Microorganisms 11 01801 v2

microorganisms
Article
Diversity and Biocontrol Potential of Endophytic Fungi and
Bacteria Associated with Healthy Welsh Onion Leaves
in Taiwan
Jian-Yuan Wang 1,† , Himanshi Jayasinghe 1,† , Yi-Tun Cho 1 , Yi-Chen Tsai 2 , Chao-Ying Chen 1 , Hung Kim Doan 3
and Hiran A. Ariyawansa 1, *
1 Department of Plant Pathology and Microbiology, College of Bio-Resources and Agriculture,

National Taiwan University, Taipei 106319, Taiwan; [email protected] (J.-Y.W.);
[email protected] (H.J.); [email protected] (Y.-T.C.); [email protected] (C.-Y.C.)
2 Hualien District Agricultural Research and Extension Station, Hualien 973044, Taiwan; [email protected]
3 Small Farms & Specialty Crops Advisor, University of California Cooperative Extension, 2980 Washington
Street, Riverside, CA 92504, USA; [email protected]
* Correspondence: [email protected]
† These authors contributed equally to this work.
Abstract: Foliar diseases caused by Stemphylium and Colletotrichum species are among the major biotic
factors limiting Welsh onion production in Taiwan. Owing to concerns about the environment and
the development of pathogen resistance to existing fungicides, biological control using endophytes
is emerging as an eco-friendly alternative to chemical control. The aim of the present study was
to isolate endophytes from healthy Welsh onion leaves and investigate their antagonistic potential
against the major phytopathogenic fungi associated with Welsh onion plants in Taiwan. A total
of 109 bacterial and 31 fungal strains were isolated from healthy Welsh onion leaves and assigned
to 16 bacterial and nine fungal genera using morphological and molecular characterization based
on DNA sequence data obtained from nuclear internal transcribed spacer (nrITS) (fungi) and 16S
rRNA (bacteria). Evaluation of these endophytic isolates for biocontrol activity against leaf blight
Citation: Wang, J.-Y.; Jayasinghe, H.;
pathogens Colletotrichum spaethianum strain SX15-2 and Stemphylium vesicarium strain SX20-2 by dual
Cho, Y.-T.; Tsai, Y.-C.; Chen, C.-Y.; culture assay and greenhouse experiments resulted in the identification of two bacterial isolates
Doan, H.K.; Ariyawansa, H.A. (GFB08 and LFB28) and two fungal isolates (GFF06 and GFF08) as promising antagonists to leaf
Diversity and Biocontrol Potential blight pathogens. Among the four selected isolates, Bacillus strain GFB08 exhibited the highest
of Endophytic Fungi and Bacteria disease control in the greenhouse study. Therefore, Bacillus strain GFB08 was further evaluated to
Associated with Healthy Welsh Onion understand the mechanism underlying its biocontrol efficacy. A phylogenetic analysis based on six
Leaves in Taiwan. Microorganisms genes identified Bacillus strain GFB08 as B. velezensis. The presence of antimicrobial peptide genes
2023, 11, 1801. https://doi.org/
(baer, bamC, bmyB, dfnA, fenD, ituC, mlna, and srfAA) and the secretion of several cell wall degrading
10.3390/microorganisms11071801
enzymes (CWDEs), including cellulase and protease, confirmed the antifungal nature of B. velezensis
Academic Editor: Gary A. Strobel strain GFB08. Leaf blight disease suppression by preventive and curative assays indicated that B.
velezensis strain GFB08 has preventive efficacy on C. spaethianum strain SX15-2 and both preventive
Received: 2 June 2023
and curative efficacy on S. vesicarium strain SX20-2. Overall, the current study revealed that healthy
Revised: 7 July 2023
Accepted: 12 July 2023
Welsh onion leaves harbour diverse bacterial and fungal endophytes, among which the endophytic
Published: 13 July 2023 bacterial strain, B. velezensis strain GFB08, could potentially be used as a biocontrol agent to manage
the leaf blight diseases of Welsh onion in Taiwan.
Keywords: 16S rRNA; antimicrobial peptide genes; curative assay; integrated disease management;
Copyright: © 2023 by the authors. ITS; preventive assay
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons 1. Introduction
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
Welsh onion (Allium fistulosum L.) belongs to the family Alliaceae [1]. This veg-
4.0/).
etable crop is an important cooking ingredient and traditional medicine in several Eastern
Microorganisms 2023, 11, 1801. https://doi.org/10.3390/microorganisms11071801 https://www.mdpi.com/journal/microorganisms

Microorganisms 2023, 11, 1801 2 of 20
countries, including China, Japan, Korea, and Taiwan [2–5]. One of the major Welsh
onion-growing areas in Taiwan is Sanxing Township, in Yilan County, and Welsh onion
in Sanxing is famous for the unique flavour of Welsh onion cultivar grown in this area
named ‘Si-Ji-Cong’. However, Welsh onion cultivation in Sanxing is severely affected by
two major foliar pathogenic fungi: Stemphylium vesicarium and Colletotrichum spp., causing
Stemphylium leaf blight (SLB) and anthracnose, respectively [6]. The current manage-
ment of foliar diseases of Welsh onion mainly relies on fungicides in Taiwan. Fungicides
such as QoIs (Quinone outside inhibitors), SDHIs (Succinate dehydrogenase inhibitors),
demethylation inhibitors, and dicarboximide are widely used on Allium crops in Sanxing to
control foliar diseases, but the amount of fungicide application has continuously increased
over time [6]. In a recent study, Wang et al. reported that foliar pathogens, especially
S. vesicarium strains, showed resistance to fungicides such as strobilurin plus azoxystrobin
and kresoxim-methyl that have been used to control SLB in Taiwan [6]. This scenario has
been observed in many other countries including the USA and Canada [7]. Moreover, ex-
cessive use of certain chemical fungicides has a negative impact on both environmental and
human health. Thus, finding alternative approaches is essential to control foliar diseases of
Welsh onion.
Applying potential biological-control agents (BCAs) is a powerful tool to control plant
pathogens in agricultural systems [8]. The diversity of plant-associated microbes can be
explored to identify new effective microorganisms as BCAs [9,10]. Fungi and bacteria natu-
rally occur as endophytes in plants and have been identified as potential BCA candidates
to control various plant diseases [11,12]. Endophytes generally protect plants by exhibiting
antagonistic behaviour against phytopathogens, which can be direct (hyper-parasitism,
production of antibiotics and lytic enzymes) or indirect (inducing systemic resistance,
competing for space and nutrients) [13–16]. In addition, endophytes can improve plant
growth through various mechanisms such as fixation of biological nitrogen, solubilization
of phosphate and potassium, and production of siderophores [17–19]. Moreover, most
endophytes can synthesize one or several phytohormones such as auxins, cytokinins, and
gibberellins, that can enhance plant growth while moderating the plant hormonal bal-
ance [20–22]. Several studies have provided the groundwork for controlling foliar diseases
of various Allium crops such as onion, garlic, and Welsh onion by using BCAs. For example,
Zapata-Sarmiento et al. reported that the inoculation of Trichoderma asperellum significantly
reduced the disease severity of SLB on onions (A. cepa) [23]. Roylawar et al. showed that
applying Piriformospora indica significantly reduced SLB severity in onions by inducing sys-
temic resistance [24]. Similarly, it has been found that the application of potential BCAs can
suppress Colletotrichum species causing anthracnose in onion. For instance, Galindez et al.
demonstrated that three Trichoderma species exhibited significant antifungal activity against
C. gloeosrioides under in vitro conditions [25].
Allium species are an abundant source of endophytic microorganisms including bac-
teria and fungi with many beneficial properties to plants, such as growth promotion and
pathogen control [26,27]. For instance, Murtado et al. isolated 40 endophytic bacteria from
onions (A. cepa) and among them, bacterial strain BBP5.2 exhibited promising inhibition of
Colletotrichum sp., one of the major foliar pathogens of onions in Brebes, Central Java [28].
In another study, the endophytic bacterium Serratia plymuthica isolated from Chinese leek
(A. tuberosum) significantly reduced the growth of Botryosphaeria dothidea causing apple ring
rot in post-harvest apples [29]. Furthermore, by both in vitro and in vivo experiments, Rat-
nawati et al. demonstrated that three endophytic Trichoderma strains (T1FLS, T3RZR, and
T2RZS) isolated from shallot (A. cepa var agregatum) in the Palu Valley, showed significant
inhibitory activity against Alternaria porri, the pathogen of shallot purple blotch disease [30].
Welsh onion also harbours numerous endophytes that can be used to develop various
BCAs [31–33]. For instance, Sasaki et al. demonstrated that Streptomyces sp. TP-A0569
isolated from Welsh onion stem produced fistupyrone, which significantly inhibited in-
fection by Alternaria brassicicola in Chinese cabbage [34]. In a recent study, Rashad et al.
indicated that endophytic Bacillus amyloliquefaciens isolated from garlic plants together with
arbuscular mycorrhizal fungi can reduce the severity and incidence of white rot of garlic
caused by Sclerotium cepivorum by inducing the activity of defence-related enzymes [35].
Nevertheless, studies related to the use of beneficial endophytes of Welsh onions against
phytopathogens are still limited in Taiwan. Thus, beneficial endophytic microorganisms
with biocontrol potency should be identified so they can be used as an alternative and
eco-friendly method to control phytopathogens of Welsh onions.
Eco-friendly management strategies to control major foliar diseases of Welsh onions
are lacking in Taiwan. In the present study, we hypothesized that cultivable endophytic
microbes associated with leaves of healthy Welsh onion plants may have great potential for
biocontrol potency against emerging phytopathogens of Welsh onions. Thus, experiments
were designed to (i) isolate and identify fungal and bacterial endophytes inhabiting Welsh
onions; (ii) evaluate their potential antagonism against the major leaf blight pathogens
of Welsh onions in vitro and in planta; and (iii) determine the potential mechanisms
underlying disease suppression.
2. Materials and Methods

