Khóa luận tốt nghiệp Công nghệ sinh học: Biocontrol of Ralstonia solanacearum by the plant-promoting Bacillus spp

in preventing thebacterial wilt disease caused by Ralstonia solanacearum and promoting plant growththrough selecting the antagonistic Bacillus strains against R.. solanacearum showed tha

MINISTRY OF EDUCATION AND TRAINING

NONG LAM UNIVERSITY HO CHI MINH CITY

FACULTY OF BIOLOGICAL SCIENCES

BIOCONTROL OF Ralstonia solanacearum BY THE PLANT-PROMOTING Bacillus spp.

Major : BIOTECHNOLOGY

Student : NGUYEN THI BUU CHAU

Student ID : 19126017

Academic year : 2019 - 2023

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCES

because they were always willing to support me and give me useful advice.

Moreover, I would like to thank all the members of Laboratory of MolecularPathogenesis and Diagnostics - Room RIBE 304 and Laboratory of Functional

Genomics - Room BT306 - who always supported, helped and encouraged me duringthis thesis

Finally, I would like to thank my friends, who always followed, supported andencouraged me everywhere

AFFIRMATION AND COMMITTMENT

My name is: Nguyen Thi Buu Chau, student ID: 19126017, class: DH19SHB

of Biotechnology, Nong Lam University Ho Chi Minh city I guarantee that thisgraduate thesis which was conducted by myself All the data and information arecompletely honest and objective I take full responsibility in front of the committeefor these commitments

Ho Chi Minh city, February 28", 2024

Student’s signature

This study aimed to evaluate the effectiveness of Bacillus spp in preventing thebacterial wilt disease caused by Ralstonia solanacearum and promoting plant growththrough selecting the antagonistic Bacillus strains against R solanacearum on Capsicum

annuum L plants under greenhouse conditions To select the Bacillus strains which have

the highest activity against R solanacearum, 14 Bacillus strains were tested antibacterialactivity R solanacearum on TTC agar by agar diffusion method According to the result,five out of 14 Bacillus strains including: Asl-1, Asl-2, Asl-5, 62 and T2-1, were selected.Among these, two strains showed the highest antibacterial activity, with the largestinhibition zone diameters in both suspension and supernatant, which were: Asl-5 (40.667

mm), (37.000 mm) and Asl-2 (38.333 mm), (34.333 mm), respectively Experimented

Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC)

of five Bacillus cell-free supernatant against R solanacearum showed that cell-free

supernatant of Bacillus strains were active at so high concentrations, three out of five

Bacillus cell-free supernatant were Asl-1, Asl-2, Asl-5 can suppressed bacterial growth of

R solanacearum with the MIC and MBC values of: 125 and 1000 wL/mL; 250 and 1000uL/mL, 7.8125 and 500 wL/mL For the strain 62 and T2-1, only the minimum inhibitoryconcentration was found The final result showed that pretreatment with the five Bacillusstrains selected can reduce the incidence of bacterial wilt

Keywords: Bacillus spp., R solanacearum, Baterial wilt disease, plant-promoting,

Solanaceous crop plants

TÓM TAT

Nghiên cứu này nhằm đánh giá hiệu quả của Bacillus spp trong phòng ngừa bệnh héo xanh vi khuân do Ralstonia solanacearum gây ra và kích thích sinh trưởng cây trồng

thuộc họ cà bằng cách chọn lọc các chung Bacillus có khả năng đối kháng sử dụng

phương pháp khuếch tán trên đĩa thạch, sau đó đánh giá hoạt tính kích thích sinh trưởng

và kháng khuân của các chủng Bacillus với R solanacearum trên cây Capsicum annuum

L trong điều kiện nhà lưới Dé chon lọc các chung Bacillus có hoạt tính khang Ralstonia solanacearum tốt nhất, 14 chung Bacillus đã được thử nghiệm hoạt tính khang R solanacearum trên môi trường thạch TTC bằng phương pháp khuếch tán trên đĩa thạch Theo kết quả, năm trong số 14 chủng Bacillus bao gồm Asl-1, Asl-2, Asl-5, 62 và T2-1

đã được chọn lọc Trong đó, hai chủng có hoạt tính kháng khuẩn cao nhất với đường kính vùng ức chế lớn nhất ở cả huyền phù, dịch nổi lần lượt là Bacillus cereus Asl-5(40.667 mm), (37.000 mm) va Bacillus amiloliquefaciens Asl-2 (38.333 mm), (34.333mm) Thi nghiệm đánh giá nồng độ ức chế tôi thiểu (MIC) và nồng độ diệt khuẩn tối thiêu (MBC) của dịch nổi năm chủng Bacillus kháng lại R solanacearum cho thay dich nỗi của các chủng Bacillus hoạt động ở nồng độ cao, dịch nỗi của ba trong số năm chủng Bacillus là Asl-1, Asl-2, Asl-5 có thé ức chế sự phát triển của vi khuẩn R solanacearumvới giá tri MIC va MBC là: 125 và 1000 pL/mL; 250 và 1000 w/mL, 7.8125 và 500uL/mL Về chủng 62 và T2-1 chi tìm thấy nồng độ ức chế tối thiểu Kết quả cuối cùng cho thay tiền xử lý năm chủng Bacillus được chon lọc có thé làm giảm mức độ nghiêm

trọng của bệnh héo xanh do vi khuân R solanacearum gây ra

Từ khóa: Bacillus spp., R solanacearum, Bệnh héo do vi khuẩn, kích thích sinh trưởng cây trồng, cây họ Cà.

