Introduction
The cruciferous vegetable is a large family of
vegetables. There are more than 300 genera divided into more than 3,000 species
originating and distributed globally. Cruciferous vegetables are one of the
most important vegetables in Thailand because they are used for daily
consumption, have commercial value and are also exported to various countries.
The cabbage is a plant in the family Cruciferae (Brassicaceae) that is
important to the food system and economy of the country. Cultivation of cabbage is
distributed throughout Thailand with the quantity and quality of production
dependent on the terrain and climate. The northern region of Thailand is
considered more suitable for growing this vegetable than other regions (Nath et al. 1999).
Currently, the cultivation of cruciferous vegetables in Thailand is facing a
serious problem from microbial infection, resulting in substantial economic
losses (Nath et al. 1999).
Plant diseases are important factors affecting plant growth and agricultural
productivity and may result in reduced production that is insufficient to meet
the demands of the world's ever-increasing population. It may also cause a
shortage of food products for export.
Black
rot disease of cruciferous plants is a common disease that is caused by Xanthomonas
campestris pv. campestris. The infection begins in the seedling
stage, causing the plant to appear stunted and, the lower leaves wither and
V-shaped yellow lesions are formed on the leaf margins. The yellowing spreads
until the midrib and the veins turn black. The contaminated area will become
brown and dry, with the injured leaves falling off before they reach maturity.
When the leaf tissues are cross sectioned, the alimentary canal is black and a
large number of bacteria are present in yellow slime and occasionally in the
mid-stem gap (Agrios 1997). When
infected seeds germinate, the bacterium on the seed coat penetrate to the
cotyledons and juvenile leaves through natural openings or wounds on the roots
and leaves. This causes considerable damage to crops during warm weather and
high rainfall during seedling growth (Français 2022). The exopolysaccharide produced by pathogens is
called xanthan a sticky substance leading to clogging of the xylem and phloem
and damaged cells turn black (Qian et al. 2006). The bacterial pathogen can migrate from
leaves to stems through the xylem under hot and humid conditions. Consequently,
it can move up and down the plant stem (Français 2022).
Pesticide
residues in vegetables are now recognized as having a direct impact on the
consumer health and the country's economy (Ruanpanun and Nimnoi 2020). However,
the chemicals are necessary to control the spread of diseases caused by
microorganisms, where often the spread is rapid, having a severe impact on
crops across a vast area. As a result, farmers choose to apply pesticides for
pest control and prevention because it is a convenient, fast and highly
efficient control method. However, the increased use of chemicals coupled with
a lack of knowledge and suitable care regarding the choice and application by
farmers, ecosystems and natural environments are being destroyed or disrupted
(Abo-Elyousr et al. 2022). The plant pathogen's natural enemies and
microorganisms are eliminated until their numbers decline, resulting in
outbreaks of severe plant disease. There are numerous issues to consider
regarding the long-term effects of applying chemicals to control plant diseases
(Sánchez-Hernández et al. 2022).
Biological control of plant
pathogens is the use of living organisms to reduce the population of plant
pathogens or to reduce the activity of pathogenic plant pathogens to a level
that does not cause economic damage (Khan and Javaid 2020, 2021, 2022). There
are principles that can be carried out to control plant pathogens using
biological approaches by: (1) introducing beneficial microorganisms from the
local source of disease causative agents (Javaid et al. 2021; Sharf et
al. 2021); (2) optimization of the environment for population growth and
its role in the antagonism; (3) maintaining the population and its role by
adding beneficial microorganisms or improving the surroundings; and (4) using
biological control integrated with other plant disease management practices
(Maleki et al. 2010; Ali et al. 2020). Therefore, it is necessary
to study biological control using antagonistic microorganisms that can inhibit
plant pathogenic bacteria in solving the problem of excessive chemical use.
The
Asian cavity-nesting honey bee Apis cerana is widespread in temperate
and tropical Asia including Thailand. A. cerana naturally occurs on an
Asian landscape of some 30 M km2 encompassing a series of climatic
zones from tropical moist rainforest, wet-dry tropical savanna, mid-latitude
steppe, dry mid-latitude grasslands, moist continental deciduous forest, and
taiga (Radloff et al. 2010). A. cerana is one of the rare native
bees found in Thailand. There are few studies on antagonistic bacteria associated
with honey bee (Apis) species in Thailand. Some actinobacteria were
isolated from giant honey bee (Apis dorsata) showed the ability to
inhibit the growth of Xanthomonas oryzae pv. oryzae, Xanthomonas
campestris pv. campestris, Ralstonia solanacearum and Pectobacterium
carotovorum (Promnuan et al. 2020). It is interesting that novel
bacterial species with high antibacterial activities have been found in A.
cerana.
Successful
application of biological control agents can improve the health of farmers and
consumers with the advantage of being a low-cost method that encourages
environmental sustainability (Abo-Elyousr et al. 2022). However, to
successfully apply knowledge of biological control in practical plant
agriculture, product formulations must be developed that are effective at
managing the plant. Therefore,
the current study investigated antagonistic microorganisms that can suppress
plant pathogenic bacteria, delved into the appropriate media for antagonistic
bacteria to grow, used the antagonists to control plant disease in plots, and
developed them to be useful to farmers.