2.1. Fungal Stains and Plant Materials
Pathogenic fungal strains, Colletotrichum spaethianum strain SX15-2 and Stemphylium
vesicarium strain SX20-2, were isolated during our previous studies from infected Welsh
onion plants with leaf blight symptoms [6]. For inoculation experiments, the healthy and
mature Welsh onion seedlings (70–90 days after planting) were obtained from Welsh onion
fields in Wan-Fu Village, Sanxing, Taiwan.
2.2. Endophyte Isolation

Based on our previous study and preliminary results, two commercial Welsh onion
fields in Sanxing, Taiwan (24◦ 400 50.800 N 121◦ 400 04.900 E and 24◦ 410 36.400 N 121◦ 400 46.200 E)
that were mostly affected by leaf blight fungal pathogens Stemphylium vesicarium and
Colletotrcihum spaethianum were chosen for this study. In total, five samples of healthy Welsh
onion plants of Si-Ji-Cong cultivar at fourth-true-leaf stage, not showing any apparent
disease symptoms, were collected from each field from June to December 2020. The
collected plants were packed immediately into sterilized polyethylene bags and transferred
to the laboratory within 24 h, and stored at 4 ◦ C prior to isolation. Before isolation from
leaves, surface disinfection of leaves was carried out by following the procedure described
by Espinoza et al. [36]. In brief, the leaf samples were washed thoroughly with running tap
water, followed by soaking in 75% ethanol for 30 s and rinsing in sterile distilled water for
one minute [36]. To confirm that the disinfection process was successful, a 0.1 mL aliquot
of the water used for the last washing step was spotted on potato dextrose agar (PDA)
(supplemented with 100 mg/L ampicillin) and nutrient agar (NA) plates, and incubated
under the same conditions in parallel.
Two isolation techniques were performed to isolate endophytic microbes (i) Di-
rect plate impression of tissues: The disinfected leaf tissues were cut into small pieces
(1 cm × 1 cm) and placed on different media (five to six tissue segments on one plate) [37].
(ii) Spread and pour plate technique: The disinfected leaf tissues were macerated using
sterile mortar and pestle and re-suspended in 5 mL of sterile distilled water [37]. Serial di-
lution of the macerated tissue was made up to 10−3 dilution by taking 1 mL of well-shaken
original suspension and adding into 9 mL of sterile distilled water. Aliquots of 100 µL from
each dilution were plated on media. NA and tryptic soy agar (TSA) plates were used for
bacterial endophyte isolation and incubated at 28 ◦ C for five days [38]. PDA and water
agar (WA) plates were used to obtain fungal endophytes and incubated at 25 ◦ C for seven
days [23]. The cultures were monitored every day for the growth of endophytes and each
emerging colony was sub-cultured to NA or PDA, and brought into pure culture by single
colony isolation.
All strains isolated in this study were initially re-inoculated to Welsh onion plants at
the fourth-true-leaf stage to observe whether they caused any visible necrotic lesions on
healthy plant leaves. For each isolate, three replicated plants were used. The isolates that
caused lesions on leaves were removed and were not used for further studies. For bacterial
strains, 20 µL of bacterial suspensions were inoculated on leaves and the inoculated sites
of leaves were wrapped with autoclaved cheesecloth. For fungi, 4 mm mycelium plugs
were cut from the 7-day-old culture and inoculated on leaves. Later, the sites inoculated
with bacterial or fungal strains were wrapped with Parafilm (Bemis® , Neenah, WI, USA)
to retain moisture. All the plants under treatment were placed in sealed plastic boxes to
maintain high humidity, and the cheesecloth and parafilm were removed five days after
inoculation. The plants were grown for 12 days, in the growth chamber at 20–25 ◦ C under
a 16/8 h light/dark photoperiod to promote disease development.
2.3. Identification of Bacterial and Fungal Isolates

For molecular identification of fungal and bacterial isolates, genomic DNA were
extracted using an EasyPure genomic DNA kit (Bioman® , Bioman Scientific Co., Ltd., New
Taipei, Taiwan) following the manufacturer’s protocol. Polymerase chain reactions (PCR)
were performed to amplify 16S rRNA of bacteria and ITS of fungi, using universal barcoding
primer pairs 27F/1492R and ITS4/ITS5, respectively [39,40]. PCR was conducted in 50 µL
microtubes containing 10 ng DNA, 0.8 units Taq polymerase, 1× PCR buffer, 0.2 mL dNTP,
0.3 µL of each primer, and 1.5 mM MgCl2 . The PCR products were checked for the expected
size on 1% agarose gels and sequenced at the Genomics company (New Taipei, Taiwan).
All sequences acquired from this study were preliminarily identified to genus level using
the BLASTn search engine (http://blast.ncbi.nlm.nih.gov, accessed on 4 April 2021) at the
National Center for Biotechnology Information (NCBI).
2.4. Antifungal Activity of Endophyte isolates