TABLE OF CONTENT Pare S

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CHAPTER 3 MATERIALS AND METHODS 153.1 Time and location 153.2 Materials and methods 153.2.1 Samples 153.2.2 Equipments and tools 163.2.2.1 Equipments 163.2.2.2 Tools 163.2.3 Bacteria culture media 163.2.4 Testing antibacterial activities R solanacearum by Bacillus spp in vitro

conditions 16

3.2.5 Determination of minimum inhibitory concentration (MIC) and Minimum

bactericidal concentration (MBC) of Bacillus strains against R solanacearum 173.2.5.1 Prepare the ELISA microplate 17

3.2.6 Determination of plant-promoting and antibacterial activities of Bacillus strains

with R solanacearum on Capsicum annuum L plants under greenhouse conditions 173.2.6.1 Preparation of bacterial inoculum 173.2.6.2 Bacterial inoculation to the plants 183.2.6.3 Evaluation of disease level 18

3.2.6.4 Statistic analysis 18

CHAPTER 4 RESULTS AND DISCUSSION 194.1 Testing antibacterial activities R solanacearum by Bacillus spp in vitro conditions

194.1.1 Zone of inhibition growth of R solanacearum with 14 Bacillus strains by agardiffusion method 194.1.2 Zone of inhibition growth of R solanacearum with 5 Bacillus strains highestactivity against R solanacearum 204.1.3 Results of inhibition zone made by 5 Bacillus strains used as biocontrol agentsagainst R solanacearum using agar diffusion method 204.2 Determination of Minimum inhibitory concentration (MIC) and Minimumbactericidal concentration (MBC) of Bacillus strains against R solanacearum 224.2.1 Bacillus velezensis (Asl-1) - MIC, MBC 224.2.2 Bacillus amyloliqueficiens (Asl-2) - MIC, MBC 23

4.2.3 Bacillus cereus (Asl-5) - MIC, MBC 24

4.2.4 Bacillus pumilus (62) - Bacillus licheniformis (12-1) 26

4.2.4.1 Bacillus pumilus (62) 264.2.4.2 Bacillus licheniformis (12-1) 24.2.5 Ampicillin (Positive control) - MIC, MBC 294.3 Determination of plant-promoting and antibacterial activities of Bacillus strainswith Ralstonia solanacearum on Capsicum annuum L plants under greenhouse

conditions 30

4.3.1 The plant-promoting efficiency of Bacillus on Capsicum annuum L plants undergreenhouse conditions 30

4.3.2 Effectiveness of plant-promoting Bacillus as a biological control against

Ralstonia Bacterial Wilt 34CHAPTER 5 CONCLUSIONS AND SUGGESTIONS 37

5.1 Conclusions 37

5.2 Suggestions 37REFERENCES 38APPENDIX

: Colony forming unit: Exo-polysaccharides: and others

: Hydrogen cyanide: Induced systemic resistance: Minimum Bactericidal Concentration: Minimum Inhibitory Concentration: National Pingtung University of Science and Technology: Optical density

: Polymerase Chain Reaction: Plant growth-promoting Rhizobacteria: Polyhydroxybutyrate

: Reactive oxygen species: Ralstonia solanacearum: Ribosomal ribonucleic acid: Sustainable development goals: species (plural)

: Tryptic Soy Agar: Tryptic Soy Broth: Triphenyl Tetrazolium Chloride: Tetrazolium chloride

: Ultraviolet

LIST OF TABLES

PageTable 2.1 Morphology and cultural characters of R solanacearum on different media

5Table 3.1 14 Bacillus strains used in this study were cultured on TSA media 15Table 4.1 7z vitro antibacterial activity of 14 Bacillus strains against R solanacearum

19Table 4.2 (next) Jn vitro antibacterial activity of 14 Bacillus strains against R

solanacearum 19Table 4.3 Inhibition zone diameters of 5 Bacillus strains against R solanacearum 20Table 4.4 Minimum inhibitory concentration (MIC) and Minimum bactericidal

concentration (MBC) of 5 Bacillus cell-free supernatant against R solanacearum 28Table 4.5 Effect of plant-promoting Bacillus on the height (cm) of Capsicum annuum

L plants under greenhouse conditions 30Table 4.6 Effect of plant-promoting Bacillus on the number of leaves of Capsicumannuum L plants under greenhouse conditions 32Table 4.7 Effect of plant-promoting Bacillus on the root length, number of flowersand number of fruits of Capsicum annuum L plants under greenhouse conditions 33Table 4.8 Effectiveness of plant-promoting Bacillus as a biological control againstRalstonia bacterial wilt 34

LIST OF FIGURES

PageFigure 2.1 Colony morphology of Ralstonia solanacearu m on different media 4Figure 2.2 Virulent and avirulent strains of Ralstonia solanacearum 5)Figure 2.3 Cell morphology of R solanacearum 6Figure 2.4 Endophytic bacteria inhibit the pathogenesis of Ra/stonia sp., over theSolanaceus plants 7

Figure 2.5 An overview of mechanisms employed by Bacillus spp in the mitigation

of biotic and abiotic stresses 10Figure 3.1 The 14 Bacillus strains were used in this study 15Figure 4.1 Zone of inhibition growth of R solanacearum with suspension and

supernatant of 14 Bacillus strains 19Figure 4.2 Zone of inhibition growth of R solanacearum with suspension and

supernatant 20

Figure 4.3 MIC result of Asl-1 with R solanacearum 22Figure 4.4 Growth inhibition ability of Asl-1 with R solanacearum 22Figure 4.5 MIC result of Asl-2 with R solanacearum 23Figure 4.6 Growth inhibition ability of Asl-2 with R solanacearum 23Figure 4.7 MIC result of Asl-5 with R solanacearum 24