Materials and Methods
Plant pathogenic bacteria
Xanthomonas campestris TISTR 1100 was obtained as a
standard culture from Thailand Institute of Scientific and Technological
Research (TISTR). It was inoculated in cabbage pots (Brassica oleracea)
before its use in this study. The lesions of black rot disease were shown after
pouring the 24 h pathogen culture onto of 24 day-old cabbage seedlings.
Sample collection
Combs of the Asiatic cavity-nesting honeybee (Apis
cerana) were collected from the Mae-rim district, Chiang Mai province,
Thailand in April 2014. The three hive samples were collected from local
villages in private areas. Adult bees, brood cells, pollen and honey were
collected, kept in sterile tubes and stored at -20°C.
Isolation of bacteria from bees
Bacterial strains were isolated from 1 g of each bee
sample (adults, brood cells and pollen using the serial dilution technique as
described by Promsai et al. (2018) with some modifications. The bee
samples were ground, added with 10 mL sterile water and vortexed for 1 min,
before being diluted in about 10 times sterile water and vortexed again for 1
min. One milliliter of successive decimal dilutions was spread on nutrient agar
(Merck®, Germany) containing 25 mg mL–1 cycloheximide and
then incubated at 37°C for 24–48 h. One colony was
randomly picked for streaking on agar plates.
Characterization of isolated bacteria
The bacterial strains that were isolated from native-Thai bees were
incubated at 37°C for 24 h using nutrient
broth. They were basically characterized for surface, color, Gram staining,
cell arrangement and spore formation under a light microscope (CX31; Olympus®,
Japan) as described by Forbes et al. (2002). The catalase activity was
tested for oxidative metabolism using the method of Holt et al. (1994).
Screening of antagonistic bacteria capable of growth
inhibition of plant pathogens
The inhibition of pathogen growth was evaluated using the agar disc
diffusion method (Balouiri et al. 2016). The pathogenic bacterium was
inoculated in 50 mL of yeast extract-malt extract (YM) broth and incubated at
30°C until the turbidity of the
culture equaled a standard McFarland value of 0.5 (approximately 107–108
CFU mL-1). The culture was spread on the surface of YM agar.
Concomitantly, the isolated bacteria were inoculated in NB and incubated at 37°C for 24 h. The culture broths
were centrifuged at 8,000 rpm and 4°C for 15 min. Then, 40 mL of supernatants were dropped
on a paper disc that was placed on a YM agar surface that contained pathogenic
culture. NB was used as a negative control and 1 mg mL–1 of
streptomycin was used as a positive control. Then, the agar plates were
incubated at 30°C for 48 h. The inhibition
zones were observed by measuring the diameter of the clear zone in millimeters.
The experiment was conducted in triplicate.
Hemolytic activity of antagonistic bacteria
The selected bacteria from the growth inhibition testing were evaluated
for hemolytic activity using the stab inoculation method (Chumphon et al.
2016). The strains were cultured on blood agar, consisting of 1% tryptose, 0.5%
sodium chloride, 1.5% agar and supplemented with 7% bovine blood, for 48 h at
37°C. Bacillus cereus was
used as the positive control. Strains which showed green-hued zones around the
colonies (α-hemolysis) or did
not produce any effect on the blood agar (γ-hemolysis)
were classified as displaying non-hemolytic activity. Strains displaying blood
lyses zones around the colonies were classified as hemolytic (β-hemolysis).
Molecular identification of antagonistic bacteria using
16S rDNA sequencing
Genomic DNA samples of 12 bacterial isolates capable of inhibiting the
plant pathogen were extracted (Promsai et al. 2018). Almost-complete 16
Svedberg units of the ribosomal ribonucleic acid (16S rRNA) gene (1.5 kb) were
amplified using the universal primer pair 20F
(5′AGTTTGATCCTGGCTC-3′) and 1540R
(5′-AAGGAGGTGATCCAGCC-3′) (Nakajima et al. 1999). The 16S
rDNA gene was amplified using polymerase chain reaction (PCR; Mulyigne Optimax;
Labnet®; USA). The PCR products were purified using Nucleo Spin® Gel and a PCR
Clean-up Kit (Invitrogen; USA), following the manufacturer’s protocol. The
purified PCR products were directly sequenced by the First Base Company,
Malaysia using primers 20F and 1540R as sequencing primers. The identities of
nucleotide sequences of the 16S rRNA gene obtained were subjected to BLAST
analysis using the NCBI database (http://www.ncbi.nlm.nih.gov). The sequence
information was supplied regarding the deposition of DNA sequences. The
almost-complete 16S rRNA genes sequences are accessible via GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The
relationships and diversity were studied in the evolutionary lines of the
protease-producing bacteria isolated from bee samples. The bacterial sequences
were studied using the Bio Edit Sequence Alignment Editor version. 7.0.5.3
Primer with Clustal_W multiple alignment and 1,000 bootstraps. The phylogenetic
tree was developed by calculating the distance between the molecular sequences
using the maximum-likelihood method with the MEGA X program, version 10.1.8 to
present an evolution chart.
