Introduction
Sclerotinia stem rot
(SSR) caused by Sclerotinia sclerotiorum (Lib.) de Bary is an important disease of
canola causing significant crop losses worldwide (Kamal et al. 2016;
Smolinska and Kowalska 2018) including West Australian (WA)
(Khangura and MacLeod 2012; Khangura et al. 2014, Khangura
and Van Burgel 2021). Strategies to reduce production
losses
in canola usually rely on the application of fungicides (Rimmer et al. 2007; Khangura
and Van Burgel 2021). However, consumer concern on the impact of
chemical fungicides has increased the demand for eco-friendly products
which are relatively free from chemical residues (Raaijmakers
et al. 2002).
Biological control is an alternative approach
for disease management that is environmentally safe and reduces the amount of
human contact with harmful chemicals and their residues. A variety of
biocontrol agents, including fungi and bacteria, have been identified but
further development and deployment is required (Sharma et
al. 2017). The limited availability of commercial BCAs has been a major
constraint to the development of eco-friendly and sustainable disease
management worldwide (Vincent et al. 2007). The key factor in developing effective
and efficient BCAs is the exploration for potential BCAs across agricultural
production regions globally. Therefore, isolation, screening and identification
of local BCAs are needed, including in WA which enforces its own biosecurity
and quarantine Act.
Several investigations have been conducted to
explore potential BCAs from a wide range of niches such as the rhizosphere, phyllosphere, sclerotia and other habitats (Whipps et al. 2008). Research has been conducted to
test the potential of fungal BCAs against S.
sclerotiorum such as the use of antagonistic Coniothyrium minitans to
control SSR disease in some countries (Whipps et
al. 2008; Yang et al.
2011). Some success stories also
include the use of antagonistic Trichoderma
spp. in bean crops against SSR in Brazil (Lopes et al. 2012).
There is an
opportunity to obtain BCAs from local agricultural regions because potential BCAs have already become established with the pathogen
in the ecosystem. However,
the concentration and distribution of potential BCAs in ecosystems can be very limited and therefore research to
discover new and potential BCAs is
invaluable (Lopes et
al. 2012). In WA, this research is the first to explore the potential of BCAs in managing SSR in canola.
Research on sustainable management of SSR on
canola has been conducted including the use of cultural practices (Kharbanda and Tewari 1996) and screening for resistant genotypes (Barbetti et al. 2013; Taylor et al. 2015).
However, due to the quantitative
nature of host resistance, it is very difficult to develop completely resistant
canola genotypes (Li et al. 2006). Currently, no commercial canola
variety in
Australia is
resistant to S. sclerotiorum.
Therefore, disease control mainly relies on the use of
fungicides in combination with cultural practices (Khangura
and McLeod 2012). Control of SSR disease by fungicide alone is less effective
due to mismatch in spraying time and ascospore release (Bolton et al.
2006). Some fungicides gradually
lose efficacy as resistant strains of S. sclerotiorum emerge (Zhang et al. 2003). Decreasing
fungicide efficacy over time has reduced
the chemical
control cost-benefit
ratio, at the same period that concern about environmental impacts from
chemical fungicides has increased. This has led to research on alternative
strategies for controlling S. sclerotiorum on canola. Interest in biological control
of SSR diseases on canola has increased over recent decades
(Saharan and Mehta 2008).
The objectives of this research were: (1) to isolate potential BCAs from WA canola growing areas to control S. sclerotiorum; (2)
to investigate
the efficacy of potential BCAs against S. sclerotiorum and their
ability to reduce sclerotial formation by the pathogen in vitro; (3) to identify the species of potential BCAs; and (4) to
investigate the efficacy of newly identified local F-BCAs and B-BCAs in
controlling SSR disease on canola under WA field conditions.
Materials and Methods
Isolation and maintenance of potential biological
control agents
Potential
BCAs were isolated from canola plants, sclerotia of S. sclerotiorum and soil. Root, stem and pod samples from
approximately 500 healthy and diseased
canola plants from the Southern Region of WA were collected and cut into pieces
3 mm in length. Samples were surface sterilised using 1% aqueous NaHClO3
for two minutes and rinsed three times with sterilized distilled water. Samples
were dried on tissue paper before being placed into a Petri dish on PDAA medium (Potato
dextrose agar + 10 ppm/L Aureomycin) and incubated in a 21oC
growth room with 12 h photoperiod. After 48 h
of incubation, potential BCAs that grew from the samples were observed and replated onto new Petri dishes.
Soil samples were collected from canola fields
during the WA canola growing season of 2012–2013 in order to isolate the BCAs from soil. The soil
samples were placed in small plastic pots
(diameter 10 cm) along with ten surface sterilised sclerotia in order to be
infected by the potential BCAs. The pots were
incubated in a 21oC growth room for three months. Afterwards, sclerotia were removed and transferred to Petri dishes
containing PDAA medium and placed in a
growth room. Three days after inoculation, Petri dishes were monitored for
potential fungal growth around the sclerotia. Potential F-BCAs, which grew from
or around the sclerotia, were isolated onto PDAA medium
for further investigation.
Petal samples (approximately 2000 petals) from
healthy and diseased canola plants were collected from producers’
fields. Petal samples were directly inoculated onto
PDA + 10 mL Streptomycin (0.5 mL) and 10 mL
Ampicillin (0.5 mL)
(PDSA) medium in Petri dishes then incubated in a 21oC growth room
with 12 h photoperiod. After 48 h
of incubation, all colonies produced from petal samples were isolated for further
investigation.
Approximately 2000 sclerotia were collected from inside the stems of diseased canola plants in 2013. Samples were sterilized using 1% NaHClO3 solution for two minutes and rinsed three times with sterilized distilled water. Sclerotia were directly inoculated on PDAA medium in Petri dishes then
incubated in a 21oC growth room with 12 h photoperiod.
After 48 h of incubation, all colonies growing from sclerotia were isolated for further investigation. Potential F-BCAs, which grew
from or around the sclerotia, were isolated for further investigation onto PDAA
plates and potential B-BCAs were transferred to peptone yeast dextrose agar
(PYDA). Potential F-BCAs and B-BCAs were maintained on PDAA and PYDA respectively at 4oC
in a cold room for further investigation.