Figure 4.8 Growth inhibition ability of Asl-5 with R solanacearum 25

Figure 4.9 MIC result of 62 with R solanacearum 26Figure 4.10 Growth inhibition ability of 62 with R solanacearum 27Figure 4.11 MIC result of T2-1 with R solanacearum 27Figure 4.12 Growth inhibition ability of T2-1 with R solanacearum 28Figure 4.14 Growth inhibition ability of Ampicillin with R solanacearum 29Figure 4.13 MIC result of Ampicillin (Positive control) with R solanacearum 29Figure 4.15 Effect of plant-promoting Bacillus on the root Capsicum annuum L

plants under greenhouse conditions 33Figure 4.16 Capsicum annuum L at the 28-day period after treated with R

solanacearum 34

CHAPTER 1 INTRODUCTION

1.1 Problem statement

The sustainability of agriculture is related to the development of the nationaleconomy and society’s stability By 2050, the crop output will be far lower than theneeds of the growing population (Ray ef al., 2013) Plant diseases are the main factorsaffecting crop losses, reducing the yield by 14% (Vyska ef al., 2016) It is consideredthat soil-borne diseases are more restrictive than seed-borne or air-borne diseases,accounting for 10 - 20% of the annual yield loss (Kurabachew ef al., 2017) Ralstoniasolanacearum 1s one of the ten most harmful plant bacteria and 1s distributed all overthe world, especially in subtropical and tropical regions (Mansfeld ef a/., 2012) Thissoil-borne bacterium can infect more than 450 plant species of 54 botanical families(Wicker ef al., 2007) and cause huge direct economic losses every year (Yuliar ef al.,

2015) The pathogenic genes of R solanacearum have coevolved with the external

environment and are characterized by multiple toxic factors (Genin and Denny, 2012).Difficulties are associated with controlling this pathogen due to its ability to grow

endophytically, its relationship with weeds, and its survival in soil (Yuliar ef a/., 2015)

Therefore, it is of great significance to research how to effectively mhibit R.solanacearum for agricultural sustainability

Yield losses caused by bacterial wilt pathogen in many Solanaceous crops areranged between 15 - 55% around the globe (Kim ef a/., 2016) In Uganda, the tomatocrop was badly affected by R solanacearum, and yield losses were recorded at 88%;and in Ethiopia, the incidence of pepper bacterial wilt disease was recorded at about

100% (Mamphogoro ef a/., 2020) Potato bacterial wilt disease caused by is the 2"4 most

important disease of potato after late blight caused by Phytophthora infestans, and yield

losses reached up to 50 - 75% (Felix ef al., 2010; Chamedjeu ef al., 2019) It wasestimated that 1.7 MH of a potato field in more than 80 countries is affected by R

solanacearum, with $950 million loss annually (Charkowski e al., 2020)

In Vietnam, bacterial wilt caused by R solanacearum, is a major threat to manyimportant field crops Bacterial wilt control still uses fungicides and technical culturesbut has not been able to reduce the incidence of disease in the field To overcome this,

it is necessary to develop biocontrol methods that are safer and more environmentallyfriendly Biological control is directed as research on antagonist agents develops.Bacillus spp 1s a genus of bacteria that is reported to be able to increase plant resistance.Morphological and physiological characteristics as well as the inhibition mechanism ofBacillus spp need to be studied to determine their effect on bacterial wilt infection.Recent studies on microbial siderophores in the rhizosphere are associated with theirbiocontrol activity due to their competitive effects on plant pathogens Bacteria produce

a wide variety of siderophores under iron-limiting conditions: hydroxamates,

phenol-catecholates, and carboxylates Therefore, this study is mainly aimed to evaluate thepotential of plant-promoting Bacillus spp as biocontrol agents against Ralstoniasolanacearum bacterial wilt disease

1.2 Objective

The research aimed to was to determine the effectiveness of the plant-promotingBacillus spp 1n suppressing R solanacearum wilt disease 1n Solanaceous crop plants.1.3 Contents

To achieve the mentioned objective, the researcher conducted studies on the 3

contents:

Content 1: Testing antibacterial activities R solanacearum by Bacillus spp in

vitro

conditions

Content 2: Determination of Minimum inhibitory concentration (MIC) and

Minimum bactericidal concentration (MBC) of Bacillus strains against R

solanacearum

Content 3: Determination of plant-promoting and antibacterial activities ofBacillus strains with R solanacearum on Capsicum annuum L plants under greenhouseconditions

CHAPTER 2 LITERATURE REVIEW

2.1 Introduction of Rastonia solanacearum

2.1.1 Taxonomy

R solanacearum was first described by Smith in 1896 and was first named Bacillussolanacearum (Smith, 1896) In 1914, Smith changed its name to Pseudomonassolanacearum (Smith, 1914) P solanacearum was included in the Approved Lists ofBacterial Names (Skerman ef al., 1980), and a substantial amount of literature waspublished under this name Later, Yabuuchi ef a/ (1992) transferred P solanacearumand related nonfluorescent pseudomonads to a novel genus called Burkholderia.Subsequently, Yabuuchi ef a/ (1995) concluded that Burkholderia solanacearum wasdistinct enough from other members within the genus to be assigned in the newlyproposed genus Ralstonia To date, the pathogen’s preferred scientific name is Ralstoniasolanacearum (Yabuuchi eft ai, 1995) R solanacearum is a member of the

nonfluorescent pseudomonads rRNA homology group II (Prior ef a/ (2016); Remenant

et al (2011); Safni et al (2014))

2.1.2 Characteristics

Ralstonia solanacearum 1s a gram-negative, rod-shaped, strictly aerobic bacteriumwhich is 0.5 - 0.7 x 1.5 - 2.0 mm in size This pathogen is very sensitive in desiccationand is inhibited in culture by low concentrations (2%) of sodium chloride (NaCl) (Feganand Prior, 2005) Majority of the strains have optimal growth temperature 820 - 900°F,however some of the strains have low optimal temperature 80.50°F The commonly usedgrowth media for culture of the bacterium are liquid and solid (Lambert, 2002) When

in solid agar medium, the individual bacterial colonies are commonly observable after