2.4.1. In Vitro Antagonistic Assay
Antagonistic activity of the isolated endophytes against the major leaf blight pathogens
of Welsh onion C. spaethianum strain SX15-2 and S. vesicarium strain SX20-2 was evaluated by
dual culture assay. Agar plugs of each pathogen (4 mm diameter) were placed on one side
of PDA. After 24 h of incubation at 25 ◦ C, an endophytic bacterial strain was streaked 4 cm
away from the pathogen disk to evaluate the inhibition efficacy of the bacterial strain [41].
For endophytic fungal strains, the mycelial plug of each fungus was placed 4 cm away
from the pathogen disk [42]. Control plates were prepared with only the pathogen. The
inhibition rate of mycelial growth (IRM) was evaluated using the formula below [6]:
IRM (%) = (Control colony diameter − Treatment colony diameter/Control colony diameter) × 100%
For initial screening, all the non-pathogenic endophytes isolated from Welsh onion
leaves were evaluated for their antagonistic ability with duplicates per treatment. Based
on the outcome of the dual culture assay, the top four strains with the highest inhibitory
activity were selected for the greenhouse pot experiment to evaluate their in planta biocon-
trol ability.
2.4.2. In Planta Antagonistic Assay

Greenhouse experiments were conducted to test the efficacy of the selected biocontrol
candidates on leaf blight pathogens. Bacterial inocula for application on Welsh onions were
prepared by culturing bacterial strains in Luria-Bertani (LB) broth (Himedia® , Mumbai, In-
dia) at 28 ◦ C with 150 rpm shaking overnight and cells were collected by centrifugation (Al-
legra X-13R Centrifuge, Beckman Coulter, Inc., Brea, CA, USA) at 3250 rpm, 25 ◦ C for 10 min.
The supernatant was discarded, and the pellet was re-suspended in sterile distilled water
supplemented with 0.1% carboxymethyl cellulose (Showa Chemical Co., Tokyo, Japan) and
adjusted to OD600 = 1.0 (~1 × 108 cells/mL) using a spectrophotometer [43,44]. For fungal
inocula, strains were cultured on PDA for seven days at 25 ◦ C. Cultures were flooded
with sterile distilled water combined with 0.05% Tween 20 (Sigma-Aldrich Co., St. Louis,
MO, USA), and the resulting suspensions were filtered through sterilized single-layered
cheesecloth with a pore size of 100 µm. Concentrations of the conidial suspensions were
determined using a haemocytometer and adjusted to 106 spore/mL concentration [45,46].
Preparation of the pathogenic inocula of C. spaethianum strain SX15-2 and S. vesicarium strain
SX20-2 was performed following Wang et al. [6]. The spore suspensions of S. vesicar-
ium strain SX20-2 and C. spaethianum strain SX15-2 with 0.05% Tween 20 (Sigma-Aldrich
Co., St. Louis, MO, USA) were filtered through one-layered cheesecloth and adjusted to
5 × 104 spores/mL and 106 spores/mL, respectively [6,47].
Welsh onion plants at the fourth-true-leaf stage were selected for the experiment and
the plant material for the inoculation was prepared following Wang et al. [6]. Welsh onion
plants were sprayed until run-off with a suspension of bacterial isolates (OD600 = 1.0, 30 mL)
and fungal isolates (106 spores/mL, 30 mL) using an airbrush connected to an air compres-
sor (ASAHI Co., Saitama, Japan) at 30 psi. One day after applying biocontrol candidates,
plants were inoculated with 30 mL of a spore suspension of the pathogen (5 × 104 spores/mL
for, S. vesicarium SX20-2 and 106 spores/mL, for C. spaethianum strain SX15-2) following
the same procedure. The suspension of each biocontrol candidate was re-supplied at three
and ten dpi (days after pathogen inoculation). Plants were kept in sealed plastic boxes for
five dpi to boost disease development. Plants inoculated with the pathogen and sterile
distilled water containing 0.05% Tween-20 were used as positive and negative controls,
respectively [48]. Plants were grown at 20–25 ◦ C under natural sunlight in the greenhouse
during the entire process. The inoculated leaves were photographed and recorded at 12 dpi.
Diseased leaf areas were measured using ImageJ software (http://rsbweb.nih.gov/ij/,
accessed on 28 June 2021) and diseased leaf area (DLA) was calculated as follows [6]:
DLA (%) = (Diseased leaf area of the oldest two leaves/The surface area of the oldest two leaves) × 100%
The experiment was repeated in two independent trials with four replicated plants
per treatment.
2.5. Phylogeny-Based Identification of the Bacterial Biocontrol Candidates

To correctly identify the bacterial endophytes with the highest biocontrol potential, a
phylogenetic tree was generated using maximum likelihood (ML). In total, six gene regions
including gyrase subunit A (gyrA), heat-shock protein groEL (groEL), DNA polymerase
III subunit alpha (polC), phosphoribosylaminoimidazolecarboxamide formyltransferase
(purH), RNA polymerase subunit B (rpoB), and 16S rRNA were amplified to show the
phylogenetic relationships of the bacterial endophytes following Rooney et al. and Dun-
lap [49,50]. NCBI BLASTn was initially used to find the closest matches in GenBank, and the
sequences of the closely related matches were downloaded from GenBank following recent
publications [51,52] (Table S3). Multiple sequence alignment was performed using MAFFT
version 7 (https://mafft.cbrc.jp/alignment/server/, accessed on 12 April 2023). The evolu-
tionary model of each gene locus was evaluated using MEGA v. 7.0.26. A ML analysis with
1000 bootstrap replicates was constructed using raxmlGUI v. 1.5b [53]. The resulting phylo-
genetic trees were visualized in FigTree v. 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/,
accessed on 12 April 2023).
2.6. Biocontrol Potential of Bacillus Velezensis GFB08

Out of the four isolates showing promising results during the antagonistic assays, Bacillus
velezensis strain GFB08, which had the highest antagonist potential from the greenhouse
experiment, was selected to further investigate its mechanisms underlying biocontrol efficacy.
2.6.1. Inhibition of Fungal Mycelial Growth by Extracellular Metabolites

In an attempt to understand the mechanism involved in the in vitro interaction, the
secondary metabolites produced by B. velezensis strain GFB08 were extracted and evalu-
ated for their antibiosis effect on the radial growth of C. spaethianum strain SX15-2 and
S. vesicarium strain SX20-2 using a cell-free filtrate assay as described by Jeong et al. [54].
In brief, bacterial isolates were grown in a shaker incubator (28 ◦ C) at 180 rpm for 3 days.
Subsequently, the supernatant was obtained and centrifuged at 4000 rpm for 10 min at
room temperature followed by filtration through a sterile membrane with 0.22 µm pore size
to obtain cell-free culture filtrate. The cell-free filtrate was added to a warm PDA medium
(60 ◦ C) in a fixed ratio (1:1). The PDA medium mixed with LB only was used as the control.
Mycelial plugs of C. spaethianum strain SX15-2 and S. vesicarium strain SX20-2 were placed
in the centre of the agar plate and incubated at 25 ◦ C. After seven days of incubation, the
radial mycelial growth of the pathogens was measured, and the morphological change in
the mycelium was observed under the microscope (Olympus® BX51, Olympus Co., Tokyo,
Japan). IRM was calculated using the same formula as in Section 2.4.1.
2.6.2. Detection of Proteolytic, Cellulolytic, and Chitinolytic Activity