Identification of potential BCAs
Fifteen
potential F-BCAs and
three potential B-BCAs were
identified to species level at the Australian Genomics Research Facilities
(AGRF) laboratories through molecular techniques. For the F-BCAs, the Sanger Sequencing method was used to sequence the purified PCR products. The samples were
prepared based on DNA sample preparation instructions by the AGRF
(www.agrf.org.au). Each reaction mixture contained 6–12 ng of PCR product and 0.8 pmol/µL of the specific primer in 12 µL
with H2O. Purified PCR products were Sanger-sequenced with Big-Dye
3.1 (PerkinElmer, Waltham, MA), using PCR primers ITS1-f forward and reverse (Gardes
and Bruns 1993; de la Cerda et al. 2007) and
ITS4 (White et al. 1990) and analysed using an ABI3730xl analyzer
(Thermofisher).
For the B-BCAs, the Australian Genomics Research Facilities 16S sequencing process employed
universal primers to interrogate an approximate 800 bp region of the 16S
ribosomal RNA (www.agrf.org.au). Bacterial samples were subjected to an initial
amplification using the universal 16S primers (www.agrf.org.au). The process
included
Mycelial and colony growth of potential BCAs
F-BCAs: A 3-day-old
on PDAA grown, 5 mm of mycelial disc from each F-BCA was placed in the centre
of each of 3 Petri dishes and then incubated in
growth room at 21oC with 12 h photoperiod.
Radial mycelial growth was recorded at 24 and 48 h after
incubation. All potential F-BCAs were grown up to 7 days to
determine colony colour and
photographs were then taken.
B-BCAs: A 1-day-old, PYDA grown
bacterial BCA was streaked onto new PYDA in Petri dishes and incubated in a
growth room at 21oC with 12 h photoperiod. Three replicates were
prepared for each isolate. Growth and colony colour
were observed 24 h after incubation and photographs were taken. The relative
extent of colonization of the Petri dish was used to rank the colony growth
rate.
In vitro biological control of S. sclerotiorum by potential BCAs
Dual culture test of BCAs: A 3-day-old mycelial plug (5 mm diameter) of
each F-BCA isolate was incubated on PDAA. A mycelial plug of an aggressive isolate (SS12) of S. sclerotiorum was removed from the colony margin of a
3-day-old culture grown on PDAA and placed 7 cm distance from the plug of the F-BCA isolate in the same
Petri dish. Three replicates were prepared. Petri dishes inoculated similarly, but with F-BCAs or S. sclerotiorum alone, were used as controls. Plates were
incubated in a 21oC growth room and observed after 72 h for inhibition zones between F-BCAs and
the S. sclerotiorum
isolate. After incubation period, radial mycelial growth
was recorded. Reduction in the radial mycelial growth
was calculated as follows:
Inhibition percentage of radial mycelial
growth = %20215-227_files/image002.png){kind=small}
Where C is
the pathogen radial mycelial growth
measurement in control plates, and T is the pathogen radial mycelial growth in presence of F-BCAs (Simonetti et
al. 2012). After incubated for two
weeks, the number of sclerotia formed by S. sclerotiorum
in each Petri dish was recorded and the percentage inhibition of sclerotia
formation was calculated with the same formula above with adjustment for
sclerotia formation.
Three isolates of B-BCAs were tested for their
antagonistic effect in dual culture tests. For dual culture tests, two
inoculation methods were used for the pathogen, either mycelial plugs or
sclerotia. In the mycelial plug method, a 3-day-old mycelial plug
with 5
mm diameter of S. sclerotiorum isolate SS12 was
placed on PYDA about 1 cm away from the edge of each Petri dish.
In the sclerotia method, a sclerotium produced by S. sclerotiorum isolate SS12 (after 2
weeks of incubation in Petri dishes) was incubated on PYDA about 1 cm
away
from the edge of each Petri dish. A 3-day-old culture of B-BCAs grown on PYDA
was streaked 7 cm away from the plug/sclerotia of the pathogen isolate in the
same petri dish. Petri dishes inoculated similarly with each B-BCA or S.
sclerotiorum isolate SS12 alone were used as
controls. There were three Petri dishes replication for each treatment.
Plates were incubated in a 21oC growth room and were
observed after 48 h for calculation of inhibition zones between
B-BCAs and S. sclerotiorum.
Radial growth was calculated after incubation period. The numbers of sclerotia
formed by the pathogen were recorded two weeks after incubation. Inhibition in
radial growth and sclerotial production by each B-BCA
was calculated as described for the F-BCAs.
Hyper-parasitisation of S. sclerotiorum sclerotia by
BCAs under artificial inoculation in pots
Ten inoculum discs of each of the 15 F-BCAs were mixed in separate 100 mm diameter plastic pots
(Burnell Agencies Pty Ltd) which were filled with a commercial bulk potting mix
produced by Baileys (http://www.baileysfertiliser.com.au/). Each F-BCA was
replicated in three plastic pots. The plastic pots were placed in a growth room
at 21oC with a 12 h photoperiod for about one week. Ten sclerotia of S.
sclerotiorum isolate SS12 were placed at a depth of 5 cm in each plastic pot. The
plastic pots were watered by hand every morning. After a week, two sclerotia
from each pot were plated onto PDAA and grown for seven days to test the viability of
sclerotia.
In planta testing the efficacy of BCA’s
Glasshouse experiment: Canola plants were grown in 30 cm diameter plastic pots that were
arranged in a Randomized Complete Block Design
(RCBD). Each pot had one canola plant
and there were four replicate
pots per treatment. A potting mix
(http://www.baileysfertiliser.com.au/) was used as the growth medium in the
glasshouse; each pot was mixed with 10 g of NPK (19:19:19) fertilizer before sowing. The glasshouse trial was undertaken at the
same time as the 2015 field experiments. The B-BCA1 (S.
proteamaculans), B-BCA2 (O. anthropi), F-BCA1 (T. koningiopsis) and F-BCA2 (T. atroviride) were sprayed at the green bud stage. The S. sclerotiorum isolate
SS12 was sprayed at 10, 30 or 50% flowering stages. The aim of the glasshouse
experiment was to evaluate the effectiveness of the BCAs in suppressing SSR for comparison with the field experiments.