36 to 48 hours of growth at 82.40°F, and the two main types of colonies which differs

in morphology can be distinguished: colonies of the normal or virulent type are white

or cream-colored, irregularly-round, fluidal, and opaque; and colonies of the mutant ornonvirulent type are uniformly round, smaller, and butyrous (dry) (Jones ef a/., 1991)

This shift from virulent to non-virulent bacterial cells occurs when in storage orunder oxygen stress in liquid media In order to differentiate between the two colonytypes, Tetrazolium chloride (TZC) medium was developed in such a way that virulent

colonies appear white with pink centers and nonvirulent colonies appear dark red

(Gitaitis et al., 1992) For detection of R solanacearum in water and soil samples and

in plant extracts, a semi-selective medium known as modified SMSA was developed

A typical bacterial colonies appear fluidal, irregular in shape, and white with pink

centers in this medium just after 2 to 5 days incubation at 82.4°F (Ji et al., 2005)

R solanacearum is prevalent in the tropics and subtropics around the world and

many strains of the pathogen have been identified and characterized so far, which

reveals a significant variability within the species

Ralstonia solanacearum is a causal agent of vascular wilt disease in more than 200crop species R solanacearum is a strict soil-borne pathogen and thrives in moist soils

(Van der Wolf et ạ., 1998) The bacterium can live for years in an infected field, andhas been reported to persist for 12 months in potato fields (van Elsas ef z/., 2000) The

sources of inoculum for agricultural fields are irrigation and surface water, weeds,

infested soil, latently infected propagative plant material, and contaminated farm tools

and equipment The bacteria exhibit subterranean movement and spread from the

infected plants’ roots to the healthy ones (Hayward, 1991)

2.1.2.1 Culture characteristics

Figure 2.2 _Virulent and avirulent strains O

Ralstonia solanacearum A Virulemt, B Avirulent (Anitha et

al YA1ENV

Table 2.1 Morphology and cultural characters of R solanacearum on different media

Media Colony morphology

Very small colony, creamy white, round, slightlyNutrient Agar medium ` : :

8 mucoid, translucent, slightly raised surface

Medium sized colonies, highly mucoid, round,

King’s B medium opaque, slightly raised surface

Semi Selective South Moderately fluidal, regularly shaped, white colonyAfrica Agar (SMSA) with dark red coloured center and the colonies were

medium relatively small rigid

Irregular with smooth margin, highly fluidal, largecolonies, pink centered with creamy white border,

solanacearum R solanacearum on NA, SMSA, CPG, KB and TTC where the colony

characters varies from regular to irregular margin, light to dark pink centre, creamy to

white colour from small to big size and variation in EPS production were also observedand the results are described in Figure 2.1; Table 2.1 The virulent and avirulent colonies

were obtained during culturing of bacteria shown in Figure 2.2 Among the different

media tested TTC medium was found to be an excellent nutrient medium for growth and

EPS production of R solanacearum According to Rohini ef al (2017) TTC medium

when used for R solanacearum, it shows the difference between avirulent colonies that

look dark red from fluidal virulent that are white with pink centre

2.1.2.2 Morphological characteristics

R solanacearum is non-spore-forming, short rod-shaped and rounded at the ends

Bacteria are usually found in single, paired or quadruplet form, in the range of 1.0 - 1.5

x 0.5 - 0.6 um in size, moved by one to several (flagella) (Nguyen Tat Thang ef ai.,2011)

lipase activity test and casein hydrolysis test; for the negative reaction for gram staining,

Levan production from sucrose test, Arginine dihydrolase test and fluorescent pigment

production test (Popoola et al., 2015; Zubeda and Hamid, 2011)

2.2 Bacterial wilt

2.2.1 Description

Bacterial wilt, caused by Ralstonia solanacearum, is a serious threat to

Solanaceous plants production in Africa (Were ef al., 2013; Uwamahoro ef al., 2020;

Otieno, 2019; Muthoni ef a/., 2020; van der Waals and Kruger, 2020) This soil-borne

disease can be spread by water, farm tools, infected seeds, previously infested cropresidue, and volunteer potato crops When infected, the whole plant wilts and dries up,

and tubers develop brown-black stains and rot, resulting in reduced marketable potato

yield and quality Yield losses ranging from 50% to 100% have been reported in some

countries in Central and East Africa (Muthoni ef a/., 2012) The pathogen has several

host plants such as pepper, tomato, tobacco, eggplant, and ornamentals, as well asseveral species of weeds that are commonly found in or near potato farmers' fields

Management of this disease using available agrochemicals is difficult

eg

S3uE|d 3usIS4 31M J8I1332!

inoculated with EB

Figure 2.4 Endophytic bacteria inhibit the pathogenesis of Ralstonia

sp., over the Solanaceus plants (Achari and Ramesh, 2014)

2.2.2 Symptoms and Signs

Plants infected with Ralstonia solanacearum can show symptoms a few days after

infection and are characterized by sudden wilting and yellowing of the leaves, followed

by undersized growth and eventually death of the plants In early stages of the disease

the first symptoms are usually seen on the foliage of plants These symptoms occurthrough in the hottest part of the day which shows wilting of the youngest leaves

(Elphinstone, 2005) In this stage only a few leaflets may wilt, and at night when the

temperature cools down, the plants will again recover very soon Under the unfavorable

conditions the entire plant may wilt and dry quickly, although dried leaves remain green,

leading to general wilting and yellowing of foliage and plant dies eventually (Lambert,2002)

Another common symptom of bacterial wilt in the field is stunting of plants Thesesymptoms may appear at any stage of plant growth even though in the field it is common

for healthy appearing plants that wilting occurs suddenly when fruits are expandedrapidly (Jones et al., 1991) In young stems of Solanaceous crops, vascular bundles areaffected showing visible appearance like long, narrow, dark brown streaks Collapse ofthe stem may be seen in young succulent plants which belong to the varieties that are of

highly susceptible (Pradhanang ef a/., 2005)

