated hyphae of S. rolfsii showed normally
72
9 Sterile water control 100
0  0
0 a 76
6  2
0 a –
Carbendazim control 68
6  3
0 b 41
4  2
5 b 45
9  2
8 a
77
7 LHSB1 treatment A 50
8  2
4 c 31
7  1
7 c 62
6  2
0 b
70
6 LHSB1 treatment B 41
2  2
2 d 21
9  1
3 d 70
8  1
5 c
72
9
70
6 Values are the mean  SD. Different lowercase letters in the same
column indicated a significant difference between the treatments
(P < 0
05).
distributed branching, uniform thickness and even sur-
faces, while the treated hyphae turned to distorted
branching, nonuniform thickness, deformity and obvious
swelling at the top under the action of LHSB1 culture fil-
trate (Fig. 4), indicating LHSB1 culture filtrate caused the
abnormalities of S. rolfsii hyphae. Furthermore, PI stain-
ing showed that obvious red fluorescence was observed in
the treated hyphae of S. rolfsii, while red fluorescence was
absent in the untreated hyphae (Fig. 4), indicating that
membrane integrity in the treated hyphae was destroyed
by the action of LHSB1 culture filtrate. Collectively,
LHSB1 culture filtrate resulted in the abnormalities of S.
rolfsii hyphae and destroyed membrane integrity of S.
rolfsii hyphal, which might be the possible mode of action
of the seed-borne endophytic B. velezensis LHSB1 against
S. rolfsii.
Antifungal metabolites from the seed-borne endophyte
By using UPLC-ESI-MS, three kinds of antifungal
lipopeptides (bacillomycin L, surfactin A and fengycin A)
were successfully found in the culture extracts of LHSB1
culture broth. As shown in Fig. 5, ions of m/z values
1021
5166, 1035
5329, 1049
5495 were detected,

98 Strain LHSBI (MN044879)

100 Bacillus velezensis strain Y9 (KY887761)
100 Bacillus velezensis strain WGB4 (KY962348)

73 Bacillus subtilis strain LKM-BL (KR560044)

Bacillus cereus strain P-25 (GU271135)

66 Bacillus megaterium strain LNL6 (GQ181059)

Bacillus coagulans strain NBRC 12583 (NR_041523)

Paenibacillus polymyxa strain IAM 13419 (NR_037006)

0·02

Figure 2 Neighbour-joining phylogenetic tree of strain LHSB1. Bootstrap values are shown at nodes. Bar indicates 2% sequence variance. [Colour
figure can be viewed at wileyonlinelibrary.com]

808 Journal of Applied Microbiology 128, 803--813 © 2019 The Society for Applied Microbiology
L. Chen et al. Seed-borne endophyte biocontrol peanut stem rot

100 2·0 recent years due to their unique ability of thriving inside
plant tissues, suppressing plant pathogens, improving
bacterial colonization and promoting plant growth (Afzal

Sclerotia diameter (mm)

Sclerotia dry weight (g)

** 1·5
et al. 2019). Several endophytes have also been obtained
Sclerotia number

75
from peanut root, stem or leave and evaluated the bio-
1·0 control activities against peanut pathogens (Luduena
et al. 2012; Wang and Liang 2014). Luduena et al.
** **
50 obtained 263 peanut endophytes from root, stem or
0·5 leave, 78% of them inhibited Sclerotinia minor growth
and 3% inhibited Fusarium solani growth (Luduena et al.
2012); Wang and Liang reported an endophytic Bacillus
25 0·0
amyloliquefaciens strain BZ6-1 from healthy peanut
l