The proteolytic activity was determined using skimmed milk agar (Himedia® , Mum-
bai, India). The bacterial suspension (10 µL) was placed on the medium and incubated at
25 ◦ C for two days. Protease production was identified by the formation of a clear zone
around colonies [55].
The cellulase enzyme activity test was performed using a medium containing 1% pep-
tone, 1% yeast extract, 1% carboxymethyl cellulose, 0.5% sodium chloride, 0.1% monopotas-
sium phosphate, and 1.6% agar (pH 7) [56]. The bacterial suspension (10 µL) was placed on
the centre of the medium and incubated at 28°C. After two days of incubation, the plates
were flooded with Congo red solution (5 mg/mL, Sigma-Aldrich Co., St. Louis, MO, USA).
The clear zone around the colony indicated a positive result for cellulase production.
Chitin detection media was prepared by following the protocol described by Agrawal
and Kotasthane [57]. Colloidal chitin and indicator dye bromocresol purple were combined
to prepare the media for testing chitin production. Plates containing B. velezensis GFB08
were incubated at 28 ± 2 ◦ C for five days. The appearance of colour changes from yellow
to purple nearby the colony showed a positive result for chitinase production.
Five replicates were used for each experiment and each experiment was repeated in
two independent trials.
2.6.3. Analysis of Antibiotic Biosynthesis Genes

B. velezensis strain GFB08 was characterized for the presence of antibiotic biosynthesis
genes (bac, baer, bamC, bmyB, dfnA, fenB, fenD, ituC, ituD, mlna, mycC, and srfAA) using
specific primers as listed in Table S2 [58–62].
2.7. Preventive and Curative Action

To evaluate the preventive and curative action of B. velezensis strain GFB08, the strain
was applied (30 mL, OD600 = 1.0) on Welsh onion plants one day prior (preventive) and
one day after (curative) inoculation with each pathogen (106 spores/mL for C. spaethianum
strain SX15-2 and 5 × 104 spores/mL for S. vesicarium strain SX20-2). Strain GFB08 was
re-applied three and ten dpi following the methods illustrated above. A fungicide mixture
of pyraclostrobin and boscalid (Wonderful® , Sigma-Aldrich Co., St. Louis, MO, USA)
was used as the positive control. Plants inoculated with sterile distilled water containing
0.05% Tween-20 and 0.1% carboxymethyl cellulose (Showa Chemical Co., Tokyo, Japan)
were used as the negative controls. Diseased leaf areas were measured by ImageJ and DLA
was calculated by the methods described above.
The experiment was repeated in two independent trials with four replicated plants
per treatment.
2.8. Statistical Analysis

Statistical analysis was performed with the R statistical software version 4.2.2 [63].
Student’s t-test (α = 0.05) was used to compare the means of pathogen mycelial growth
inhibition by extracellular metabolites of B. velezensis GFB08. Data for dual culture assays,
in planta assays and extracellular enzyme assays were analysed using one-way analysis
of variance (ANOVA), followed by Tukey’s HSD (honestly significant difference) test