Field experiments: Field
experiments were conducted in 2014 and 2015 to evaluate the effectiveness of
selected BCAs under field conditions at the
Department of Primary Industries and Regional Development field experimental
area in South Perth. The aim of the 2014 field experiments was to investigate
the efficacy of newly identified local F-BCAs and B-BCAs in controlling SSR
disease on canola by
co-inoculating at 50% flowering. The 2014 field experiment was established as a
RCBD comprising seven treatments and three replicate
plots. Individual plot size
was 2 x 2 m2. Seed (cultivar Crusher) was
sown by hand to a depth of 1 cm on the 29 May 2014. Fertilizer was applied
based on common practice for canola in Australia: 110 kg N/ha,
15 kg P/ha,
12 kg K/ha
and 20 kg S/ha (Norton
et al. 2011). The BCA and pathogen treatments
were
applied at 50%
flowering (Table 1).
To prepare the pathogen inoculum, ten agar
plugs (5 mm diameter) were cut from the
margin of actively growing 3-day-old colonies and transferred to a
250 mL conical flask contained
sterile liquid medium (24 g/L potato dextrose broth with 10 g/L peptone) and shaken at 250 rpm. The
inoculated medium was incubated for 3 days at 21°C. The S. sclerotiorum
colonies
were
harvested and rinsed three times with sterile deionized water. Before inoculation on plants, the
harvested mycelial mats were transferred into 150 mL of liquid medium and homogenized at medium speed in a blender for 1 min.
The macerated mycelia were filtered through three layers of cheesecloth and
suspended in the same liquid medium. Then, the concentration of mycelia was counted using a
haemocytometer and adjusted to the required
concentration for the experiment. Similar procedures were applied to produce inocula of the B-BCAs. Suspensions (100 mL) containing
either mycelia of F-BCAs or colonies of B-BCAs were sprayed at concentrations
of ~106 fragment mL-1 and ~1010 CFU
mL-1, respectively, in each treatment plot. Mycelia of the pathogen were also sprayed at 100 mL per plot at a concentration
of ~106 fragment mL-1.
The fungicide Prosaro® was sprayed based on the recommended dose of
450 mL ha-1 (equal to 0.2 mL plot-1) to compare the
efficacy of potential BCAs with a fungicide recommended for Sclerotinia control
in canola in Australia. The control treatment plots were sprayed with water
only. The
number of infected plants in every plot was counted and disease incidence was
calculated for each treatment two weeks after inoculation. Plots were harvested
at maturity and seed yield was obtained for each plot.
In 2015, two field experiments were carried out to optimise the
timing of application of BCAs against S. sclerotiorum. In the first experiment, the BCAs were
sprayed at the
green bud stage, while in the second experiment; the BCAs were sprayed
one week before the pathogen and at the same time as the pathogen at
30% bloom. In
experiment 1, a Randomized Complete Factorial
Design (RCFD) was used consisting
of sixteen treatments with
pseudo-replication inside the treatment due to limited space (Table 2). Rows were 7 m long.
There were 3 buffer rows on each side of the treatment
rows to
prevent inoculum drift. Cultivar
Hyola 404 was hand sown @ 0.8 g (approximately 150
seeds) per row on 1st June 2015. In
this experiment, the BCAs (S. proteamaculans, O. anthropi, T. koningiopsis, and T. atroviride) were applied as
foliar sprays at the green bud stage and
at the same concentrations and water volume as in 2014. Mycelia of the pathogen isolate SS12 were sprayed at 100 mL per plot at a concentration of ~1012 fragment mL-1 at 10% and 50% bloom
stages, respectively.
The design of field experiment 2 was the same as experiment 1 except there were
fifteen treatments (Table 3). Row length, row
spacing, cultivar, time of seeding, fertilization, treatment application
(method and rate) were as in experiment 1. In this experiment, each BCA (S.
proteamaculans, T. atroviride) was applied one week prior to pathogen inoculation at 10 and 50% bloom
stages or both the BCA and the pathogen were applied at the same time at 10 and 50% bloom stages. Spray
application of the fungicide Prosaro® at the same rate as the previous field
experiment was included as a positive control and applied at the same timings
as the two BCAs.
For field
experiments 1 and 2, the
number of infected plants in each treatment row was counted 2 weeks after
inoculation and disease incidence (DI) and disease control efficiency for each
treatment were calculated
as:
Disease control efficiency = (Mean of DI in pathogen
treated plots-Mean of DI in treatment plots)*100/Mean of DI in pathogen
treated plots.
Seed was harvested from each treatment row
and middle buffer row at maturity.
Statistical analysis
Analysis
of variance (ANOVA) of radial mycelial growth and inhibition by F-BCAs at 24
and 48 h after incubation, sclerotial formation by F-BCAs and data
of infected plants were performed using
GenStat 16® software (Release 16, Lawes Agricultural Trust –
Rothamsted Experimental Station) followed by mean separation by
LSD (P≤0.05). Percentage of disease incidence data for the glasshouse
experiment were analysis. Data of field experiments with pseudo-replication were predicted using
Restricted Maximum Likelihood (REML) analysis.
Results
Isolation and mycelial growth of potential F-BCAs and B-BCAs
In this
study we excluded fungal isolates that were not of interest (genera other than Trichoderma). The percentage of Trichoderma spp. isolated by all methods was
5–10%. In total, fifteen
potential Trichoderma species were isolated. Mycelial colour of the Trichoderma species showed wide
variation, being dark green, light green, green, whitish green, brownish green,
yellowish white, and white (Fig. 1).