d
plants, which significantly decreased the disease incidence
tro

tro

tro
te

te

te
on

ea

on

ea

on

ea

of peanut bacterial wilt from 84
5 to 12
1% (Wang and

C

Tr

Tr

Tr

Liang 2014). However, the endophytes from peanut seeds

Figure 3 Inhibition effect of LHSB1 culture filtrate on sclerotia forma-
were less reported. In this study, we obtained 37 endo-
tion. Error bar represents the standard error of means of replicates.
phytes from peanut seeds, and 6 of which exhibited
‘**’ represents significant difference at 0
05 level. ( ) number; ( )
dry weight; ( ) diameter. [Colour figure can be viewed at wileyonline stronger antagonistic potential against S. rolfsii. Strain
library.com] LHSB1, the prominently antagonistic B. velezensis isolate,
demonstrated up to 93
8% growth inhibition of S. rolfsii
hyphae in vitro, and achieved 62
6 and 70
8% of control
corresponding to C14-16 bacillomycin L [M + H]+ as pre- efficacy against peanut stem rot by two different LHSB1
viously reported (Roongsawang et al. 2002), and ions of treatments under greenhouse conditions, which were both
m/z values 1043
4989, 1057
5138, 1071
5314 correspond significantly higher than that of fungicide control
to C14-16 bacillomycin L [M + Na]+; ions of m/z values (P < 0
05). This study proved that plant seed-borne
994
6393, 1008
6555, 1022
6712, 1036
6879, 1050
7036, endophytic bacteria could be a good candidate resource
1064
7201 were detected, corresponding to C12-17 sur- for biocontrol strains, utilizing such seed-borne endo-
factin A [M + H]+ as previous reported (Dhanarajan phytic strains offers a feasible and eco-friendly choice to
et al. 2016), and ions of m/z values 1044
6530, 1062
6694, control peanut stem rot caused by S. rolfsii.
1072
6852, 1086
7016 correspond to C14-17 surfactin A Plant endophytes are reported to suppress plant patho-
[M + Na]+; ions of m/z values 1449
7831, 1463
7978, gens and protect plants by multiple mechanisms, for
1477
8133,1491
8289, 1505
8454 were also detected, corre- example, antibiosis by producing various antimicrobial
sponding to C15-19 fengycin A [M + H]+ as previous metabolites (Backman and Sikora 2008; Chen et al. 2019).
reported (Dhanarajan et al. 2016), and ions of m/z values Among these reported metabolites, Bacillus lipopeptides
1499
7956, 1513
8109, 1527
8266 correspond to C17-19 were considered as versatile weapons for plant disease bio-
fengycin A [M + Na]+. control, acting as ‘antagonists’ to inhibit the growth of
Bacillomycin L, surfactin A and fengycin A were both many phytopathogens through their membrane permeabi-
proved to have the strong antifungal activity against a lization, disruption or solubilization (Ongena and Jacques
wide variety of fungi, especially for filamentous fungi 2008; Hazarika et al. 2019). Many research have reported
(Ongena and Jacques 2008; Falardeau et al. 2013; Jiang the excellent antibiosis of Bacillus lipopeptides, for exam-
et al. 2016). The three antifungal metabolites from ple, B. amyloliquefaciens PGPBacCA1 was shown to be
LHSB1 could be responsible for its antifungal activity able to coproduce the lipopeptides surfactin, iturin and
against S. rolfsii. Additionally, the lipopeptides were fengycin, which were responsible for the effective antifun-
revealed to act in a synergistic manner towards fungi, gal effects against S. rolfsii, Sclerotinia sclerotiorum and F.
and the synergistic action effectively enhanced the bio- solani (Torres et al. 2017), while surfactin was proved to
control efficiency against fungi (Ongena and Jacques be responsible for the biocontrol activity of B. subtilis
2008; Luo et al. 2015). It could be inferred that so does SCB-1 against diverse fungi including the genera Sacchari-
LHSB1 act against S. rolfsii. cola, Cochliobolus, Alternaria and Fusarium (Hazarika
et al. 2019). Specially, reports indicated that coproduction
of various lipopeptides in a biocontrol strain may generate
Discussion
a synergistic effect to increase its biocontrol efficacy, sig-
Plant endophytes have been considered as an excellent nificantly broadening the application potential (Luo et al.
resource of biocontrol strains and bio-inoculants in 2015; Kaur et al. 2017). In this study, we found that the

Journal of Applied Microbiology 128, 803--813 © 2019 The Society for Applied Microbiology 809
Seed-borne endophyte biocontrol peanut stem rot L. Chen et al.