2.3 Bacillus spp

2.3.1 Introduction

Over the past decades, genetic engineering and plant breeding approaches havebeen employed to develop new cultivars with desired traits, such as high yield andresistance to environmental stresses (Radhakrishnan ef a/., 2017) To obtain better crop

yield, applications of chemical fertilizers have been the opted strategy However, over

time, studies and empirical evidence have shown that this traditional method the use ofchemical fertilizers is not sustainable due to the inherent negative effects these productshave on the environment

The incorporation of biostimulants, such as PGPR - based formulations, incropping systems has increasingly shown to be a promising strategy for sustainableagriculture and global food security, aligning with the United Nations sustainabledevelopment goals (SDGs) (Backer ef a/., 2018) A broad array of bacterial species hasbeen reported to possess plant growth-promoting attributes with the prominent speciesbelonging to the genus Bacillus

Members of the genus Bacillus are ubiquitous, gram-positive, and aerobic bacteria(Grover ef al., 2011; Vejan ef al., 2016) Bacillus species produce a multitude of

enzymes, antibiotics, and metabolites which give them prominent applications in

various sectors such as pharmaceuticals and agriculture

When applied to the plant as dormant cells, these Bacillus spores must germinate

to form metabolically active cells Following germination, these bacteria could beattracted by chemotaxis and leading to the root colonization process (which is

mechanistically complex) and exerting growth promotion potential (Beauregard ef al.,2013)

Upon colonization, Bacillus elicit direct (eg, siderophore production, nitrogenfixation, phytohormone production, and nutrient solubilization) and indirectmechanisms (such as the production of exo-polysaccharides (EPS), biofilm formation,

hydrogen cyanide (HCN) and lytic enzymes) to promote plant growth and yield, undervarious environmental conditions (Vejan ef a/., 2016).

2.3.2 Applications

Many strategies have been proposed to counteract the negative effects ofenvironmental stress, and these include, for example, the overexpression of singleenzymes that encode transport ions and remove the most common reactive oxygenspecies (ROS) in the world The application of this method is limited due to the

multidirectional effects on plant growth and the multiple pathways involved in the

response to environmental stress (Xie et al., 2019) Another strategy is to use based fertilizers and pesticides which are becoming increasingly harmful to the

chemical-environment and some of these xenobiotics have become highly toxic in the agriculturalfood chain Therefore, non-harmful, eco-friendly, sustainable, and nature inspired

solutions and strategies are needed, including the use of PGPR formulations specificallyBacillus spp The latter can assist in reducing the effects of environmental stress Severalstudies have demonstrated that biostimulants based on Bacillus spp activates plantdefense mechanisms against both abiotic and biotic stress

2.3.2.1 Bacillus spp against Biotic stress

Biological control, utilizing beneficial microbes, is an excellent approach tolimiting the adverse effect of disease-causing microbes on plant health and productivity

Considerable effort has been placed on identifying microbial biocontrol agents that can

repress phytopathogens, especially those that are responsible for soil- borne diseases,and that can enhance agricultural productivity (Hashem ef a/., 2019; Cazorla et al.,2007) Many strains of Bacillus exhibit the ability to act as biocontrol agents againstpathogens and thus can be used to suppress diseases (Wang ef al., 2015) Several

mechanisms, both direct and indirect, are responsible for their ability to control

pathogenic microbes as shown in Figure 2.5 These include the production of a widearray of antibiotic compounds (lipopeptides), the ability to form endospores, the ability

to form biofilms on root surfaces, and the ability to induce host systemic host resistance,

and stimulate plant growth (Kinsinger ef al., 2003)

Abiotic stresses Biotic stresses

Bacillus-colonised roots › | cf

Bacillus

Roots exudates Bacillus exudates

Amino acids

Amino acids Rhizhosphere 6 , sa

Flavonoids chanical ormones ( )

Organic acids intercommunication VOCsPhytohormones AHL

Sugars Lipopeptides

Mechanisms of action

* Biocontrol (competition) » Metal chelation

¢ ISR ¢ Favourable microbiota

¢ Nutrient acquisition * Osmoprotection

¢ Root development and growth ¢ Redox balance

¢ Shoot growth * Soil health

Figure 2.5 An overview of mechanisms employed by Bacillus spp in the

mitigation of biotic and abiotic stresses (Tsotetsi, 2022)

Biotic stresses often lead to the production of reactive oxygen species (ROS) whichlead to oxidative stress and are toxic to the cells The inoculation of plants with Bacillus

- based formulations has shown to elicit the production of antioxidant defense enzymes

such as superoxide dismutase and peroxidase, which scavenge the ROS (Yasmin ef ai.,

2016) In a study by Zebelo ef a/ (2016), cotton plants inoculated with Bacillus spp.demonstrated an increase in gossypol and jasmonic acid levels and secretion reducinglarval feeding by spodoptera exigua There was also upregulation of genes involved in

synthesis of allelochemicals and jasmonates in the inoculated plants Furthermore,

Bacillus spp secrete various catabolic enzymes such as proteases, chitinases, andglucanases, as well as peptide antibiotics and secondary metabolites that contribute topathogen suppression (Tyagi ef a/., 2018) Bacillus spp secrete cyclic lipopeptides such

as iturin and surfactin (Figure 2.5) which play a role in disease

suppression by acting as bifunctional molecules through their antifungal activity andelicitation of ISR The latter involves pathogen recognition at plant cell surface,stimulation of early cellular immune-related events, systemic signaling through a fine-tuned hormonal cross talk and activation of defense mechanisms (Ongena, 2020).Bacillus spp can elicit ISR in plants, which switches on pathogenesis related genes,mediated by phytohormone signaling pathways and defense regulatory proteins toprecondition plants against future pathogen ambush (Pieterse ef al., 2014).