Spore potential of antagonistic bacteria
According
to the results of antibacterial activities, hemolytic activities and molecular
identification, Bacillus species which had no pathogenicity and showed
potent antibacterial effects, were selected for further studies. Among
KUKPS-C3BN2 and KUKPS-C16HM4, the isolate KUKPS-C16HM4 were used as
representative. In
this study, the isolates KUKPS-C3AN5 and KUKPS-C16HM4 were then evaluated the
spore potential. The method of spore potential was modified
from Foldes et al. (2022) and Oscariz et al. (1999). The
isolates KUKPS-C3AN5 and KUKPS-C16HM4 were cultivated in Luria Bertani (LB)
broth at 37°C for
48 h. Then, the culture broth samples were incubated in a water bath at 80°C for
15 min. Viable cell counts were determined using the dilution plate technique
on LB agar and incubated 37°C for
48 h. The survival rate percentages were calculated using the following
equation:
Survival rate (%) = Cell
number in treated (CFU mL-1) / Cell number in control (CFU mL-1)
× 100
Where treated is the treatment that was incubated at 80°C for 15 min and control is
the treatment that was incubated at 37°C for 15 min.
Development of antagonist product
Preparation of antagonist
inoculum: The
antagonistic bacteria were inoculated in NB and incubated at 37°C for
24 h. Each culture broth sample was measured using a spectrophotometer to
adjust the optical density to 1.0 at 540 nm (approximately 108 CFU
mL-1), followed by centrifugation at 8,000 rpm at 4°C for
10 min to obtain a cell pellet. The obtained cell pellets (20–30% moist) were
used for mixing with carriers.
Preparation of antagonist product
using soybean and mung bean extract: The patented soybean and mung bean extract
medium (Promsai and Chumphon 2021) was prepared and sterilized at 121°C for
15 min. The medium was cooled for 30 min prior to mixing with the cell pellets
of the antagonistic bacteria. The antagonist products were kept at room
temperature for 60 days.
Preparation of antagonist
product using rice grains as carrier: The rice grains were prepared and sterilized
at 121°C for
15 min. After cooling, the rice grains were mixed with cell pellets of the
antagonistic bacteria and the antagonist products were kept at room temperature
for 60 days.
Investigation of cell number
survival in antagonist products: The viable cell count of antagonistic
bacteria was evaluated using the serial dilution plate count technique. Briefly,
a sample of 1 g or 1 mL of antagonist products was added in 0.85% NaCl and
diluted in 0.85% NaCl until the proper dilution was achieved. The decimal
dilution was spread on NA at 37°C for
48 h. The viable cell number was determined weekly and the survival rate was
calculated at day 60 using the equation (Bao et al. 2010):
Survival rate (%) = Log N1
/ Log N0 × 100
Where, N1 is cell number of antagonistic bacteria at
day 60 (CFU mL-1 or CFU g-1) and N0 is
cell number of antagonistic bacteria at day 0 (CFU mL-1 or CFU g-1).
Evaluation of capacity of pathogenic inhibition of
antagonist products
The
inhibition of X. campestris by antagonist products was investigated
using the paper disc diffusion method. The overnight culture of pathogenic
bacteria (approximately 107–108 CFU mL-1) was
spread on YM agar. One milliliter of soybean-mungbean antagonist product was
centrifuged at 8,000 rpm and 4°C for 10 min to obtain the supernatant. After that,
10 mL of supernatant were dropped onto a paper disc (6
mm diameter) and the immersed paper disc was placed on the YM agar. The
evaluation of the rice grains used 1 g of the rice grain antagonist product
with 5 mL of sterile 0.85% NaCl and was mixed well. After that, 10 mL of solution were dropped onto a paper disc (6 mm
diameter) and the immersed paper disc was placed on the YM agar. Untreated
soybean-mungbean extract medium or rice grains were used as the respective
negative controls. Then, the agar plates were incubated at 30°C for 48 h. The inhibition zones were observed by
measuring the diameter of any clear zone in millimeters. The experiment was
conducted in triplicate.
Statistical analysis
The results of the inhibition of pathogenic bacteria were statistically
analyzed in IBM SPSS Statistics software by analysis of variance (ANOVA),
followed by a post hoc comparison of means by Duncan Multiple Range’s test.
Results
Re-infection of X. campestris in cabbages
From the
results of re-infection, the infected plants showed V-shaped yellow-brown
lesions on the tips of the cabbage leaves that is a unique characteristic of
black rot disease (Fig. 1).
Isolation of bacteria from native Thai bees
Using the serial dilution technique, 106 pure isolates of bacteria were
recorded from bees, pollen, brood cells and honey (32, 33, 20 and 21 isolates,
respectively). Most of the bacterial isolates grew well at 37°C on NA. The morphological
characteristics of all 106 isolates were examined using Gram staining and
observed under a microscope as well as using a catalase test and investigating
growth characteristics on the medium. It was found that the growth
characteristics on culture medium showed the colony sizes in the range 1–7 mm,
the colony shape was round and the colonies of some isolates were amorphous.
The colony surfaces were both glossy and matte. Two types of colony colors were
observed: translucent white and opaque white. Cell shapes were round and
rod-shaped. Some bacterial isolates produced endospores inside cells.