DIC PI Merged

Control

Treated

Figure 4 Microscopy analyses of Sclerotium rolfsii hyphae treated with LHSB1 culture filtrate. By using confocal laser scanning microscope, when
under the white light (DIC), the untreated hyphae of S. rolfsii (Control) showed normal distributed branching, uniform thickness and even sur-
faces, while the treated hyphae (Treated) appeared distorted branching, nonuniform thickness, deformity and obvious swelling at the top (black
arrow), indicating the cell membrane integrity in treated hyphae was destroyed by the action of LHSB1 culture filtrate; When under the green
light (PI), the treated hyphae of S. rolfsii (Treated) appeared with obvious red fluorescence, while the untreated hyphae showed absent distribu-
tion of red fluorescence, indicating the cell membrane integrity in treated hyphae was destroyed by the action of LHSB1 culture filtrate. Bar indi-
cates 50 lm. [Colour figure can be viewed at wileyonlinelibrary.com]

endophyte LHSB1 was able to produce three lipopeptides
bacillomycin L, surfactin A and fengycin A, and under the
pressure of LHSB1 cultural filtrate, hyphal growth, sclero-
tia formation and germination of S. rolfsii were signifi-
cantly inhibited, accompanied by the presence of hyphae
abnormalities and membrane integrity damage. We attrib-
uted the results to the antifungal action of the lipopep-
tides, consistent with previous reports. On the other
hand, reports showed that surfactin can trigger ISR in
peanut plants, reducing the disease incidence and severity
of S. rolfsii and providing an enhanced protection against
S. rolfsii (Rodrıguez et al. 2018). Collectively, according to
the reports and our studies, antibiosis elicited by the three
lipopeptides produced by LHSB1 provided an enhanced
protection against S. rolfsii in peanut and extensive appli-
cation. Nevertheless, plant endophytes can also protect
plants against phytopathogens by ISR, plant growth pro-
motion, production of siderophores, ammonia, lytic
enzymes, plant hormones or hydrogen cyanide. We
believe that it is necessary to evaluate the above perfor-
mances of LHSB1 in the future, so as to further explore
its action mechanism and broaden its application.
Sclerotium, the special asexual reproductive structure
of S. rolfsii, is the main reason why S. rolfsii is difficult to
prevent and control, as it is also the principal overwinter-
ing structure and the primary infection source of S. rolfsii
(Mehan et al. 1994; Le et al. 2018). Once conditions are
favourable, sclerotia could germinate, develop to hyphae
and infect the plants (Mehan et al. 1994). Errakhi et al.
(2009) reported that the Streptomyces isolate J-2 exhibits
100% inhibition of sclerotia germination and 80% inhibi-
tion of hyphal growth by the action of its culture filtrate,
while the inhibition of sclerotia formation was not
showed. Li et al. (2019) observed that Penicillium griseo-
fulvum strain CF3 could considerably limit the sclerotia
formation and germination of S. rolfsii through the cul-
ture filtrate, the IRs of sclerotia formation and germina-
tion were 33
1–66
3% and 44
2–100% respectively. While

810 Journal of Applied Microbiology 128, 803--813 © 2019 The Society for Applied Microbiology
L. Chen et al. Seed-borne endophyte biocontrol peanut stem rot

47·83
100
43·26 49·79

8·94 32·57 37·21

50
15·81
1·53 12·53 26·10
7·38 21·09
0
(a) m/z = 50.00–2000.00
1057·5138 1022·6712
100 100

90 90
1035·5329
80 80

70 70 1036·6879
Relative Abundance

Relative Abundance
60 60 1008·6555

50 1043·4989 50
1044·6530
40 1021·5166 40
1058·6694 46·81
Relative Abundance

994·6393
100 30 30
1072·6852
20 1049·5495
1071·5314
20 43·26
10 10 1086·7016
980·6242
0 0
960 980 1000 1020 m/z 1040 1060 1080 1100
50 1000 1020 1040 m/z 1060 1080 1100

15·81
12·43 20·31
0
(b) m/z = 900·00–1100·00
1463·7978
100
90
1477·8133
80
Relative Abundance