Table 1: Details of treatments for field experiment in 2014
|
No |
Treatment |
Code |
|
1 |
Pathogen only (mycelia
of S. sclerotiorum) |
PO |
|
2 |
Pathogen + Fungicide (Prosaro®) |
P-Fc |
|
3 |
Pathogen + F-BCA1 |
P-FBCA1 |
|
4 |
Pathogen + F-BCA2 |
P-FBCA2 |
|
5 |
Pathogen + B-BCA1 |
P-BBCA1 |
|
6 |
Pathogen + B-BCA2 |
P-BBCA2 |
|
7 |
Untreated Control |
Control |
Table
2: Details
of treatments of field experiment 1 in 2015
|
No |
Treatment |
|
1 |
F-BCA1
at
green bud followed by pathogen at
10% flowering |
|
2 |
F-BCA1
at
green bud followed by pathogen at
30% flowering |
|
3 |
F-BCA1
at
green bud followed by pathogen at
50% flowering |
|
4 |
F-BCA2
at
green bud followed by pathogen at
10% flowering |
|
5 |
F-BCA2
at
green bud followed by pathogen at
30% flowering |
|
6 |
F-BCA2
at
green bud followed by pathogen at
50% flowering |
|
7 |
B-BCA1
at
green bud followed by pathogen at
10% flowering |
|
8 |
B-BCA1
at
green bud followed by pathogen at
30% flowering |
|
9 |
B-BCA1
at
green bud followed by pathogen at
50% flowering |
|
10 |
B-BCA2
at
green bud followed by pathogen at
10% flowering |
|
11 |
B-BCA2
at
green bud followed by pathogen at
30% flowering |
|
12 |
B-BCA2
at
green bud followed by pathogen at
50% flowering |
|
13 |
Pathogen at 10% flowering |
|
14 |
Pathogen at 30% flowering |
|
15 |
Pathogen at 50% flowering |
|
16 |
Un-inoculated
control |
Table
3: Details
of treatment of field experiment 2 in 2015
|
No |
Treatment |
|
1 |
F-BCA at 1 week before pathogen
10% flowering |
|
2 |
F-BCA + pathogen together at 10% flowering |
|
3 |
B-BCA at 1 week before pathogen
10% flowering |
|
4 |
B-BCA + pathogen together at10% flowering |
|
5 |
F-BCA at 1 week before pathogen at 30% flowering |
|
6 |
F-BCA + pathogen together at 30% flowering |
|
7 |
B-BCA at 1 week before pathogen at 30% flowering |
|
8 |
B-BCA + pathogen together at 30% flowering |
|
9 |
Pathogen a week before Prosaro® at
10% flowering |
|
10 |
Pathogen a week before Prosaro® at
30%
flowering |
|
11 |
Pathogen + Prosaro® at
10% flowering |
|
12 |
Pathogen + Prosaro® at
30%
flowering |
|
13 |
Pathogen only at 10% flowering |
|
14 |
Pathogen only at 30% flowering |
|
15 |
Un-inoculated
control |
There were significant differences
(P≤0.001) in growth rate among
isolates at 24 and 48 h of incubation. At 24 h, Isolate F-BCA9 had the highest radial mycelial growth with
diameters of 3.17 cm (24 h) and 8.5 cm (48 h), followed by isolates F-BCA11 (2.83 cm at 24 h, 7.2 cm at 48 h) and F-BCA14 (2.63 cm at 24 h, 7.53 cm at 48 h). Isolate F-BCA12 had the smallest radial mycelial growth at 24 h (2.03 cm) but had accelerated growth at 48 h (6.53 cm) (Fig. 2).
Three isolates of potential B-BCAs were obtained. Colony colour of isolates of
B-BCA1, B-BCA2 and B-BCA3 were light yellow, yellow, and whitish yellow,
respectively. Isolate B-BCA3 had the fastest colony growth rate and morphologically had
the softest and more
watery colony; whereas isolate B-BCA2 had a much drier colony compared with isolates B-BCA1 and
B-BCA3 (Fig. 3).
Molecular identification of F-BCAs and B-BCAs
F-BCAs: The Trichoderma isolates were identified as Trichoderma atroviride (four isolates), T. gamsii (three isolates), T. koningiopsis
(two isolates), T. longibrachiatum
(two isolates), T. paraviridescens (two isolates), T. pseudokoningii (one isolate) and T. viridescens (one isolate). Trichoderma atroviride, T. gamsii, T. koningiopsis, and T. viridescens are reported for the
first time from Western Australia. Accession numbers are provided in Table 6.
B-BCAs: Isolates B-BCA1 and B-BCA2 were identified as Serratia
proteamaculans and isolate B-BCA3 as Ochrobactrum anthropi.
The highest level of identity for S.
proteamaculans and O. anthropi
were 99.58 and 100%, respectively. Accession numbers are given in Table 7.
In vitro biological
control of S. sclerotiorum by potential BCAs (inhibition of mycelial growth and hyper-parasitisation of S. sclerotiorum sclerotia by
F- BCAs)
F-BCAs: All potential F-BCA isolates showed some capacity to reduce mycelial growth
and the number of
sclerotia of S. sclerotiorum SS12 in dual culture
tests in Petri dishes. The morphology of the dual cultures of the potential F-BCAs with SS12 is shown in Fig. 4. There
were significant differences (P≤0.001) in inhibition of both radial
mycelial growth and sclerotia formation by the pathogen among the 15 F-BCAs. Mycelial growth of SS12 was inhibited by 46–60%. The
highest inhibition was caused by F-BCA9 (60%). The presence of F-BCAs decreased sclerotia formation
by 65–100%. Isolates F-BCA12 and F-BCA15 completely inhibited the formation
of sclerotia by the pathogen, while isolates F-BCA13 and F-BCA10 had the least potential to
inhibit sclerotia formation, reducing the number of sclerotia by 65 and 70%,
respectively. There was an antagonistic effect
of F-BCA against sclerotia in soil in pots. After a week on PDAA, no new
sclerotia were formed (Fig. 7).
%20215-227_files/image004.jpg)
Fig. 1: Mycelial
growths of fifteen isolates of potential F-BCAs from WA on PDAA media 7 days
after incubation. From left to right: top row F-BCA1, F-BCA2, F-BCA3, F-BCA4,
F-BCA5; second from top row F-BCA6, F-BCA7, F-BCA8, F-BCA9, F-BCA10; and bottom
row F-BCA11, F-BCA12, F-BCA13, F-BCA14, F-BCA15
%20215-227_files/image006.png)
Fig. 2: Radian mycelial
growth
of fifteen potential F-BCAs on PDAA media at 24 and 48 hours after incubation.
Least significant difference of means (LSD) at 5% level at 24 hours after
incubation was 0.1909 and at 48 hours was 0.3014. Columns having different
letters at each incubation time are highly significant different. Values are
means (n=3)
%20215-227_files/image008.jpg)
Fig. 3: Bacterial colonies from three isolates of potential B-BCAs from WA on PYDA media after 24
hours incubation. From
left to right: B-BCA1, B-BCA2 and B-BCA3
B-BCAs: Using the mycelial plug method, the three
potential B-BCAs inhibited in vitro radial
mycelial growth of SS12 by 57–79% and sclerotia formation by 89–95% (Fig. 5), but there was no significant difference among
isolates in inhibition of mycelial growth (P=0.934)
or sclerotia (P=0.78). The three isolates also inhibited mycelial
growth and sclerotia formation using the sclerotium inoculation method (Fig. 6). There
were significant differences (P=0.029)
in inhibition of radial mycelial growth among the three B-BCAs but there were no differences (P=0.072) among the B-BCAs in the inhibition of sclerotia.