Antibacterial activity of antagonistic bacteria
All isolated bacteria were tested for their inhibition ability against
the black rot pathogen (X. campestris TISTR 1100) using the agar disc
diffusion method on YM agar medium. It was found that
12 isolated bacteria could inhibit growth of X. campestris and each of
these isolates had a slightly different capacity to inhibit the growth of the
plant pathogen (Table 1).
Hemolytic activity of isolated bacteria
The selected bacteria were tested for hemolytic activity to consider the
microbiological safety of the bacterial strains before any development of
antagonist products. The results indicated that 3 isolates, namely KUKPS-C3BN8,
KUKPS-C17AN4 and KUKPS-C17AN6 showed hemolytic activity, whereas 9 isolates,
namely KUKPS-C2AN1, KUKPS-C2AN2, KUKPS-C2AN3, KUKPS-C3AN5, KUKPS-C3BN2,
KUKPS-C16HN7, KUKPS-C16HM4, KUKPS-C17AN3 and KUKPS-C18AN6 did not show any
hemolytic activity (Table 2).
Molecular identification and construction of
phylogenetic tree
After the 16s RNA gene analysis of selected bacteria, it was found that
the isolates KUKPS-C3BN2, KUKPS-C3AN5 and KUKPS-C16HM4 had similarity with Bacillus
spp. of 96, 97 and 96%, respectively. The similarity values of the antagonistic
bacterial isolates KUKPS-C2AN2, KUKPS-C16HN7 and KUKPS-C17AN3 to Kluyvera
spp., Enterobacter spp. and Enterobacter spp. were 98%, 99% and
96%, respectively. Subsequently, the 16s RNA gene sequences of the antagonistic
bacteria Bacillus spp. were evaluated closely using phylogenetic tree
construction (Fig. 2).
Based on the phylogenetic tree construction, the maximum likelihood tree
confirmed the placement of the isolate KUKPS-C3AN5 to B. tequilensis, while
the isolates KUKPS-C3BN2 and KUKPS-C16HM4 were B. thuringiensis.
Spore production of antagonistic bacteria
From the results of the study of the morphological characteristics under
a microscope at 1,000x magnification, B. tequilensis KUKPS-C3AN5 and B.
thuringiensis KUKPS-C16HM4 formed intracellular endospores. Thus, efficacy
of spore tolerance to heat was investigated in this study. The LB culture broth
was incubated at 80°C for 15 min to test the
ability of spore production following heat. As shown in Table 3, B.
tequilensis KUKPS-C3AN5, B. thuringiensis KUKPS-C16HM4 and B.
subtilis (the comparable standard culture) had spore survival rates of 21,
38 and 71%, respectively.
%20265-273_files/image002.png)
Fig. 1: Lesions on cabbage leaves
of re-infection
of X. campestris TISTR 1100 after inoculation at leaf germination stage.
Arrows indicate the V-shaped
yellow lesions
Table 1: Growth
Inhibition of bacterial isolates against X. campestris
|
Antagonistic bacteria |
Gram staining |
Diameter of inhibition zone (mm)* |
|
KUKPS-C2AN1 |
Positive |
17.7 ± 0.6 BC |
|
KUKPS-C2AN2 |
Negative |
17.3 ± 0.6 BC |
|
KUKPS-C2AN3 |
Negative |
17.3 ± 0.6 BC |
|
KUKPS-C3AN5 |
Positive |
22.0 ± 1.0 B |
|
KUKPS-C3BN2 |
Positive |
32.3 ± 1.5 A |
|
KUKPS-C3BN8 |
Positive |
12.0 ± 10.4 C |
|
KUKPS-C16HN7 |
Negative |
22.3 ± 3.5 B |
|
KUKPS-C16HM4 |
Positive |
21.0 ± 1.7 B |
|
KUKPS-C17AN3 |
Negative |
22.3 ± 4.0 B |
|
KUKPS-C17AN4 |
Negative |
20.3 ± 5.5 B |
|
KUKPS-C17AN6 |
Negative |
33.3 ± 2.3 A |
|
KUKPS-C18AN6 |
Negative |
24.3 ± 2.1 B |
* Data are presented as the mean
± standard deviation. Values within the column followed by different uppercase
superscripts are significantly different at P
< 0.05 level
Table 2: Hemolytic activity of antagonistic bacteria on blood
agar
|
Antagonistic bacteria |
Hemolytic activity |
||
|
Beta-hemolysis |
Alpha-hemolysis |
Gamma-hemolysis |
|
|
KUKPS-C2AN1 |
|
|
ü |
|
KUKPS-C2AN2 |
|
|
ü |
|
KUKPS-C2AN3 |
|
|
ü |
|
KUKPS-C3AN5 |
|
|
ü |
|
KUKPS-C3BN2 |
|
|
ü |
|
KUKPS-C3BN8 |
ü |
|
|
|
KUKPS-C16HN7 |
|
|
ü |
|
KUKPS-C16HM4 |
|
|
ü |
|
KUKPS-C17AN3 |
|
|
ü |
|
KUKPS-C17AN4 |
ü |
|
|
|
KUKPS-C17AN6 |
ü |
|
|
|
KUKPS-C18AN6 |
|
|
ü |
ü Positive result
Table 3:
Efficacy of endospore survival of antagonistic bacteria after incubation at 80°C for 15 min
|
Isolates |
Viable cell count (cells) after incubation at |
Endospore survival rate (%) |
|
|
37°C |
80°C |
||
|
B. tequilensis KUKPS-C3AN5 |
48 |
10 |
21 |
|
B. thuringiensis KUKPS-C16HM4 |
34 |
13 |
38 |
|
B. subtilis (control) |
31 |
22 |
71 |
Feasibility study of development of antagonist product
B. tequilensis KUKPS-C3AN5 and B. thuringiensis KUKPS-C16HM4 were selected for
the development of antagonist products using rice grains and soybean-mung bean
extract as carriers. From the results of survival ability and efficacy against
plant pathogenic bacteria (X. campestris) after 60 days of storage, the
survival rates of antagonistic bacteria cultivated in rice grain carrier were
higher than for the soybean-mungbean extract carrier (Fig. 3). Additionally,
the efficacy against plant pathogens showed that B. tequilensis
KUKPS-C3AN5 was more effective than B. thuringiensis KUKPS-C16HM4 (Fig.