70 1491·8289
1499·7956
60
1513·8109
50
1505·8454
40 1527·8266
32·57
100 30
20 1449·7831
34·66
10
0
50 1440 1460 1480
m/z
1500 1520 1540
36·41

43·15 52.10
28·88
0
0 5 10 15 20 25 30 35 40 45 50 55
(c) m/z = 1400·00–1600·00 Time (min)

Figure 5 Detection of antifungal metabolites from strain LHSB1 by UPLC-ESI-MS. (a) The total ion flow spectra of m/z 50–2000; (b) the extracted
ion flow spectra of m/z 900–1100; (c) the extracted ion flow spectra of m/z 1400–1600. The blue ions peaks ( ) in (b) cover the reported
lipopeptide bacillomycin homologues, ions of m/z values 1021
5166, 1035
5329, 1049
5495 correspond to C14-16 bacillomycin L [M + H]+, and
ions of m/z values 1043
4989, 1057
5138,1071
5314 correspond to C14-16 bacillomycin L [M + Na]+ respectively; The yellow ions peaks ( ) in (b)
cover the reported lipopeptide Surfactin homologues, ions of m/z values 994
6393, 1008
6555, 1022
6712, 1036
6879, 1050
7036, 1064
7201
correspond to C12-17 Surfactin A [M + H]+, and ions of m/z values 1044
6530, 1062
6694, 1072
6852, 1086
7016 correspond to C14-17 surfactin
A [M + Na]+ respectively; The red ions peaks ( ) in (c) cover the reported lipopeptide Fengycin, ions of m/z values 1449
7831, 1463
7978,
1477
8133,1491
8289, 1505
8454 correspond to C15-19 fengycin A [M + H]+, and ions of m/z values 1499
7956, 1513
8109, 1527
8266 corre-
spond to C17-19 fengycin A [M + Na]+ respective. [Colour figure can be viewed at wileyonlinelibrary.com]

Cheng et al. (2019) showed that Selenium can reduce the In conclusion, this study described a seed-borne endo-
pathogenicity of S. sclerotiorum by inhibiting sclerotia for- phytic B. velezensis strain LHSB1 from peanut seeds effec-
mation and germination, the IRs of sclerotia number and tive against S. rolfsi, evaluated its biocontrol efficacy under
weight were 43
8–54
6% and 25
7–42
3% respectively. greenhouse conditions and revealed its action mechanism.
Herein, we found that B. velezensis LHSB1 could consid- We found that the LHSB1 treatment not only inhibited
erably inhibit the formation and germination of S. rolfsii hyphae growth, sclerotia formation and germination of S.
sclerotia. The inhibition of sclerotia germination could rolfsii but also brought about the abnormalities and mem-
effectively decrease the infection rate of such disease, brane integrity damage of S. rolfsii hyphae. Furthermore,
while the suppression of sclerotial formation could effec- three antifungal lipopeptides bacillomycin A, surfactin A
tively reduce the overall available inoculum of S. rolfsii and fengycin A, were detected in LHSB1 culture extracts
and re-infection rate (Manjula et al. 2004), indicating the by UPLC-ESI-MS. In a greenhouse experiment, applying
great potential of LHSB1 to control S. rolfsii. the LHSB1 treatments significantly reduced the disease

Journal of Applied Microbiology 128, 803--813 © 2019 The Society for Applied Microbiology 811
Seed-borne endophyte biocontrol peanut stem rot
incidence and severity of peanut stem rot (P < 0
05),
obtaining significantly higher biocontrol efficacy compared
with the fungicide control. In the future, field trials should
be conducted to investigate the biocontrol efficacy of
LHSB1 against peanut stem rot caused by S. rolfsii and to
explore its application strategy.
Acknowledgements
This work was supported by National Natural Science
Foundation Project of China (31401543), National Key
R&D Program of China (2017YFC1600800), National
Top Youth Talent Support Program for Grain Industry
(LQ2016101), Natural Science Foundation Project of
Henan Province (182300410042), National Modern Agri-
cultural Industry Technology System Construction Pro-
gram (CARS-13).
Conflict of Interest
No conflict of interest declared.
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