(p ≤ 0.05) for mean separation.
3. Results
3.1. Field Survey and Endophyte Isolation
A total of 109 bacterial and 31 fungal strains were isolated from the leaves of healthy
Welsh onion plants. Isolated strains were classified into taxonomic groups based on
DNA sequence data of ITS (fungi) and 16 rRNA (bacteria). Based on the BLASTn results,
endophytic strains were grouped into sixteen bacterial and nine fungal genera. Among the
identified bacterial genera, Bacillus, Burkholderia, and Klebsiella were the most dominant,
Microorganisms 2023, 11, x FOR PEER REVIEW
representing 27%, 19%, and 12% of the total, respectively. Among the fungal isolates,
Chaetomium (30%), Colletotrichum (23%), and Aspergillus (13%) were identified as the most
dominant genera (Figure 1).
Figure 1. Composition of endophytes isolated from healthy Welsh onion leaves at the genus level.
Figure 1. Composition of endophytes isolated from healthy Welsh onion leaves at the g
(A) Proportion of bacterial endophytes. (B) Proportion of fungal endophytes.
(A) Proportion of bacterial endophytes. (B) Proportion of fungal endophytes.
3.2. Dual Culture and Pot Assays for the Selection of Promising BCAs
3.2.1. Dual Culture Assay
To identify the most promising BCAs for further study and to understand their poten-
tial biocontrol mechanisms, several screening experiments were conducted and the strains
without significant biocontrol potential were eliminated. Strains were selected as follows.
Initially, all the strains isolated from Welsh onion leaves were inoculated to healthy Welsh
onion plants at the four-leaf stage to check whether they caused any necrotic lesions. Based
on the initial screening, nine strains were identified as pathogenic isolates as they caused
visible necrotic lesions on healthy leaves; these were excluded from further analysis.
Out of 131 non-pathogenic endophytes, four strains (GFB08, LFB28, GFF06 and GFF08,
Table S1) that exhibited significant inhibitory activity against leaf blight pathogens in dual
culture assay were selected for further investigation. Out of the four strains, Bacillus strains
GFB08 and LFB28 showed the highest inhibitory activity against C. spaethianum strain
SX15-2 by reducing the mycelial growth rate up to 66% and 71%, respectively. Compared
to Bacillus strains, two fungal strains (Fusarium GFF06 and Chaetomium GFF08) exhibited
moderate activity against C. spaethianum strain SX15-2, respectively, reducing the mycelial
growth rate by 59% and 56% (Figure 2A,C). With S. vesicarium strain SX20-2, Bacillus strains
Figure
GFB08 and 1. LFB28
Composition of endophytes
exhibited inhibitory activities of isolated from
63% and 70%, healthywhile
respectively, Welsh
fungalonion l
strains
(A) Fusarium GFF06
Proportion and Chaetomium
of bacterial GFF08 exhibited
endophytes. inhibitory activities
(B) Proportion of 71%endophyt
of fungal and
40%, respectively (Figure 2B,D).
Figure 2. Inhibition of pathogen mycelial growth by biocontrol candidates (dual culture assay). (A,C)
Figure 2. Inhibition
C. spaethianum ofvesicarium
SX15-2. (B,D) S. pathogenSX20-2.mycelial growth
Control, cultures by biocontrol
with pathogen only. Columnscandida
(A,C) C.means
represent spaethianum SX15-2.
of four technical repeats(B,D)
and twoS. vesicarium
biological SX20-2.
repeats and Control,
the vertical cultures w
bars indicate
standard error. Columns with different letters are significantly different according to Tukey’s HSD
umns represent means of four technical repeats and two biological repea
(p ≤ 0.05).
indicate standard error. Columns with different letters are significantly different according to
Tukey’s HSD (p ≤ 0.05).
3.2.2. Disease Suppression under Greenhouse Conditions
The results of the greenhouse study suggested that the application of Bacillus strains
(LFB28 and
3.2.2. GFB08)
Disease and Fusarium
Suppression under GFF06
Greenhousereduced the DLA caused by C. spaethianum strain
Conditions
SX15-2 up toresults
The 52%, of 48%,
the and 62%, respectively
greenhouse study suggested(Figure 3A).
that the The DLAofcaused
application by S. vesicar-
Bacillus strains
ium (LFB28
strain and GFB08)
SX20-2 and Fusarium
decreased up toGFF06
15%, reduced
14%, theandDLA15%caused
afterbytheC. application
spaethianum strain
of Bacillus
SX15-2 up to 52%, 48%, and 62%, respectively (Figure 3A). The DLA caused
(LFB28 and GFB08) and Fusarium GFF06, respectively (Figure 3B). The application of Chae- by S. vesicarium
strain SX20-2 decreased up to 15%, 14%, and 15% after the application of Bacillus (LFB28 and
tomium GFF08 did not show a significant reduction in infection rate compared to the pos-
GFB08) and Fusarium GFF06, respectively (Figure 3B). The application of Chaetomium GFF08
itivedid
control when
not show the plantreduction
a significant was inoculated
in infectionwith
rate C. spaethianum
compared strain SX15-2
to the positive and S. vesi-
control when
carium
thestrain SX20-2.
plant was Basedwith
inoculated on the results of strain
C. spaethianum this inSX15-2
plantaandassay, Bacillusstrain
S. vesicarium GFB08 strain was
SX20-2.
considered the most promising BCA and used for further studies.
Based on the results of this in planta assay, Bacillus GFB08 strain was considered the most
promising BCA and used for further studies.
Figure 3. Disease suppression assay of potential BCAs on foliar pathogens under greenhouse
Figure 3. Disease suppression assay of potential BCAs on foliar pathogens under greenhouse con-
conditions. (A) C. spaethianum strain SX15-2. (B) S. vesicarium strain SX20-2. Un, un-inoculated plants;
ditions. (A) C. spaethianum strain SX15-2. (B) S. vesicarium strain SX20-2. Un, un-inoculated plants;
Cs, plants inoculated with C. spaethianum strain SX15-2 only; Sv, plants inoculated with S. vesicarium
Cs, plants inoculated with C. spaethianum strain SX15-2 only; Sv, plants inoculated with S. vesicarium
strain SX20-2 only. Columns represent means of four technical repeats and two biological repeats
strain SX20-2 only. Columns represent means of four technical repeats and two biological repeats
and the vertical bars indicate standard error. Columns with different letters are significantly different
and the vertical bars indicate standard error. Columns with different letters are significantly differ-
according to Tukey’s HSD (p ≤ 0.05).
ent according to Tukey’s HSD (p ≤ 0.05).
3.3. Identification of Bacillus Biocontrol Candidates
3.3. Identification of Bacillus
Bacillus isolates Biocontrol
(LFB28 Candidates
and GFB08) were further analysed to determine their correct
taxonomic identity. (LFB28
Bacillus isolates Several datasets were organized
and GFB08) to infer
were further phylogenies
analysed of bacterial strains
to determine their correct
based on ML analysis. The strains selected for the phylogenetic analysis were based on
taxonomic identity. Several datasets were organized to infer phylogenies of bacterial
Dunlap [49]. The dataset consisted of 5560 characters including genes encoding gyrA, groEL,
strains
polC,based on ML
purH, rpoB, andanalysis.
16S rRNA.TheA beststrains
scoringselected for isthe
RAxML tree phylogenetic
shown in Figure 4, analysis
with the were
based on Dunlap
likelihood value[49].
of −The dataset consisted
37,688.462084. The ML treeof obtained
5560 characters
from this including
study showedgenes encoding
overall
gyrA, groEL, polC, purH, rpoB, and 16S rRNA. A best scoring RAxML tree is shown in Fig-
topologies of species level relationships in agreement with previous work based on ML [50].
The
ure 4, two the
with promising Bacillus
mostlikelihood value of strains used in this study
−37,688.462084. The formed
ML treea well-supported
obtained fromclade this study
within the clade containing the ex-type strain B. velezensis NRRL B-41580.
showed overall topologies of species level relationships in agreement with previous Therefore, the work
Bacillus strains (LFB28 and GFB08) were identified as B. velezensis.
based on ML [50]. The two most promising Bacillus strains used in this study formed a
well-supported clade within the clade containing the ex-type strain B. velezensis NRRL B-
41580. Therefore, the Bacillus strains (LFB28 and GFB08) were identified as B. velezensis.
Microorganisms2023,
Microorganisms 2023,11,
11,1801
x FOR PEER REVIEW 10 of
of20
20
Figure4.4.Maximum-likelihood
Figure Maximum-likelihood(GTR+G+I
(GTR+G+Imodel)
model)phylogenetic
phylogenetictree
treeof
ofBacillus
Bacillussubtilis
subtilisgroup
groupbased
based
onsix
on sixgenes
genes(16S,
(16S,groEL,
groEL,gyrA,
gyrA,polC,
polC,purH,
purH,and
andrpoB).
rpoB).BS
BSgreater
greaterthan
than70%
70%are
aremarked
markedat atthe
thenodes.
nodes.
Isolatesobtained
Isolates obtainedininthe
thepresent
presentstudy
studyare
areininred,
red,the
theex-type
ex-typesequences
sequencesareareindicated
indicatedininbold,
bold,and
and
registered commercial Bacillus strains are in purple. Bacillus cereus ATCC 14579 was used as the out-
registered commercial Bacillus strains are in purple. Bacillus cereus ATCC 14579 was used as the
group. The scale bar shows the number of estimated mutations per site.
outgroup. The scale bar shows the number of estimated mutations per site.
3.4.Biocontrol
3.4. BiocontrolPotential
PotentialofofB.B.velezensis
velezensisGFB08
GFB08
3.4.1.Effect
3.4.1. EffectofofExtracellular
ExtracellularMetabolites
MetabolitesofofB.B.velezensis
velezensisGFB08
GFB08on
onMycelium
MyceliumGrowth
Growth
Asmentioned
As mentioned previously, velezensis strain
previously, B. velezensis strainGFB08
GFB08showed
showedsignificant
significant inhibitory ef-
inhibitory
effects onmycelium
fects on mycelium growth of both C. spaethianum
growth of spaethianum strain
strain SX15-2 and S.S.vesicarium
SX15-2 and vesicariumstrain
strain
SX20-2
SX20-2(Figure
(Figure2).
2).To
Todetermine
determinewhether
whetherthe thesuppression
suppressionofofthe
thepathogens
pathogenswaswasdependent
dependent
Microorganisms 2023, 11, x FOR PEER REVIEW 11 of 20