%20215-227_files/image010.jpg)
Fig. 4: Dual cultures of fifteen
potential F-BCAs against S. sclerotiorum (isolate 12) on PDAA media. Each F-BCA is
on the left side and the pathogen is in the right side of the Petri dish. From
left to right: top row: F-BCA1, F-BCA2, F-BCA3, F-BCA4, F-BCA5; second from top
row: F-BCA6, F-BCA7, F-BCA8, F-BCA9, F-BCA10; and bottom row: F-BCA11, F-BCA12,
F-BCA13, F-BCA14, F-BCA15
%20215-227_files/image012.jpg)
Fig.
5: Dual culture tests
of three potential B-BCAs against S. sclerotiorum isolate 12 on PYDA media: (A) B-BCA1, (B)
B-BCA2, (C) B-BCA3 and (D) Control pathogen only. Inoculum source of pathogen
was from a mycelial agar plug placed in left side of each Petri dish
In-planta testing of BCAs against S. sclerotiorum
Glasshouse experiment: In the glasshouse experiment, all BCA’s were applied at the green bud
growth stage of canola and the pathogen was inoculated at 10, 30 or 50% bloom
stages. No disease developed in the un-inoculated
control. Significantly higher levels (100%) of disease developed when the
pathogen was applied at 10% bloom compared with at 30 and 50% bloom. F-BCA1
(T. koningiopsis) was very effective against the pathogen at 10% bloom. Likewise, incidence of the disease was reduced significantly with O. anthropi
when the pathogen was inoculated at 10% bloom. The fungal F-BCA1 (T. koningiopsis) and F-BCA2 (T. atroviride) were significantly more effective in reducing incidence
of the disease when the pathogen was applied at 30% bloom
compared with the B-BCAs. Due to very low disease incidence with pathogen
application at 50% bloom, only T. atroviride and O.
anthropi provided complete suppression of disease
incidence (Fig. 8).
Field
experiments
2014 field
experiment: Very
low levels of disease (<5% disease incidence in S. sclerotiorum inoculated plots)
developed in the 2014 field experiment (data not shown). However, there was a
clear difference in the appearance of each treatment plot. Plots sprayed with
fungicide Prosaro® and O. anthropi
were much greener with denser
foliage and greater leaf area compared to other treatment plots. There were no
significant yield differences, but the yield of Fungicide Prosaro®+Pathogen and O. anthropi+Pathogen treatments trended higher compared to
pathogen only and other treatments where
yield was increased by 19 and 18% with O.
anthropi and Prosaro®,
respectively, compared with the pathogen only treatment (Fig. 9).
%20215-227_files/image014.jpg)
Fig. 6: Dual culture tests of three potential B-BCAs
against S. sclerotiorum
isolate 12 on PYDA media: (A) B-BCA1, (B) B-BCA2, (C) B-BCA3, (D) Control sclerotium only. Inoculum source of pathogen was sclerotium placed in left side of each
Petri dish
%20215-227_files/image016.jpg)
Fig. 7: Plating of S. sclerotiorum
sclerotia after being colonized by each isolate of F-BCAs in soil for a week.
From left to right: top row (sclerotia in the presence of F-BCA1, F-BCA2,
F-BCA3, F-BCA4); second from top row (F-BCA5, F-BCA6, F-BCA7, F-BCA8); third
from top row (F-BCA9, F-BCA10, F-BCA11, F-BCA12); bottom row (F-BCA13, F-BCA14,
F-BCA15, control sclerotia only)
2015 field
experiments: Effect of each treatment and their interaction in field experiment 1
were predicted using Restricted Maximum Likelihood (REML) analysis (Table 4). There were
significant differences (P<0.001)
among the BCAs in controlling SSR disease. There were significant differences (P<0.001) in disease incidence of SSR
with application of the pathogen at different flowering stages. In addition,
there were significant (P<0.001)
interactions between BCAs and pathogen application at various flowering stages.
There was significantly less disease when the pathogen was applied at 30 and
50% compared with 10% flowering. Both bacterial B-BCA1 (S. proteamaculans) and B-BCA2
(O. anthropi) were significantly more effective than the
fungal F-BCA1 (T. koningiopsis) and F-BCA2 (T. atroviride) in reducing the disease incidence when the pathogen
was applied at 10% flowering. The maximum disease control efficiency (89%) was
achieved with O. anthropi (Fig. 10). However, when the pathogen was applied at 50% flowering stage, both the
fungal BCAs were significantly more effective than the bacterial BCAs in
reducing the disease incidence.
For experiment 2,
predicted treatment and interaction effects from REML analysis are given in
Table 5. There was a significant difference (P<0.001) among the BCAs. Time of
application of S. sclerotiorum was highly
significant (P<0.001) but application
time of fungicide
was not significant (P=0.901>0.001). There were also
highly significant differences between time of application of BCAs (P<0.001), but no significant
difference (P=0.382>0.001) between interaction of BCAs and time of
spraying the pathogen. In addition, there was a significant (P=0.002<0.005) interaction of BCAs
and timing of application. High level of disease developed when the S. sclerotiorum was applied at 10% flowering. Disease
development was negligible when the S. sclerotiorum was applied at
30% flowering. There was a significant reduction in disease
incidence when T. atroviride
was applied either one week before the pathogen or at the same time as the
pathogen at 10% flowering, with the disease control efficiency being 86 and
98%, respectively. However, S. proteamaculans and fungicide Prosaro® were more effective when applied at the same time as the pathogen at
10% flowering resulting in disease control efficiencies of 75 and 100%,
respectively (Fig. 11).
There was a
significant (P=0.12) interaction of
BCA and time of application of the pathogen on yield. Significant yield
responses were achieved when T. atroviride and S.
proteamaculans were applied one week before S. sclerotiorum and when S. proteamaculans
and S.
sclerotiorum were applied together at 10% flowering. Despite negligible
levels of disease with S. sclerotiorum inoculation at
30% flowering, there was a significant yield response (19.6 and
19.8% enhancement respectively)
to the
fungal BCA (T. atroviride)
when applied a week before or at the same time as the pathogen
(Fig. 12).