4).
Discussion
The result of a re-infection in cabbage of X. campestris TISTR
1100, which is a standard culture obtained from the Thailand Institute of
Scientific and Technological Research %20265-273_files/image004.jpg)
Fig. 2: Maximum likelihood phylogenetic tree of antagonistic bacteria and Bacillus
species based on 16S rRNA sequences analysis. Scientific names in parentheses
are the originally proposed names of the strains and numbers in parentheses are
GenBank EMBL database accession numbers
%20265-273_files/image006.png)
Fig. 3: Viable cell count of antagonistic bacteria after 8 weeks of storage in 2
carriers (soybean-mungbean and rice grains)
(TISTR), confirmed that this pathogen could cause the severe lesion in
cabbage. Other studies reported that bacteria in the genus Xanthomonas
were the major causative agents on vegetable crops, especially X. campestris
pv. campestris, regarding black rot in cabbage, cauliflower and lettuce.
Rubel et al. (2017) reported the occurrence of black rot disease in
seeds infected with X. campestris pv. campestris as one of the
most common diseases in cruciferous plants and that it could reduce the crop
yield by more than 50 percent under the proper growth conditions.
In general, the application of antagonistic products is often used in the
form of a spray or seed coating which means that there is a chance that
consumers and farmers may come in direct contact with the microorganisms.
Therefore, it is necessary to study the risk characteristics that might cause
harm to consumers and farmers. Red blood cell lysis is one of the distinctive
features of human pathogens. Human blood is made up of a solid part (45%) that
is mostly blood cells and platelets, whereas the liquid part (55%) is plasma (Tuchin
et al. 2004). Most human pathogens are capable of lysis of red blood
cells and these pathogens can be divided into 3 groups: (1) alpha hemolytic
group incomplete lysis of red blood cells (a greenish-brown zone around the
colonies of the pathogens on blood agar), %20265-273_files/image008.png)
Fig. 4: Efficacy of growth inhibition of X. campestris by antagonistic
bacteria cultured on 2 carriers (soybean-mungbean and rice grains). The storage
of antagonistic products was examined for 8 weeks, and the agar diffusion
method was performed every week. Values within the
week followed by different uppercase letter are significantly different at P < 0.05 level
for example Streptococcus
viridans; (2) beta hemolysis the complete lysis of red blood cells (a clear
zone around the colony), for example, B. cereus and (3) gamma hemolysis
or non-hemolytic group no red blood cell lysis and no zone around the colony
(Chumphon et al. 2021). From the current study, the 6 isolates that were
non-hemolytic strains were selected for molecular identification. Some strains
which belonged to the genus Bacillus, were selected for construction of
phylogenetic tree to correctly identify the bacterial strains. The evolutionary
tree illustrates how different living things have evolved from members of the
same species due to variations in genes or proteins. The results of these
investigations are frequently represented in a graphic that shows the
connection between or the site of the recent gene modifications. In the current
study, based on the results of hemolytic activity and molecular identification,
2 strains of antagonistic microorganisms (B. tequilensis KUKPS-C3AN5 and
B. thuringiensis KUKPS-C16HM4) that were expected to have potential,
were used for the development of antagonist product.
This research was consistent
with several studies in finding that several species of Bacillus was
often used in the development of antagonistic microorganisms for the control of
various plant diseases. Wulff et al. (2002) investigated the biochemical
and molecular characteristics of antagonistic bacteria (Bacillus
amyloliquefaciens, B. subtilis and B. pumilus) capable of
producing secondary metabolites and inhibiting the black rot pathogen X.