2.3.2.2 Bacillus spp against Abiotic stress

Being sessile organisms, plants have to withstand various adverse abiotic stressessuch as drought, salinity, heat or cold, and heavy metal toxicity which pose a majorthreat to agriculture by negatively impacting plant growth and yield worldwide (Tiwari

et al., 2017) These stresses elicit stress responses in plants, including an accumulation

of reactive oxygen species (ROS) and reduced photosynthetic activity, which ultimatelyleads to reduced plant growth and crop yield PGPR such as Bacillus spp can mediatethe induction of abiotic stress responses in plants These responses to abiotic stresses areattributed to metabolic regulations which often require wide changes in theconcentration, composition, and distribution of both primary and secondary metabolites.Biostimulants containing Bacillus strains have shown the potential to stimulate abioticstress tolerance (Meena ef al., 2017)

2.4 Recent related research in Vietnam and in the world

2.4.1 In the world

In 2016, Singh ef a/ isolated fifty seven rhizobacteria from rhizospheric soil of

wilted tomato plants and among them two strains of rhizobacteria, having better

antagonistic and plant growth promoting ability were characterized them as Bacillusamyloliquefaciens DSBA-11 and DSBA-12 based on morphological, biochemical,partial gene sequence analysis of 16S rRNA and fatty acid methyl ester analysis.Antagonistic activity of these strains DSBA-11, DSBA-12 was compared with otherBacillus species such as B subtilis DTBS-5, B cereus JHTBS-7, B pumilus MTCC-

7092 strains, against Ralstonia solanacearum race 1, bv 3, phylotype I, inciting bacterialwilt of tomato under in vitro conditions B amyloliquefaciens DSBA-11 showed

maximum growth inhibition of R solanacearum (4.910cm? ) followed by strains

DSBA-12 (3.310cm? ) and B subtilis (3.070 cm?) Biocontrol efficacy and plant growth ability

of these bacterial antagonists was tested against bacterial wilt of tomato cv Pusa Rubyunder glasshouse conditions Minimum bacterial wilt disease incidenceincultivar PusaRuby (17.950%) was recorded in B amyloliquefaciens DSBA-11followed by B.amyloliquefaciens DSBA-12 after 30 days of inoculation.The biocontrol efficacy washigher in B amyloliquefaciens DSBA -12 treated plants, followed by B pumilus MTCC-7092.

In 2020, Jinal ef al investigated the evaluation of biologically controlled Bacillus

species in promoting plant growth and resistance to systemic fungal and bacterial wilt

pathogens Its focuses on the biocontrol potential of two species belonging to the samegenera, as Bacillus subtilis (SSR2I) and Bacillus fexus (AIKDL) have contrasting

activity under in vivo and in vitro conditions In this study, two medicinal

plants-associated bacteria showing antagonistic activity against wilt-causing pathogens wereselected and identified as B subtilis (SSR2]) and B fexus (AIKDL) based on 16S rRNAgene sequencing The results indicated that even though the isolates had strongantagonistic potential under in vitro conditions, their biocontrol efficiency differed 1n invivo conditions

Recently, there have been several studies related to biocontrol of plant diseases byBacillus spp According to Hasinu (2021), the use of biocontrol agents such as Bacillusspp 1s an alternative method of controlling R solanacearum 1n bananas The study used

Bacillus subtilis strain SW116b and Bacillus subtilis strain HPC2-1 isolates The results

showed that the SW116b stain Bacillus subtilis has the highest activity against R.solanacearum, which is 10.500 mm, so it has the potential as a biological control agent

in suppressing the development of Ralstonia wilt disease in bananas

In 2022, Singh ef a/ invesgated the suppression of Tomato bacterial wilt incited

by Ralstonia pseudosolanacearum Using polyketide antibiotic-producing Bacillus spp

isolated from rhizospheric soil The results show that about in vitro study on antagonisticability of Bacillus spp against R pseudosolanacearum Five Bacillus species viz.,Bacillus amyloliquefaciens DSBA-11, B cereus JATBS-7, B pumilus MTCC-7092, B.subtilis DTBS-5 and 8B licheniformis DTBL-6 were used against R.pseudosolanacearum UTT-25 under in vitro conditions A significant variation (CDvalue (5.000%): 0.355, CV: 7.140%) was recorded among the species of Bacillus toform an inhibition area against R pseudosolanacearum Maximum inhibition area

against R pseudosolanacearum was recorded in the treatment of B amyloliquefaciens

DSBA-11 (3.300 cm”) followed by B subtilis DTBS-5 and B licheniformis DTBL-6

after 48 hours of incubation at 28 + 1°C

2.4.2 In Vietnam

After 1945, plant disease research in Vietnam became increasingly successful careabout Particularly, diseases caused by R solanacearum bacteria have manydepartments interested in studying domestically Especially: Institute of Plant

Protection, University Hanoi Agriculture, Vietnam Academy of Science and

Technology (former), Fruit and Vegetable Research Institute, Institute of AgriculturalGenetics, Institute of Agrochemical Soils Overall, there is not much research on wiltdisease caused by R solanacearum 1n Vietnam, not yet comprehensive and not deep.Currently, research on R solanacearum also has become an important issue in plantprotection

In 2014, Le Thi Thanh Thuy isolated two strains of R solanacearum bacteria (LH3

and YH3) from soil samples and plants with a high risk of causing green wilt disease on

peanuts, chili peppers, and strains LH3 belongs to biovar 3, strain YH3 belongs to biovar