on toxic metabolites, culture filtrate of B. velezensis strain GFB08 was assessed for its effects
on mycelium growth of both pathogens. Cell free culture filtrate from B. velezensis GFB08
significantly inhibited
on toxic metabolites, the mycelium
culture filtrate of B. growth
velezensisof bothGFB08
strain C. spaethianum
was assessedstrain
for itsSX15-2
effects and S.
vesicarium strain SX20-2 (Figures 5 and S1). Moreover, hyphae and conidia
on mycelium growth of both pathogens. Cell free culture filtrate from B. velezensis GFB08 of C. spaethi-
anum strain SX15-2
significantly became
inhibited swollengrowth
the mycelium and distorted
of both C. when grown on
spaethianum medium
strain SX15-2 mixed
and with
S. vesicarium strain SX20-2 (Figure 5 and Figure S1). Moreover, hyphae and
filtrate. Unlike C. spaethianum strain SX15-2, hyphae of S. vesicarium strain SX20-2 did not conidia of
C. spaethianum
show strain SX15-2
any significant becameinswollen
difference and distorted
morphology when grown
compared to theoncontrol.
mediumHowever,
mixed cell
with filtrate. Unlike C. spaethianum strain SX15-2, hyphae of S. vesicarium strain SX20-2
free filtrate of B. velezensis strain GFB08 significantly reduced mycelium growth and spore
did not show any significant difference in morphology compared to the control. However,
germination of S. vesicarium strain SX20-2 (Figure S1). The results of the cell free filtrate
cell free filtrate of B. velezensis strain GFB08 significantly reduced mycelium growth and
assay
sporesuggest that of
germination theS. antagonistic
vesicarium strainmechanisms
SX20-2 (Figureof B.S1).
velezensis strain
The results GFB08
of the against C.
cell free
spaethianum strain SX15-2 and S. vesicarium strain SX20-2 may be related
filtrate assay suggest that the antagonistic mechanisms of B. velezensis strain GFB08 against to extracellular
metabolites
C. spaethianum produced by B.and
strain SX15-2 velezensis GFB08.
S. vesicarium strain SX20-2 may be related to extracellular
metabolites produced by B. velezensis GFB08.
Figure 5. The inhibitory effect of cell culture filtrates of B. velezensis GFB08 on colony diameter of
Figure 5. The inhibitory effect of cell culture filtrates of B. velezensis GFB08 on colony diameter of
foliar pathogens. (A) C. spaethianum SX15-2 (Cs). (B) S. vesicarium SX20-2 (Sv). Data are presented as
foliar pathogens. (A) C. spaethianum SX15-2 (Cs). (B) S. vesicarium SX20-2 (Sv). Data are presented as
means and standard error of four technical replicates and two biological repeats. Means labelled with
means and standard error of four technical replicates and two biological repeats. Means labelled
asterisks are significantly different (p < 0.05) compared with the control according to student’s t test.
with asterisks are significantly different (p < 0.05) compared with the control according to student’s
(***, p < 0.001).
t test. (***, p < 0.001).
3.4.2. Extracellular Enzyme Activity of B. velezensis GFB08
3.4.2. Extracellular Enzyme
Hydrolytic enzyme tests Activity of cellulase,
of protease, B. velezensis
and GFB08
chitinase were performed to check
the Hydrolytic
extracellular enzymatic activity
enzyme tests B. velezensis
of ofprotease, and B.
strain GFB08.
cellulase, velezensiswere
chitinase strain performed
GFB08 to
produced protease and cellulase, but not chitinase (Figure S2).
check the extracellular enzymatic activity of B. velezensis strain GFB08. B. velezensis strain
GFB08 producedofprotease
3.4.3. Detection Antibioticand cellulase,
Coding Genes but
in B.not chitinase
velezensis (Figure S2).
GFB08
Specific primer pairs encoding genes for the biosynthesis of dipeptides, lipopeptides,
3.4.3.
and Detection
polyketidesof Antibiotic
(Table Coding
S2) were used toGenes in B.the
determine velezensis
presenceGFB08
of antibiotic biosynthesis
genes of B. velezensis
Specific strainencoding
primer pairs GFB08. The amplification
genes results suggested
for the biosynthesis that B. velezensis
of dipeptides, lipopeptides,
strain GFB08 is able to synthesize antibiotics such as bacillaene, bacillomycin,
and polyketides (Table S2) were used to determine the presence of antibiotic bacilysin,
biosynthesis
difficidin, fengycin, iturin, macrolactin, and surfactin (Figure 6). The presence of genes
genes of B. velezensis strain GFB08. The amplification results suggested that B. velezensis
encoding the above antibiotics might indicate their involvement in the mechanism of
strain GFB08the
suppressing is able
growthtoofsynthesize antibiotics
both C. spaethianum such
strain as bacillaene,
SX15-2 bacillomycin,
and S. vesicarium bacilysin,
strain SX20-2.
difficidin, fengycin, iturin, macrolactin, and surfactin (Figure 6). The presence of genes
encoding the above antibiotics might indicate their involvement in the mechanism of sup-
pressing the growth of both C. spaethianum strain SX15-2 and S. vesicarium strain SX20-2.
Microorganisms
Microorganisms2023, 11,11,
2023, 1801
x FOR PEER REVIEW 12
12ofof2020
Figure 6. Detection of antibiotic biosynthesis genes in B. velezensis strain GFB08. (A) srfAA; (B) ituC;
Figure 6. Detection of antibiotic biosynthesis genes in B. velezensis strain GFB08. (A) srfAA; (B) ituC;
(C) ituD; (D) fenB; (E) fenD; (F) bamC; (G) bmyB; (H) mycC; (I) dfnA; (J) mlna; (K) baer. (L) bac. Lane L,
ituD;Bio
(C)Omic fenB;
(D)100 bp(E)
DNAfenD; (F) bamC;
ladder; Lane(G) bmyB;
1, B. (H) mycC;
velezensis dfnA; 2,
(I) Lane
GFB08; (J)B.mlna; (K) baer.
velezensis (L) bac.
LFB28; LaneLane L,
N, neg-
Omic
ativeBio 100 bp DNA ladder; Lane 1, B. velezensis GFB08; Lane 2, B. velezensis LFB28; Lane N,
control.
negative control.
3.5. Preventive and Curative Action of B. velezensis GFB08
3.5. Preventive and Curative Action of B. velezensis GFB08
The results of preventive and curative activity of B. velezensis strain GFB08 against
The results of preventive and curative activity of B. velezensis strain GFB08 against leaf
leaf blight pathogens showed that the application of strain GFB08 one day prior to patho-
blight pathogens showed that the application of strain GFB08 one day prior to pathogen
gen inoculation reduced disease severity of leaf blight caused by C. spaethianum strain
inoculation reduced disease severity of leaf blight caused by C. spaethianum strain SX15-2
bySX15-2 by upHowever,
up to 58%. to 58%. However, the application
the application of strain
of strain GFB08 oneGFB08 oneinoculation
day after day after inoculation
with the
with the same pathogen did not significantly reduce disease severity (Figure 7A). Both
Microorganisms 2023, 11, x FOR PEER REVIEW 13 of 20
preventive and curative treatments of strain GFB08 on leaves reduced disease severity
same pathogen did not significantly reduce disease severity (Figure 7A). Both preventive
caused by S. vesicarium strain SX20-2 up to 18% and 17%, respectively (Figure 7B). A com-
and curative treatments of strain GFB08 on leaves reduced disease severity caused by
mon fungicide used in Welsh onion fields for foliar pathogens (pyraclostrobin + boscalid)
S. vesicarium strain SX20-2 up to 18% and 17%, respectively (Figure 7B). A common fungicide
wasused
alsointested
Welshto compare
onion fields the efficacies
for foliar of biocontrol
pathogens candidates
(pyraclostrobin and chemical
+ boscalid) fungicide.
was also tested
Applying fungicide,
to compare respectively,
the efficacies reduced
of biocontrol 96% andand
candidates 95%chemical
of disease severityApplying
fungicide. caused by C.
spaethianum
fungicide, strain SX15-2
respectively, and S.96%
reduced vesicarium
and 95%strain SX20-2.
of disease Thecaused
severity results
byof
C.this experiment
spaethianum
suggest
strainthat B. velezensis
SX15-2 strain GFB08
and S. vesicarium exhibits
strain SX20-2. Thepreventive effects
results of this on C. spaethianum
experiment suggest thatwhile
B. velezensis
exhibiting bothstrain GFB08 and
preventive exhibits preventive
curative effectseffects
on S.on C. spaethianum
vesicarium strainwhile exhibiting
SX20-2.
both preventive and curative effects on S. vesicarium strain SX20-2.
Figure 7. Disease suppression by Bacillus velezensis strain GFB08 under greenhouse conditions. (A)
Figure 7. Disease suppression by Bacillus velezensis strain GFB08 under greenhouse conditions. (A)
C. spaethianum strain SX15-2. (B) S. vesicarium strain SX20-2. Un, uninoculated plants; Cs, plants