Table 4: Effect of applying BCAs and the pathogen S. sclerotiorum
during the different flowering stages and their interaction from field
experiment 1 in growing season 2015 based on REML analysis
|
Change |
d.f |
deviance |
Mean deviance |
Deviance ratio |
Approx. chi pr |
|
BCAs |
4 |
44.80 |
11.20 |
11.20 |
<0.001 |
|
PATH_Flowering |
2 |
283.85 |
141.92 |
141.92 |
<0.001 |
|
Residual |
8 |
101.19 |
12.65 |
|
|
|
BCAs.PATH_Flowering |
8 |
101.19 |
12.65 |
12.65 |
<0.001 |
|
Total |
14 |
429.84 |
30.70 |
|
|
Table 5: Effect of each treatment and their interaction
from field experiment 2 in growing season 2015 based on REML analysis
|
Change |
d.f |
deviance |
Mean deviance |
deviance ratio |
approx chi
pr |
|
BCAs |
3 |
59.186 |
19.729 |
19.73 |
<0.001 |
|
PATH_Flowering |
1 |
129.170 |
129.170 |
129.17 |
<0.001 |
|
Timing_Fungicide |
1 |
0.016 |
0.016 |
0.02 |
0.901 |
|
Timing_BCAs |
2 |
71.811 |
35.905 |
35.91 |
<0.001 |
|
BCAs.PATH_Flowering |
1 |
0.764 |
0.764 |
0.76 |
0.382 |
|
BCAs.Timing_Fungicide |
0 |
0.000 |
* |
|
|
|
Residual |
3 |
15.211 |
5.070 |
|
|
|
BCAs.Timing_BCAs |
3 |
15.211 |
5.070 |
5.07 |
0.002 |
|
PATH_Flowering.Timing_Fungicide |
0 |
0.000 |
* |
|
|
|
PATH_Flowering.Timing_BCAs |
0 |
0.000 |
* |
|
|
|
BCAs.PATH_Flowering.Timing_Fungicide |
0 |
0.000 |
* |
|
|
|
BCAs.PATH_Flowering.Timing_BCAs |
0 |
0.000 |
* |
|
|
|
Total |
11 |
276.157 |
25.105 |
|
|
Table 6: Accession numbers of F-BCAs used in the investigations
|
FASTA ID |
Accession # |
Gene description |
|
F_BCA1_F_A01 |
MW268857 |
Trichoderma ovalisporum
isolate MI98 internal transcribed spacer 1 |
|
F_BCA1_R_D01 |
MT529291 |
Trichoderma sulphureum
clone SF_15 small subunit ribosomal RNA gene |
|
F_BCA2_F_A02 |
MT137373 |
Trichoderma sp. strain
21F13C_AC small subunit ribosomal RNA gene |
|
F_BCA2_R_D02 |
MT529291 |
Same as F_BCA1_R_D01 |
|
F_BCA3_F_A03 |
MT529638 |
Trichoderma sulphureum
clone SF_362 small subunit ribosomal RNA gene |
|
F_BCA3_R_D03 |
MT732907 |
Trichoderma sp. PB-2018
strain 56E small subunit ribosomal RNA gene |
|
F_BCA4_F_A04 |
MW269180 |
Trichoderma neokoningii
isolate MI479 small subunit ribosomal RNA gene |
|
F_BCA4_R_E12 |
MK808808 |
Trichoderma sp. isolate
DS554 small subunit ribosomal RNA gene |
|
F_BCA5_F_A05 |
MK862245 |
Trichoderma erinaceum
isolate SWFU000006 internal transcribed spacer 1 |
|
F_BCA5_R_D05 |
MK808808 |
Same as F_BCA4_R_E12 |
|
F_BCA6_F_F01 |
MK862247 |
Trichoderma samuelsii
isolate SWFU000004 internal transcribed spacer 1 |
|
F_BCA6_R_D06 |
MK460812 |
Trichoderma atroviride
strain CSK3_13 small subunit ribosomal RNA gene |
|
F_BCA7_F_A07 |
MK910067 |
Trichoderma
longibrachiatum isolate BM12 small subunit ribosomal RNA gene |
|
F_BCA7_R_D07 |
MF076623 |
Trichoderma reesei
isolate S254 small subunit ribosomal RNA gene |
|
F_BCA8_F_A08 |
MK871246 |
Trichoderma sp. isolate
SDAS203393 small subunit ribosomal RNA gene |
|
F_BCA8_R_D08 |
MF076590 |
Trichoderma koningii
isolate S54 small subunit ribosomal RNA gene |
|
F_BCA9_F_A09 |
MN795754 |
Trichoderma atroviride
strain p18 small subunit ribosomal RNA gene |
|
F_BCA9_R_D09 |
MF076590 |
Same as F_BCA8_R_D08 |
|
F_BCA10_F_A10 |
MK910067 |
Trichoderma
longibrachiatum isolate BM12 small subunit ribosomal RNA gene |
|
F_BCA10_R_D10 |
MF076623 |
Same as F_BCA7_R_D07 |
|
F_BCA11_F_A11 |
MK407088 |
Uncultured Trichoderma
clone D1314ITS internal transcribed spacer 1 |
|
F_BCA11_R_D11 |
MK460812 |
Same as F_BCA6_R_D06 |
|
F_BCA12_F_A12 |
MN795754 |
Same as F_BCA9_F_A09 |
|
F_BCA12_R_D12 |
MF076590 |
Same as F_BCA8_R_D08 |
|
F_BCA13_F_B01 |
MK333266 |
Trichoderma
citrinoviride isolate MTAT17 small subunit ribosomal RNA gene |
|
F_BCA13_R_E01 |
EU280097 |
Trichoderma
pseudokoningii strain DAOM 167678 18S ribosomal RNA gene |
|
F_BCA14_F_B02 |
MW269180 |
Same as F_BCA4_F_A04 |
|
F_BCA14_R_E02 |
MK808808 |
Same as F_BCA4_R_E12 |
|
F_BCA15_F_B03 |
MN795754 |
Same as F_BCA9_F_A09 |
|
F_BCA15_R_E03 |
MF076590 |
Same as F_BCA8_R_D08 |
Table 7: Accession numbers of B-BCAs used in the investigations
|
FASTA ID |
Accession # |
Gene description |
|
16SQ11665-1_B_BCA1 |
MT561436 |
Serratia sp.