1 Two bacterial strains B subtilis DKB1 and P fluorescens DKP1 can resist R

solanacearum bacteria (LH3 and YH3) with inhibition zone diameters are 15 mm and

16 mm respectively and they are biosafety grade The antibiotics Phenazine have been

isolated from P fluorescens strain DKP1 and Iturin A from B subtilis strain DKB1

Both of these substances have the ability to inhibit R solanacearm bacteria that causeswilt disease in peanuts and chili peppers (LH3 and YH3) And Phenazine, IturinA andsiderophore have been identified as some of the substances involved in the process ofantagonizing R solanacearum bacteria by P fluorescens DKP1 and B subtilis DKB1

In 2020, Tran Kim Diep ef a/ evaluate the abilities and effects inhibitory of some

B subtilis strains against Rhizopus sp and Mucor sp on strawberries post-harvest The

results revealed that B subtilis B1S showed the strongest in vitro biocontrol activity

against Rhizopus sp at 56.100% and Mucor sp at 36.260% By using in vivo

inoculation method, B subtilis B1S reduce the incidence of diseases from 95.830% to66.710% for Rhizopus sp and from 66.580% to 30.740% for Mucor sp Therefore, thecontrol efficiency reached up 30.390% on Rhizopus sp and 52.400% on Mucor sp.,respectively In 2021, Le Thanh Binh ef a/ determine strains that cause bacterial wiltdisease 1n cucumber and to select some Bacillus that is capable of antagonism fo R

solanacearum,

causing the wilt disease in cucumber The result showed that the strain DLHX4bacterium was selected which based on morphological and biochemistry characteristics,pathogenic causing the wilt disease quickly in cucumber and 16S ribosomal DNA regionsequences showed 99.000% similarity to R solanacearum The resulting study was thatthe 3 strains selected were Bacillus DB8.7, DB2.1, DB9.9 which high antagonistic effect

on Ralstonia solanacearum by agar well diffusion method The antagonistic effect ofstrain DB8.7, DB2.1, DB9.9 were 31.870 mm, 21.890 mm, 21.670 mm Result of a

detailed analysis of 16S rDNA gene segments, strain DB8.7, DB2.1, DB9.9 showed

similarity to Bacillus subtilis, Bacillus velezensis, Bacillus amyloliquefaciens

CHAPTER 3 MATERIALS AND METHODS

3.1 Time and location

This study was performed from July 1%, 2023 to December 31", 2023 at

Laboratory - Room BT306, Department of Biological Science and Technology,

National Pingtung University of Science and Technology (NPUST), Taiwan

3.2 Materials and methods

3.2.1 Samples

Source of microorganisms: Ralstonia solanacearum and 14 Bacillus strains wereall molecularly identified and were provided by Functional Genomic Lab, Department

of Biological Science and Technology, National Pingtung University of Science and

Technology (NPUST), Taiwan

Table 3.1 14 Bacillus strains used in this study

No Name of strains ID No Name of strains ID

1 Bacillus amiloliquefaciens Asl-2 8 Bacillus coagulans G2

2 Bacillus subtilus NKI 9 Bacillus licheniformis T2-]

3 Bacillus megaterium ERI 10 Bacillus altitudinis T4-1

4 Bacillus pumilus 43Y 11 Bacillus altitudinis Asl-4

5 Bacillus pumilus 40 12 Bacillus cereus STI-9

6 Bacillus pumilus 62 13 Bacillus cereus Asl-5

7 Bacillus coagulans DC2-3 14 Bacillus velezensis Asl-1

3.2.2 Equipments and tools

3.2.2.1 Equipments

Microbiological incubator (HPX - 9052MBE, Shanghai Boxun industry, China),Centrifuge System (Beckman Coulter, Germany), Precision balance (ScoutPro, China),Refrigerator and Freezer, Microwave oven

3.2.2.2 Tools

Petri dishes, test tubes, glass flasks, measuring tubes, alcohol lamps, and

inoculation rods

3.2.3 Bacteria culture media

R solanacearum: Triphenyl Tetrazolium Chloride (TTC) agar and broth,Casamuino acid - Peptone - Glucose (CPG) agar and broth

Bacillus spp: Tryptone Soy Agar (TSA) and Tryptone Soy Broth (TSB)

Composition of Triphenyl Tetrazolium Chloride (TTC): Casamino acid (Caseinhydrolysate) 1 g/L, Meat peptone 10 g/L, Glucose 5 g/L, Tetrazolrum Red 0.1 g/L Forsolid medium, add 16 g/L Agar Adjust pH = 6.5 - 7.0 Autoclave at 121°C for 20minutes

Composition of Casamino acid - Peptone - Glucose (CPG): Casamino acid (Caseinhydrolysate) 1 g/L, Meat peptone 10 g/L, Glucose 5 g/L For solid medium, add 16 g/LAgar Adjust pH = 6.5 - 7.0 Autoclave at 121°C for 20 minutes

Composition of Tryptone Soy Broth (TSB): Pancreatic digest of casein 17 g/L,Papaic digest of soybean meal 3 g/L, Dibasic potasstum phosphate 2.5 g/L, Sodiumchloride 5 g/L, Glucose monohydrate 2.5 g/L For solid medium, add 15 g/L Agar.Autoclave at 121°C for 20 minutes

3.2.4 Testing antibacterial activities R solanacearum by Bacillus spp in vitroconditions

For Ralstonia solanacearum: bacteria were activated on TTC agar medium andincubated at 30°C for 48 hours Then cultured in TTC broth, incubated at 30°C for 24hours For 14 Bacillus strains: bacteria were activated on TSA medium and incubated

at 37°C for 12 hours Then grow in TSB medium, incubated at 37°C for 24 hours

Selection of antagonistic Bacillus strains by agar diffusion method Bacillus strains

were shaken at 100 rpm in TSB After 24 hours, the Bacillus proliferative solution wascentrifuged at 8000 rpm, 100 uL of supernatant was aspirated into 3 wells (7 mm in

diameter) on TTC agar plate which was equipped with 100 pL of ® solanacearumcultured in TTC broth at 30°C for 48 hours Experimental set up were repeated 3 times.Antagonist plates were incubated at 30°C for 24 hours, 48 hours (Le Thanh Binh, 2021).Observe and measure the diameter of inhibition zone (the transparent circle surrounding

the agar hole) The antagonistic activity was calculated according to the formula:Resistance zone size = D - d, where: D: the diameter of the countervailing zone(mm); d: the agar hole diameter (mm)