campestris pv. campestris in cauliflower. Their results revealed that
B. amyloliquefaciens could produce surfactin, iturin,
bacillomucine and azalomycin F, while B. subtilis could produce
surfactin and arthrobactin, whereas B. pumilus could produce surfactin,
amphomycin, arthrobactin and valinomycin that could reduce the black rot caused
by X. campestris pv. campestris. These results were in accordance
with the findings of Todorova and Kozhuharova (2010) who evaluated the
antagonistic properties and antimicrobial agents of B. subtilis
that had been isolated from soil. The isolates B. subtilis TS 01
and ZR 02 could inhibit the growth of the pathogenic fungi Alternaria solani,
Botrytis cinerea, Monilia linhartiana 869, Phytophthora
cryptogea 759/1 and Rhizoctonia spp. In addition, the isolates TS 01
and ZR 02 could inhibit Pseudomonas syringae pv. tomato and X.
campestris with zones of inhibition of 48.0 and 50.0 mm, respectively. In
addition, Mishra and Arora (2012) investigated the plant root-associated
bacteria Pseudomonas and Bacillus for the control of the black
rot pathogen X. campestris pv. campestris
(Xcc) in cruciferous plants. Their results indicated that 54 bacterial isolates had ability
to inhibit the growth of Xcc. Two isolates, namely KA19 and SE produced
inhibition zone diameters of more than 11 mm that increased to 18.1 mm when the
mixed culture was used. From the results of 16s RNA gene and phylogenetic tree
analysis, the isolates KA19 and SE were closely related with Pseudomonas
aeruginosa and Bacillus thuringiensis, respectively. When these 2
antagonistic bacteria were evaluated in field conditions by spraying the
leaves, soaking the seeds and kneading the soil, it was found that both strains
were able to reduce the symptoms of black rot disease compared to the
uninoculated plants. In addition, the growth inhibition of pathogens was more
effective when mixed cultures (KA19 + SE) were used.
Endospores are thick-walled
structures that are resistant to adverse environmental conditions, such as
radiation, stress, chemical disinfectants, extreme heat or cold, and a food
shortage. Furthermore, endospores can regenerate into vegetative cells when
grown in a suitable soil and environment; these can be highly effective at
controlling pathogens immediately. Consequently, endospore-forming bacteria
would be suitable for application in the development of endospore products to
extend product shelf life and ease of use. A B. subtilis endospore
product was developed for the control of wilt disease in ginger and the
endospores of B. subtilis were able to withstand temperatures up to
100°C (Udomsak 2009).
Notably, although B.
subtilis had a high potential spore production, it is a high risk for
humans and animals. Based on the survival rates of the endospores of the
selected isolates, they could readily be developed into live products due for
use in a real planting environment as the highest temperature found in the
plots was 38–42°C and the highest temperature
at which the pathogen X. campestris could grow was 40°C. Furthermore,
endospore-forming products would be easier to keep and to extend their shelf
life compared to the live cells of bacteria.
In this research, the focus
was on the development of formulas of ingredients suitable for the growth of
antagonistic bacteria while maintaining the ability to inhibit the growth of
the pathogen. In the study of the suitable formulation of the medium or carrier
for survival, the results showed that B. tequilensis KUKPS-C3AN5 strains
that had been stored for 8 weeks in the soybean-mungbean medium had a survival
rate of 99.10%, while the culture that was stored in rice grains had a survival
rate of 106.42%. Similarly, the survival rates for B. thuringiensis KUKPS-C16HM4
stored in the soybean-mungbean medium and rice grains, were 99.19 and 108.34%,
respectively. Accordingly, the survival rates of both strains in either of the
carriers were not different. However, soybean-mung bean cost was higher than
for the rice grains. However, to consider the effect on survival rate and
efficacy in suppressing black rot pathogens, the soybean-mungbean was more
cost-effective because of its inhibition efficacy in cases of severe black rot
disease pathogen infestation in vegetable plots. It is necessary to use
antagonists that can inhibit pathogens quickly and efficiently to achieve
effective control of the spread of disease and the resulting damage to
vegetables. From the observations on the inhibitory efficacy of B.
tequilensis KUKPS-C3AN5 and B. thuringiensis KUKPS-C16HM4 cultured
in soybean-mungbean and rice grain carriers for 2 months, both strains cultured
in the soybean-mungbean medium were more effective at inhibiting pathogens than
those cultured in rice grains. Comparing the two strains cultured in the same
medium, B. tequilensis KUKPS-C3AN5 cultured in the soybean-mungbean
medium had the better inhibition of pathogens with an inhibition zone of 16.78
mm, while the inhibition zones of B. tequilensis KUKPS-C3AN5 cultured in
rice grains, of B. thuringiensis C16HM4 cultured in soybean-mungbean
medium and of B. thuringiensis C16HM4 cultured in rice grains were
11.44, 9.78 and 8.22 mm, respectively. In addition, after 2 weeks of
inoculation, the inhibition efficacy of the black rot pathogens decreased. This
seemed to indicate that antagonist products should not be inoculated for more
than 14 days and should be re-inoculated every time before use.