strain CT197 16S ribosomal RNA gene |
|
16SQ11665-2_B_BCA2_R_E03 |
MK530301 |
Serratia proteamaculans strain Sample_92 16S ribosomal RNA gene |
|
16SQ11665-3_B_BCA3 |
MG430400 |
Ochrobactrum pituitosum strain AA2 16S ribosomal RNA gene |
Discussion
The application of biological
control agents is a
promising tool to manage the damage caused by plant pathogens. Biological
control treatments for soil-borne plant pathogens must provide enhanced levels
of disease suppression and consistency of control over diverse soils before their
wide-scale application on a commercial scale (Hu et
al. 2019). %20215-227_files/image018.png)
Fig. 8: Effect of
various BCA’s on the disease incidence of S. sclerotiorum in canola under glasshouse conditions. Tk
= T. koningiopsis, Ta = T. atroviride, Sp = S. proteamaculans, Oa = O. anthropi, gb = green bud. Values
are means (n=4). Error bars
represent Standard Error (SE)
%20215-227_files/image020.png)
Fig. 9: Effect of various
BCAs on yield of canola under field conditions during 2014. Tk-MB = T. koningiopsis isolate Mount Baker,
Tk-Kendenup = T. koningiopsis isolate
Kendenup, Sp = S. proteamaculans, Oa
= O. anthropi. The pathogen is S. sclerotiorum. Values are means (n = 3). Error
bars represent LSD
We
exploited a range of techniques to isolate naturally
occurring fungal and bacterial BCAs with view to their future use to manage S. sclerotiorum in canola. We isolated 18 taxa that are known to have biological
control potential in a number of host-pathogen systems (Ghazanfar et al.
2018; Kshetri
et al. 2019).
The fifteen isolates of Trichoderma
had moderate to fast radial
mycelial growth rates on PDAA medium.
Rapid growth is one of the important competitive advantages antagonistic fungi
have over plant pathogenic fungi. It enables them to compete for space and
nutrients. Furthermore, some Trichoderma species can induce host
resistance responses against pathogens (Harman et al. 2004). Several
studies have shown the biocontrol potential of Trichoderma species in
controlling pathogens in in vitro and in
vivo conditions (Ojaghian 2011; Saxena et
al. 2015). For example, an in vivo
seed coating test using thiophanate-methyl or Trichoderma spp. substantially improved soybean germination and
suppressed growth of S. sclerotiorum
(Macena et al. 2020). Our results are consistent
with previous research where colonies of T. longibrachiatum, T. atroviride
and T. harzianum grew faster than S. sclerotiorum in both single or mixed
cultures (Matroudi and Zamani 2009). Our studies also
showed that isolates of T. atroviride
were highly effective in reducing mycelial growth and completely inhibiting
sclerotia production by the pathogen
as also reported by Gupta et al. (2014). Furthermore, T. atroviride has been
shown to reduce colony growth by 93 and 85% in two isolates of S. sclerotiorum from canola
(Matroudi and Zamani 2009).
Knowledge on the effectiveness
of the new isolates of BCAs on
controlling the critical stages of the life cycle of a particular pathogen is
very important to determine the most effective
isolates for commercialisation. The
survival of S. sclerotiorum depends
on the production and viability of sclerotia that
can remain viable in soil for more than 7 years (Kora et al. 2008; Smolińska et al. 2018). Therefore, to control this pathogen, the key is to
reduce production and viability of
sclerotia. Since the sclerotia reside in soil, using chemical
sprays to reduce the inoculum load in broad-acre crops is not feasible. Thus,
effective biological products may be more feasible to reduce the density of sclerotia in soil. A
commercially available product Contan®WG
(a formulation of C. minitans) is reported to
control sclerotial populations in soil in canola and
other hosts including carrots and soybean (Fernando et al. 2004; McQuilken and Chalton 2008; Zeng et
al. 2012). This product was not tested in the current studies as, due to
strict quarantine regulations in WA, it was not possible to
import commercial C. minitans. Our in vitro
experiments revealed significant inhibition of sclerotia
formation by Trichoderma species both in dual plate and
soil inoculation treatments. The
nearly complete inhibition of sclerotial formation
could possibly be due to
reduced viability of mycelia. It could also be attributed to competition for space and nutrients
or mycoparasitism reducing
growth and consequently inhibiting the sclerotia
formation ability of the pathogen. Our results corroborate those of Abdullah et
al. (2008) who reported that T.
harzianum had an ability to control both mycelial
growth and sclerotial production by S.
sclerotiorum when tested on the same plate.
Molecular identification indicated that bacterial isolates B-BCA1 and B-BCA2 are species of S.
proteamaculans. Serratia
is a diverse and widely dispersed group of gamma
proteobacteria (Grimont and Grimont 2006). Some species of Serratia have beneficial effects on
economically and ecologically important crops (Kalbe et al. 1996;
Kurze et al.
2001) and others are indicated as opportunistic pathogens for humans and other
organisms (Grimont and Grimont 2006). Serratia associated with plants has considerable interest in
agriculture and some strains have been investigated as
BCAs in field crops (Kalbe et al. 1996;
Kurze et al.
2001) and as plant growth promoting
rhizobacteria (PGPR) (Bababola 2010). Furthermore, some isolates of S. proteamaculans can stimulate plant
growth and suppress growth of some important soil-borne fungal pathogens (Neupane et al.
2013). B-BCA3 was identified as Ochrobactrum anthropi, a species known for
its potential as a BCA and PGPR (Chakraborty
et al. 2009; Bababola 2010). This species is a gram-negative
bacterium that has a structure membrane composed of an outer membrane,
periplasmic space, and inner membrane (Bababola 2010). Ochrobactrum anthropi isolated from the rhizosphere
of Camellia produces
IAA and siderophores in vitro, and have potential for
biological control (Chakraborty et al. 2009).
Our in vitro
experiments showed that the B-BCAs
inhibited radial mycelial growth of the
pathogen and sclerotia production by 60 to 95%. Ability of the BCAs to reduce sclerotial production in
planta or their viability in soil can substantially curtail the primary
inoculum source of S. sclerotiorum
for susceptible crops. A possible mechanism of supressiveness by B-BCAs in vitro is
the production of antibiotics (Abdullah et al. 2008).
Raaijmakers et al. (2002) argued that antibiotics produced by
antagonistic microorganism are evidence they can play an important role in the
suppression of some soil-borne pathogens. Further investigation is needed to
determine if antibiotics were produced by the WA B-BCAs in our
study.