3.2.5 Determination of minimum inhibitory concentration (MIC) and Minimumbactericidal concentration (MBC) of Bacillus strains against R solanacearum3.2.5.1 Prepare the ELISA microplate

From the result of antagonistic Bacillus strains by agar diffusion method 5

Bacillus strains were cultured in TSB at 37°C After 48 hours, CFS were collected by0.45 um membrane with the highest concentration at 1000 pL/mL Diluted 2 times in96-wells plate to the lowest concentration at 0.4875 wL/mL Test with pathogenicbacteria Ralstonia solanacearum was prepared as previously described, adjusted to be

at the concentration 10° CFU/mL 100 uL TSB add with 100 uL diluted Ralstonia

solanacearum broth in CPG was use as a negative control, Ampicillin was use as apositive control Prepare 96-wells plates, add 100 uL pathogenic bacteria solution to thediluted 100 pL CFS in each well, incubate at 30°C for 24 hours, measure the OD at 600

nm at 0 hours and after 24 hours, recorded the value respectively The point where theabsorbance value of OD 600 nm does not change is MIC MBC were determined by theminimum concentration that where the bacteria does not exist

3.2.6 Determination of plant-promoting and antibacterial activities of Bacillusstrains with R solanacearum on Capsicum annuum L plants under greenhouse

conditions

3.2.6.1 Preparation of bacterial inoculum

Ralstonia solanacearum was streaked on TTC agar and incubated at 30°C for 48hours Colonies were harvested using distilled water, and the inoculum were made byadjusting the cell suspension to an OD = 0.5 at 600 nm, corresponding to about

approximately 108 CFU/mL Bacillus strains with the highest activity against Ralstonia

solanacearum were streaked on TSA and incubated at 37°C for 24 hours The cell

suspensions were adjusted to corresponding to about approximately 108 CFU/mL.

3.2.6.2 Bacterial inoculation to the plants

Prepare 18 samples of Capsicum annuum L seedlings The experiment is

conducted under green-house condition with natural light and dark photoperiod EachBacillus strain was grown overnight in TSB broth Each broth is centrifuged at 10000

rpm in 10 minutes, washed with phosphate buffer three times and then pellets were

dissolved in ddH2O Before being used for pot experiments Bacillus strains were each

adjusted with distilled water to an ODsoo value, respectively, giving a cell density of 108

CFU/mL The 8-week-old Capsicum annuum L seedlings were treated with a

suspension of selected Bacillus strains by poured into each plastic pot 20 mL of eachBacillus suspension immediately afterward Do the same for the control plants butreplace the suspension with distilled water After 5 days of inoculation, the roots of eachCapsicum annuum L plant was artificially mjured and infected with R solanacearum

by pouring 20 mL of bacterial suspension into each plastic pot at the base of the plant.Capsicum annuum L seedlings were treated with only R solanacearum (positivecontrol) and distilled water was used as negative control All experiments wereconducted in triplicate

3.2.6.3 Evaluation of disease level

Following inoculation with R solanacearum, the disease severity index of the

plants was observed at 7, 14, 21, 28 days post inoculation, with level 0 (-): no wiltingsymptoms, healthy; level 1 (+): 1-10% wilting leaves, level 2 (++): 11-30% wiltingleaves, level 3 (+++): 31-60% wilting leaves, level 4 (++++): > 60% wilting leaves, level

5 (Œ++r+++): 100% wilting leaves (Takikawa et al., 1994; Ateka ef al., 2001)

3.2.6.4 Statistic analysis

All experimental results reported were three independent averages of triplicates,the means and standard deviations (SD) were determined to check for errors andvariation among the triplicates One-way ANOVA with Tukey-LSD (P < 0.05) usingMini Tab version 16

CHAPTER 4 RESULTS AND DISCUSSION

4.1 Testing antibacterial activities R solanacearum by Bacillus spp in vitro

conditions

4.1.1 Zone of inhibition growth of R solanacearum with 14 Bacillus strains by agar

Figure 4.1 Zone of inhibition growth of R solanacearum with suspension andsupernatant of 14 Bacillus strains SS: Suspension; SP: Supernatant; (-): TSB; (+):Ampicillin 25 ug/mL

Table 4.1 7z vitro antibacterial activity of 14 Bacillus strains against R solanacearum

Asl-2 NKI ERI 43Y 40 62 DC2-3

+ + +, high inhibition, > 25 mm; + +, moderate inhibition, (15—25 mm); + low inhibition, (5—

15 mm); —, no inhibition Asl-2: Bacillus amiloliquefaciens; NKI: Bacillus subtilus; ERI:Bacillus megaterium; 43Y: Bacillus pumilus; 40: Bacillus pumilus; 62: Bacillus pumilus; DC2-3: Bacillus coagulans; C2: Bacillus coagulans; T2-1: Bacillus licheniformis; T4-1: Bacillusaltitudinis; Asl-4: Bacillus altitudinis; STI-9: Bacillus cereus; Asl-5: Bacillus cereus; Asl-1:Bacillus velezensis

In the primary screening step, the agar diffusion method on TTC agar medium and

using the suspension and supernatant of Bacillus strains was conducted to determine the

antibacterial activity of 14 Bacillus strains with R solanacearum (Figure 4.1) Among

the 14 Bacillus strains, five strains were selected: Asl-1, Asl-2, Asl-5, 62, T2-1