C spaethianum strain SX15-2. (B) S. vesicarium strain SX20-2. Un, Un, uninoculated plants; Cs, plants
inoculated
inoculated with C. C.
with spaethianumstrain
spaethianum strain SX15-2
SX15-2 only;
only;Sv, Sv,plants
plants inoculated with
inoculated S. vesicarium
with strainstrain
S. vesicarium
SX20-2
SX20-2 only;
only; Re,Re, cell suspension
pathogenic B. velezensis
strainsofwere GFB08
inoculated was
one dayapplied on leaves BCAs;
after potential of Welsh onion
Cur, cellone
suspen-
day prior to inoculation with the pathogen and re-applied two times at three
sion of B. velezensis strain GFB08 was applied on leaves of Welsh onion one day after inoculation and ten dpi; Cur,
withcell suspension
pathogen; Pre, B. velezensis
of cell suspensionstrainofGFB08 was applied
B. velezensis strainonGFB08
leaves was
of Welsh onion
applied onone day after
leaves of Welsh
inoculation with pathogen; Pre, cell suspension of B. velezensis strain GFB08
onion one day prior to inoculation with the pathogen; Fungicide, mixture of pyraclostrobin was applied on leaves of plus
Welshwas
boscalid onion one dayone
applied prior to inoculation
day after pathogen with inoculation.
the pathogen;Columns
Fungicide,represent
mixture ofmeans
pyraclostrobin
of four tech-
plus boscalid was applied one day after pathogen inoculation. Columns represent
nical repeats and two biological repeats and the vertical bars indicate standard error. Columns means of four with
technical repeats and two biological repeats and the vertical bars
different letters are significantly different according to Tukey’s HSD (p ≤ 0.05).indicate standard error. Columns
with different letters are significantly different according to Tukey’s HSD (p ≤ 0.05).
4. Discussion
4. Discussion
Control
Controlof ofplant
plantdiseases
diseases using beneficialmicrobes
using beneficial microbes is is
anan environmentally
environmentally friendly
friendly
andandimportant
important component
componentof of integrated pestmanagement
integrated pest management (IPM).
(IPM). Endophytic
Endophytic microbes
microbes
residing
residing in in host
host plantsare
plants arevaluable
valuable natural
naturalresources
resourcesthat
thatcan be be
can exploited as BCAs
exploited due due
as BCAs
to their beneficial effects on development, growth, and fitness of the host
to their beneficial effects on development, growth, and fitness of the host plant [64,65]. plant [64,65].
Although Welsh onion is an economically important vegetable crop in many countries,
Although Welsh onion is an economically important vegetable crop in many countries,
research exploring its endophytic communities is lacking. In the present study, antagonistic
research exploring its endophytic communities is lacking. In the present study, antagonis-
potential of bacterial and fungal endophytes isolated from healthy Welsh onion leaves were
tic potential
evaluated of
forbacterial and fungal
their antagonistic endophytes
potential against isolated from
major foliar healthyofWelsh
pathogens Welshonion
onion.leaves
were evaluated for their
In the present antagonistic
study, the majoritypotential against
of the fungal major
strains foliar
isolated frompathogens of Welsh
healthy Welsh
onion.
onion leaves belonged to the genus Chaetomium (Figure 1B). Several previous studies
In the that
indicate present study, the
Chaetomium majority
species of the
can occur as fungal strains
endophytes isolated
of Allium from
crops healthy
and show in-Welsh
onion leaves belonged to the genus Chaetomium (Figure 1B). Several previous studies in-
dicate that Chaetomium species can occur as endophytes of Allium crops and show inhibi-
tory activity against plant pathogens. For instance, C. globosum isolated from A. sativum
showed significant inhibitory activity against Fusarium oxysporum, which causes basal rot
hibitory activity against plant pathogens. For instance, C. globosum isolated from A. sativum
showed significant inhibitory activity against Fusarium oxysporum, which causes basal rot in
onion [66]. The second most abundant fungal genus was Colletotrichum, accounting for 23%
of the fungal strains isolated from healthy Welsh onion plants (Figure 1B). Colletotrichum
contains numerous phytopathogenic species and has been reported from various Allium
crops causing anthracnose on leaves and smudge on bulbs worldwide [67,68]. The lifestyles
of Colletotrichum species can be categorized as necrotrophic, hemibiotrophic, latent or qui-
escent, and endophytic [69]. Prusky et al. defined quiescence (latency) as a continued
period in the fungal life cycle in which the pathogen remains dormant within the plant host
before it switches to an active phase [70]. During latency, activity of the pathogen is almost
suspended. The quiescent stage in C. truncatum after inoculation to Capsicum annuum fruit
was reported by Ranathunge et al. to lack apparent symptoms until six dpi [71]. Thus, the
results of the present study suggest that Colletotrichum strains isolated from Welsh onion
plants without symptoms might be related to the quiescent behaviour of the Colletotrichum
species associated with host plants.
Bacillus species account for the majority (27%) of the bacterial strains isolated in the
present study (Figure 1A). Bacillus species have been reported as endophytes of Allium
crops [72,73]. According to Wang et al., B. siamensis isolated from A. sativum bulbs signif-
icantly inhibited the white rot disease caused by Sclerotium cepivorum while promoting
plant growth [16]. In the present study, isolates exhibiting the highest biocontrol potential
against foliar pathogens also belonged to the genus Bacillus. Burkholderia, the second most
predominant bacterial genus, includes approximately 19% of the total endophyte isolates.
Burkholderia species have been isolated as endophytes from various Allium crops [74]. Pelle-
grini et al. indicated that onion seeds inoculated with a consortium of B. ambifaria showed
increased plant height and crop yields [75].
Recent studies have found that strains expressing the best activities in vitro are not
always the strains showing the best results in planta and vice versa [76]. For example,
reports on B. cereus isolate BT8 showed a lack of antagonism to Phytophthora capsici by
in vitro studies, but the same organism suppressed lesion development caused by P. capsici
on cocoa (Theobroma cacao) leaves under field applications [77]. Therefore, in the present
study, we used all four isolates with promising results in vitro for the in planta study to
select the strain with best biocontrol performance for further studies. The result of the
greenhouse assay showed that applying Bacillus strains GFB08, GFB28, and Fusarium strain
GFF06 significantly reduced the disease severity caused by both C. spaethianum strain
SX15-2 and S. vesicarium strain SX20-2 (Figure 3); compared to the other tested isolates,
B. velezensis strain GFB08 showed the highest control efficacy against both C. spaethianum
strain SX15-2 and S. vesicarium strain SX20-2, even though it did not have the highest
inhibition of those pathogens in the dual culture assay. This phenomenon showed that
in vitro and in planta results do not always correlate and reflect disease suppression within
the same levels. Nonetheless, in vitro studies and their results are particularly useful for
identifying likely candidates for biocontrol and for making educated guesses concerning
the mechanisms by which they reduce pathogen damage. Finally, Bacillus strain GFB08,
which showed the highest pathogen control from the greenhouse assay, was selected for
further studies including the mechanisms underlying its bio-controlling efficacy.
In the present study, Bacillus GFB08 strain was identified as B. velezensis in a multi-
gene phylogeny based on 16S, groEL, gyrA, polC, purH, and rpoB gene regions [50]. Dunlap
recommended these six gene regions to determine the species limits of the B. subtilis species
complex, as analysis of the 16S rRNA gene alone is insufficient due to its highly con-
served nature. The B. subtilis species complex includes B. amyloliquefaciens, B. atrophaeus,
B. axarquiensis, B. malacitensis, B. mojavensis, B. sonorensis, B. vallismortis, B. tequilensis, and
B. velezensis [78–85]. Most of these species (endophytic or non-endophytic) are well-known
plant pathogen antagonists. For instance, a recent study found that B. amyloliquefaciens