From the experimental results,
it can be concluded that for inhibition efficacy of X. campestris
pathogens of both antagonists the following factors were important: a complete
supply of essential nutrients for the growth of the antagonists and water as
one of the main factors essential for bacterial growth, as each strain of
microorganism requires a different amount of moisture to grow. The moisture in
the medium that can be used by microorganisms is in the form of available water
or water activity (Aw). The free water surrounding the medium is not
absorbed by the medium or ions of other substances where bacteria have a
minimum Aw level of 0.91 (Barbosa‐Cánovas et al. 2020). The optimum Aw
value for bacterial growth is close to 1.0, which is a high value and at that
point there will be dissolved nutrients suitable for the growth of
microorganisms. Sirisoontaralak et al. (2013) reported that normal
cooked rice had Aw values in the range 0.93–0.97, which was close to
that for the rice grains used in the current study, while the soybean-mung bean
medium in liquid form had an Aw value of 1.0. It may be concluded
from the above information that the suitable medium should consider the
nutrients and have an Aw value sufficient for culturing. Future work
should include: a comparative study of the use of mixed microorganisms in the
inhibition of pathogens; optimization of the nutrients or physical factors
necessary for the growth of antagonistic microorganisms; and the
characterization of inhibiting substances produced by antagonistic
microorganisms to develop into potential products suitable to be used in
planting plots.
Conclusion
According to this research, B. tequilensis KUKPS-C3AN5 and B.
thuringiensis KUKPS-C16HM4 are antagonistic bacteria with potential
application as a growth control product for X. campestris, a causative
agent of black rot in cruciferous plants. Both strains could produce spores but
not lysis red blood cells, which confirmed they did not harm humans and
animals. From the application of soybean-mung bean and rice grains to develop
into antagonist products, it was found that the survival rates for both strains
of Bacillus were similar. Additionally, B. tequilensis
KUKPS-C3AN5 was more effective in inhibiting black rot pathogens than B.
thuringiensis KUKPS-C16HM4. Finally, the more suitable carrier for product
development was the soybean-mungbean extract medium.
Acknowledgements
This research was financially supported by the Research Promotion and
Technology Transfer Center (RPTTC), Faculty of Liberal Arts and Science,
Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom Year 2019.
Author Contributions
SP contributed to the conception and design of the experiments. SP, YP,
and SM planned the experiments, and sample collection. SP and KK conducted and
investigated the experiments. SP prepared original draft manuscript. SP, YP,
and SM performed final revision and reviewed the manuscript. SP supervised and
administered research.
Conflict of Interest
All authors declare no conflict of interest.
Data Availability
Data presented in this study will be available on a fair request to the corresponding
author.
Ethics
Approval
Not applicable in this manuscript
Funding Source
Research Promotion and Technology Transfer Center
(RPTTC), Faculty of Liberal Arts and Science, Kasetsart University, Kamphaeng
Saen Campus, Nakhon Pathom Year 2019.
References
Abo-Elyousr KAM, MASAAH Seleim, NM Almasoudi, HMMK Bagy (2022).
Evaluation of native bacterial isolates for control of cucumber powdery mildew
under greenhouse conditions. Horticulturae 8:1143
Agrios GN (1997). Plant Pathology, 4th edn. Academic
Press, San Diego, California, USA
Ali
A, A Javaid, A Shoaib, IH Khan (2020). Effect of soil amendment with Chenopodium album dry biomass and two Trichoderma species on growth of
chickpea var. Noor 2009 in Sclerotium
rolfsii contaminated soil. Egypt J
Biol Pest Cont 30:102
Balouiri M, M Sadiki, SK Ibnsouda (2016). Methods for in vitro
evaluating antimicrobial activity: A review. J Pharm Anal 6:71‒79
Bao Y, Y Zhang, Y Zhang, Y Liu, A Wanga, X Dong, Y Wang, H Zhang (2010).
Screening of potential probiotic properties of Lactobacillus fermentum
isolated from traditional dairy products. Food
Cont 21:695‒701
Barbosa‐Cánovas GV, AJ Fontana, SJ Schmidt, TP Labuza
(2020). Water Activity in Foods, 2nd edn. John
Wiley & Sons, Chicago, Illinois, USA
Chumphon T, K Pangjit, S Promsai (2021). Innovative production of
multistrain synbiotic product using Thai-pigmented rice and rice bran oil. Intl
J Food Sci Technol 56:2182‒2192
Chumphon T, P Sriprasertsak, S Promsai (2016). Development of rice as
potential carriers for probiotic Lactobacillus amylovorus. Intl J
Food Sci Technol 51:1260‒1267
Foldes T, I Banhegyi, Z Herpai, L Varga, J Szigeti (2022). Isolation of Bacillus
strains from the rhizosphere of cereals and in vitro screening for
antagonism against phytopathogenic, food-borne pathogenic and spoilage
microorganisms. J Appl Microbiol 89:840‒846
Forbes BA, DF Sahm, AS Weissfeld (2002). Bailey & Scott’s
Diagnostic Microbiology, 11th edn. Mosby Inc., St. Louis, Missouri,
USA
Français FR (2022) Black Rot of Crucifer Crops. Available at: http://www.omafra.gov.on.ca/english/crops/facts/02-025.htm
(Accessed: 1 October 2022)
Holt JG, NR Krieg, PHA Sneath, JT Staley, ST Williams (1994). Bergey’s
Manual of Determinative Bacteriology, 9th edn. Williams and
Wilkins, Philadelphia, Pennsylvania, USA
Javaid A, A
Ali, A Shoaib, IH Khan (2021). Alleviating stress
of Sclertium rolfsii on growth of
chickpea var. Bhakkar-2011 by Trichoderma
harzianum and T. viride. J Anim Plant
Sci 31:1755‒1761
Khan
IH, A Javaid (2022). DNA cleavage of the fungal pathogen and production of
antifungal compounds are the possible mechanisms of action of biocontrol agent Penicillium italicum against Macrophomina phaseolina. Mycologia 114:24‒34
Khan
IH, A Javaid (2021). In vitro
screening of Aspergillus spp. for
their biocontrol potential against Macrophomina
phaseolina. J Plant Pathol 103:1195‒1205
Khan
IH, A Javaid (2020). In vitro biocontrol potential of Trichoderma pseudokoningii against Macrophomina phaseolina. Intl J Agric Biol 24:730‒736
Maleki M, S Mostafaee, L Mokhtarnejad, M Farzaneh (2010).