Application of BCAs in the field can sometimes give unexpected results due to factors that can
be attributed to the behaviour of BCAs and environmental conditions (Saharan
and Mehta 2008). However, application
methods that are consistent with the cropping system may enhance biological
control of S. sclerotiorum (Li et
al. 2020). In our studies, selected Trichoderma
isolates including T. koningiopsis
and T. atroviride
and an isolate each of the bacterial BCA (O.
anthropi and S.
proteamaculans) significantly reduced disease
incidence of S. sclerotiorum
under glasshouse and field conditions. However, the effectiveness of the BCAs
varied with the growth stage of canola and in particular the timing of
application of the antagonist and the pathogen. Under glasshouse conditions,
the fungal BCAs were more effective than the bacterial BCAs when applied at the
green bud stage followed by inoculation of S.
sclerotiorum at 30% flowering. However, under
field conditions, bacterial BCAs were significantly more effective than the
fungal BCA’s when applied at the green bud stage followed by inoculation of S. sclerotiorum
at 10% flowering. In field experiment 2, the fungal BCA T. atroviride
was substantially superior in reducing disease incidence when applied either a
week before the pathogen or co-inoculated with the pathogen at 10% flowering
compared with the bacterial BCA treatments at the same growth stage. These
results indicate that the disease suppression window of bacterial BCA’s is
comparatively shorter than that of the fungal BCA’s.
To investigate the beneficial
effect of disease reduction by the BCA’s on canola yield, the field experiments
in 2014 and 2015 were hand harvested and seed yield for each treatment was
measured. Dry seasonal conditions in 2014 resulted in very low disease incidence
in pathogen only inoculated plots, consequently, yield responses to BCA
application were not expected. However, the greening effect with the bacterial
BCA- O. anthropi
and fungicide Prosaro® was evident by eye
and seed yield in both these treatments was marginally (19 and 18%,
respectively) higher than the S. sclerotiorum only treatment. This improvement in yield
in the absence of disease is possibly due to the growth promoting effect of O. anthropi.
Likewise, in field experiment 2 in 2015, a significant increase (21%) in yield with T.
atroviride over the pathogen only treatment at 30% flowering was
observed. These
results are encouraging in the context that these BCA’s can improve canola
yield in both the presence or absence of disease thus making it a useful
additional tool for alleviating %20215-227_files/image022.png)
Fig. 10: Effect of various BCA’s on
disease incidence of S. sclerotiorum in canola in field experiment 1 during 2015. Tk = T. koningiopsis, Ta = T. atroviride, Sp = S. proteamaculans, Oa = O. anthropi, gb = green bud. Error bars represent LSD
%20215-227_files/image024.png)
Fig. 11: Effect of various BCA’s on
disease incidence of S. sclerotiorum in canola in Field experiment 2 during
2015. Ta = T. atroviride, Sp = S. proteamaculans. Error
bars represent Standard Error (SE)
%20215-227_files/image026.png)
Fig. 12: Effect of various BCA’s and S. sclerotiorum on seed yield (g/7m row) of canola
in field experiment 2 during 2015. Ta = T.
atroviride, Sp = S. Proteamaculans. Error
bars represent LSD
both biotic and abiotic stresses in canola.
Moreover, it is noteworthy that the efficacy of the tested BCA’s in the current
studies was similar to that of a commercial product Prosaro®
implying that BCAs can potentially be used as an alternative to fungicides or
as an additional tool in the integrated management of SSR in canola. Growth
promoting and pathogen suppression ability of T. atroviride, T. koningiopsis, S. proteamaculans and O. anthropi is
well documented in other host pathogen systems (Chakraborty et
al. 2009). However, further field studies
with large field plots at multiple locations in a range of environments are
required to validate these preliminary findings and develop commercial formulations
and spray regimes. Furthermore, combinations
of some BCAs may increase the possibility of synergetic action in suppressing
the pathogen (Jain et al. 2011). For example, a triple-compatible
microbial consortium increased enzyme activities and phenol accumulation 1.4 to
4.6 times compared with individual and dual consortia (Jain et al. 2011).
Future research to screen multiple
combinations of these in field trials
may further enhance the disease control potential of these beneficial
micro-organisms.
Biological control products should be extensively
evaluated with robust testing under local conditions before deployment. Not
only must they be effective, BCAs should also be easy to use, non-toxic,
economical, environmentally safe, meet biosecurity concerns, and be acceptable
to growers, consumers, and regulatory agencies. In this regard, Western
Australia has its own biosecurity and plant quarantine regulations and
commercial BCAs from outside of the state would need to be rigorously evaluated
before release to primary producers. Hence, the identification of effective
local BCAs is a priority for research.
Conclusion
For the
first time we identified potential fungal and bacterial BCAs
from Western Australia that suppressed both
growth and sclerotial formation of S.
sclerotiorum in vitro and reduced disease incidence when applied as
foliar applications under glasshouse and field conditions. Mycelial
and sclerotial inhibition ranged from 40–60% and 65–100%
for the F-BCAs and 57–59% and 89–95% for the B-BCAs, respectively.
Selected isolates of F-BCAs (T.
koningiopsis and T. atroviride)
and of B-BCAs (O. anthropi and S. proteamaculans) significantly reduced
disease incidence of S. sclerotiorum
under glasshouse and field conditions. Under field conditions, O. anthropi provided the maximum disease control
efficiency (89%) when the pathogen was applied at 10% flowering. Field
efficacy of tested BCAs was similar or better than the commercial fungicide
Prosaro®. Further studies are required to
understand their mechanism of suppression against S. sclerotiorum
and their ability to persist and to provide protection
under field conditions. In addition, the
life-cycle of the beneficial organisms needs to be understood in an environment
where the climate has increasing variability due to
climate change.
Acknowledgements
We
are thankful to The Australian Centre for International Agricultural Research
(ACIAR) for financial support of this study through John Allwright Fellowship
(JAF) to the first author as the main contibutor of this paper. We gratefully
acknowledge the Department of Primary Industries and Regional Development
(Western Australia) for providing facilities to conduct this research. We thank Mr. Mario D’ Antuouno
and Mr. A Van Burgel,
Biometricians, Department of Primary Industries and Regional Development for
their help with the statistical analysis. Sincere
thanks are extended to Dr. Dwi Praptomo Sudjatmiko, head of Institute for
Assessment of Agricultural Technology (IAAT) West Nusa Tenggara Province –
Indonesia for giving the permission and opportunity for the first author to
continue her study.
Author
Contributions
BN Hidayah (main contributor): planned work,
conducted the experiments, and wrote the manuscript; R Khangura and B Dell
(supporting contributors): supervised work and proof read the manuscript.
Conflict
of Interest
The authors declare that they have no known
conflict of interest.
Data
Availability
Data presented in this study are available on fair
request to the corresponding author.
Ethic
Approval
Not applicable
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