YN201732, a beneficial endophyte isolated from tobacco seeds controlled the pathogenic
fungus Erysiphe cichoracearum causing powdery mildew in tobacco by inducing defence-
related gene expressions [86]. Another study found that an endophytic B. atrophaeus
strain, DM6120, isolated from Fragaria × ananassa roots produced volatile inhibitory com-
pounds and lytic enzymes to control the strawberry anthracnose pathogen Colletotrichum
nymphaeae [87]. Moreover, B. velezensis endophyte C2, isolated from the crown tissue of a
tomato, significantly reduced Verticillium wilt incidence in tomatoes by secreting antibiotics
and lytic enzymes [88].
One of the best known and most important mechanisms used by BCAs to limit
pathogen invasion in host plant tissues is antibiosis through the production of anti-pathogen
metabolites [89,90]. Strains identified as B. velezensis have been shown to exhibit remarkable
biocontrol activity against phytopathogens due to the production of the lipopeptide group
of antibiotics such as bacillomycin, fengycin, iturin, and surfactin [91]. For example,
B. velezensis isolated from soil was reported to produce surfactin and bacillomycin D
against Colletotrichum gloeosporioides, which caused anthracnose on mangoes (Mangifera
indica) [92,93]. Kim et al. (2021) reported similar findings and identified B. velezensis AK-0,
a BCA against bitter rot caused by C. gloeosporrioides in apples, encoding antimicrobial
genes of bacillaene, bacillomycin, bacilysin, difficidin, iturin, macrolactin, and surfactin [93].
In the same study, Kim et al. further reported that B. velezensis AK-0 expressed higher
levels of ituD and bacD during interaction with pathogenic C. gloeosporrioides and reduced
the disease severity [94]. Based on PCR in the present study, B. velezensis GFB08 encodes
genes of bacillaene, bacillomycin, bacilysin, difficidin, fengycin, iturin, macrolactin, and
surfactin, consistent with recent findings related to B. velezensis (Figure 6). Presence of
these genes indicated that the antagonistic effect might be due to the secretion of certain
antifungal metabolites by B. velezensis strain GFB08 against C. spaethianum strain SX15-2
and S. vesicarium strain SX15-2. However, the presence of these genes does not guarantee
that they are expressed during the interactions with pathogens. Therefore, further studies
based on qRT-PCR should be conducted to check this hypothesis of whether these genes
are expressed during the interaction between pathogen and BCA.
In addition to lipopeptides, B. velezensis is well-known for its production of CWDEs.
For example, Shin et al. demonstrated that B. velezensis HYEB5-6 inhibited the disease devel-
opment of C. gloeosporioides on Euonymus japonicus by producing cellulase and protease [95].
The in vitro enzyme tests in the present study indicated that B. velezensis strain GFB08 could
produce several CWDEs including cellulase and protease (Figure S2). This property can
play an important role in the natural environment, allowing the BCA to degrade the cell
wall material of pathogenic fungi. Therefore, secretion of these enzymes indicated that the
antagonistic effect might also be related to the production of certain CWDEs by B. velezensis
strain GFB08 against C. spaethianum strain SX15-2 and S. vesicarium strain SX15-2.
This is the first study investigating the diversity of bacterial and fungal endophytes
harboured in the leaves of healthy Welsh onions in Taiwan. Moreover, this is the first
report showing the biocontrol efficacy of the naturally occurring endophyte B. velezensis
strain GFB08 in controlling leaf blight fungal pathogens associated with Welsh onions. The
findings of this study are significant because the diversity of Welsh onion endophytes has
not been fully explored and the possibility of employing Welsh onion endophytes as BCAs
against Welsh onion foliar diseases has not been studied before.
5. Conclusions
In the present study, 109 bacterial and 31 fungal endophytic strains were isolated from
healthy Welsh onion leaves in fields with leaf blight diseases. The results indicated that
among the endophyte isolates, two bacterial isolates (GFB08 and LFB28) and two fungal
isolates (GFF06 and GFF08) could significantly inhibit leaf blight pathogens under both
in vitro and in planta conditions. Among the four antagonists tested in the greenhouse
assay, B. velezensis strain GFB08 had the highest control of the disease by reducing the
lesion area caused by Colletotrichum spaethianum strain SX15-2 and Stemphylium vesicarium
strain SX20-2 up to 48% and 14%, respectively. Various mechanisms might be involved
in biocontrol activity against leaf blight pathogens, such as production of antimicrobial
compounds and CWDEs. Taken together, the results of this study reveal that B. velezensis
strain GFB08 can be developed as a BCA to control and manage Welsh onion leaf blight dis-
eases. However, further studies should be carried out under field conditions to evaluate its
biocontrol efficacy, effect on plant growth, influence on indigenous microbial communities
as well as the effect of agronomic practices (chemical fertilizers, pesticides, fungicides, etc.)
on B. velezensis strain GFB08.
Supplementary Materials: The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/microorganisms11071801/s1, Figure S1: The inhibitory effect of cell free
filtrates of B. velezensis GFB08 on mycelium growth and spore germination of C. spaethianum SX15-2 (Cs)
and S. vesicarium SX20-2 (Sv); Figure S2: Production of extracellular enzymes by B. velezensis GFB08;
Table S1: Strain ID and genus names of the biocontrol candidates exhibited promising results in
antagonistic assays; Table S2: Primers used to detect the presence of genes synthesizing different
antibiotics (bacilisyne, bacillaene, bacillomycin, difficidin, fengycin, iturin, macrolactin and surfactin)
in B. velezensis GFB08; Table S3: Bacillus strains and GenBank accession numbers of DNA sequences
used in the phylogenetic study.
Author Contributions: Conceptualization, H.A.A.; methodology, J.-Y.W.; formal analysis, J.-Y.W. and
H.J.; investigation, J.-Y.W. and Y.-T.C.; resources, H.A.A. and Y.-C.T.; data curation, J.-Y.W. and H.J.;
writing—original draft preparation, H.J., H.A.A., C.-Y.C. and H.K.D.; writing—review and editing,
H.A.A., C.-Y.C. and H.K.D.; supervision, H.A.A. and C.-Y.C.; project administration, H.A.A. and
H.K.D. All authors have read and agreed to the published version of the manuscript.
Funding: This research is funded by the Council of Agriculture (COA), Executive Yuan, Taiwan.
(Grant No. 111AS-1.3.2-ST-aN and 112 AS-1.3.2-ST-aF).
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: The authors would like to thank Welsh onion growers in Sanxing, Guan, and
Lin for their kind assistance during field surveys and H.Y. Chen, NTU Plant Teaching Hospital for her
useful discussion and suggestions. The authors appreciate C.H. Wang, I. Tsai, Y.C. Xu for their kind
assistance and Y.C. Chang, R.F. Liu, H. Rho, Y.L. Chen, C.L. Chung and N.C. Lin for their valuable
advice. Hiran Ariyawansa is grateful to A.D Ariyawansa, D.M.K Ariyawansa, Ruwini Ariyawansa,
Amila Gunasekara and Oshen Gunasekara for their valuable suggestions.
Conflicts of Interest: The authors declare no conflict of interest.
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microorganisms

1 Department of Plant Pathology and Microbiology, College of Bio-Resources and Agriculture,

Microorganisms 2023, 11, 1801. https://doi.org/10.3390/microorganisms11071801 https://www.mdpi.com/journal/microorganisms

2. Materials and Methods

2.2. Endophyte Isolation

2.3. Identification of Bacterial and Fungal Isolates

2.4. Antifungal Activity of Endophyte isolates

2.4.2. In Planta Antagonistic Assay

2.5. Phylogeny-Based Identification of the Bacterial Biocontrol Candidates

2.6. Biocontrol Potential of Bacillus Velezensis GFB08

2.6.1. Inhibition of Fungal Mycelial Growth by Extracellular Metabolites

2.6.2. Detection of Proteolytic, Cellulolytic, and Chitinolytic Activity

2.6.3. Analysis of Antibiotic Biosynthesis Genes

2.7. Preventive and Curative Action

2.8. Statistical Analysis

of variance (ANOVA), followed by Tukey’s HSD (honestly significant difference) test

Microorganisms 2023, 11, 1801 11 of 20

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