Characterization of Pseudomonas fluorescens strain CV6 isolated from
cucumber rhizosphere in Varamin as a potential biocontrol agent. Aust J Crop
Sci 4:676‒683
Mishra S, NK Arora (2012) Evaluation of rhizospheric Pseudomonas
and Bacillus as biocontrol tool for Xanthomonas campestris pv. campestris.
World J Microbiol Biotechnol 28:693‒702
Nakajima Y, V Kitpreechavanich, K Suzuki, T Kudo (1999). Microbispora
corallina spp. nov., a new species of the genus Microbispora
isolated from Thai Soil. Intl J Syst Bacteriol 49:1761‒1767
Nath P, M Papademetriou, K Piluek, EM Herath (1999). The Vegetable
Sector in Thailand, a Review. Available at: https://www.fao.org/3/ AC145E/AC145E00.htm#TOC
(Accessed: 1 October 2022)
Oscariz JC, I Lasa, AG Pisabarro (1999). Detection and characterization
of cerein 7, a new bacteriocin produced by Bacillus cereus with a broad
spectrum of activity. FEMS Microbiol Lett 178:337‒341
Promsai S, T Chumphon (2021). Formulation and manufacturing process of
bean extract media for culturing of probiotic microorganisms. Petty Pat
Thailand 18468. Patent No. WO2016109509A1
Promsai S, P Sriprasertsak, S Meelai, Y Promnuan, T Chumphon (2018).
Selection and validation of carbohydrate-utilizing bacteria as a new probiotic
candidate to develop probiotic-supplemented Thai rice cultivar product. Chiang
Mai J Sci 45:717‒730
Qian F, L An, X He, Q Han, X Li (2006). Antibacterial activity of
xantho-oligosaccharide cleaved from xanthan against phytopathogenic Xanthomonas
campestris pv. campestris. Proc Biochem 41:1582‒1588
Radloff SE, C Hepburn, HR Hepburn, S Fuchs, S Hadisoesilo, K Tan, MS
Engel, V Kuznetsov (2010). Population structure and classification of Apis
cerana. Apidologie 41:589‒601
Ruanpanun P, P Nimnoi (2020). Evaluation on the efficiency and
persistence of Streptomyces jietaisiensis strain A034 in controlling
root knot disease and promoting plant growth in the plant-parasitic nematode
infested soils. Biol Cont 144:104221
Rubel MH, AHK Robin, S Natarajan, JG Vicente, HT Kim, JI Park, IS Nou
(2017). Whole-genome re-alignment facilitates development of specific molecular
markers for races 1 and 4 of Xanthomonas campestris pv. campestris,
the cause of black rot disease in Brassica oleracea. Intl J
Mol Sci 18:2523
Sánchez-Hernández E, P Martín-Ramos, J
Martín-Gil, A Santiago-Aliste, S Hernández-Navarro, R Oliveira, V
González-García (2022). Bark extract of Uncaria tomentosa L. for the control of strawberry
phytopathogens. Horticulture 8:672
Sharf
W, A Javaid, A Shoaib, IH
Khan (2021). Induction of resistance in chili against Sclerotium rolfsii by plant growth
promoting rhizobacteria and Anagallis
arvensis. Egypt J Biol Pest Cont 31:16
Sirisoontaralak P, A Sirapragasit, A Vititteeranont (2013). Gamma
irradiation of cooked rice. Agric Sci J 44:501–504
Todorova S, L Kozhuharova (2010). Characteristics and antimicrobial
activity of Bacillus subtilis strains isolated from soil. World J
Microbiol Biotechnol 26:207‒1216
Tuchin VV, DM Zhestkov, AN Bashkatov, EA Genina (2004). Theoretical study
of immersion optical clearing of blood in vessels at local hemolysis. Opt
Exp 12:2966‒2971
Udomsak B (2009). Development of Bacillus subtilis endospore
forming products for ginger wilt control. In: Proceedings of 47th Kasetsart University
Annual Conference. Bangkok, Thailand, 17–20 March 2009
Wulff EG, CM Mguni, K Mansfeld‐Giese, J Fels, M Lübeck, J
Hockenhull (2002). Biochemical and molecular characterization of Bacillus
amyloliquefaciens, B. subtilis and B. pumilus isolates with
distinct antagonistic potential against Xanthomonas campestris pv. campestris.
Plant Pathol 51:574‒584