Identification, characterization and mechanisms of potential rhizobacterial strains for biological control of bacterial brown stripe and bacterial sheath rot of rice, growth promotion and abiotic stress resistance in rice

Table of Contents

List of Tables

List of Figures

Abstract

MM

List of Abbreviations

Chapter 1
Introduction

Chapter 2
Literature review
2.1. Current Status and Challenges of Rice Production in China
2.3.1. Bacterial Brown sheath rot of rice
2.1.1.1. The pathogen (P. fuscovaginae) and its disease symptoms on rice
2.1.2. Bacterial brown stripe of rice
2.1.2.1. The pathogen (A. avenae subsp. avenae) and its symptoms on rice
2.1.3. Abiotic stress
2.2. Approaches to improve the health of rice plant
2.3. Plant growth-promoting rhizobacteria
2.3.1. Mechanisms involved in PGPR bioactivity
2.3.1.1. Antagonism against plant pathogen
2.3.1.1.1. Race for root colonization
2.3.1.1.2. Biofilm formation
2.3.1.1.3. Siderophores production
2.3.1.1.4. Antibiotic-mediated suppression
2.3.1.1.5. Lytic enzymes and other byproducts
2.3.1.1.6. Induced systemic resistance
2.3.1.2. Plant growth promotion
2.3.1.2.1. Free Nitrogen-Fixing
2.3.1.2.2. Phytohormone production and phosphate solubilization

Chapter 3
A novel rhizobacterium Bk7 for biological control of brown sheath rot of rice caused by Pseudomonas fuscovaginae and its mode of action
3.2. Introduction
3.3. Materials and methods
3.3.1. Isolation and culture conditions
3.3.2. Primary screening
3.3.3. Inhibitory activity of the rhizobacterial culture filtrates against P. fUscovaginae
3.3.4. Pot experiment
3.3.4.1. Seed treatment and plant growth condition
3.3.4.2. Pathogen inoculation
3.3.4.3. Data collection and analysis
3.3.4.4. Growth promotion efficacy (GPE)
3.3.5. Characterization of growth promoting traits
3.3.5.1. Indole acetic acid production
3.3.5.2. Phosphate solubilization
3.3.5.3. Siderophores production
3.3.5.4. Microtitre plate assay for biofilm formation
3.3.6. Mechanism of biological control activity
3.3.6.1. Nature of culture filtrates
3.3.6.2. Multiplex PCR assay for distribution of antimicrobial peptide markers
3.3.6.3. Gene expression analysis with real time quantitative PCR
3.3.7. Identification of selected bacterial strains
3.3.7.1. Fatty acid methyl esters (FAMEs) analysis
3.3.7.2. Sequence analysis for 16S rRNA gene
3.4. Results
3.4.1. Primary selection of antagonists
3.4.2. Extracellular metabolites efficacy
3.4.3. Pot experiment
3.4.3.1. Growth promotion efficacy
3.4.4. Characterization of plant growth promoting traits
3.4.5. Mechanism of biological control activity
3.4.5.1. Molecular nature of the antibacterial metabolites
3.4.5.2. Detection of lipopeptide biosynthetic gene markers
3.4.5.3. Quantitative expression of antimicrobial peptide coding genes
3.4.6. Identification of rhizobacterial strains 3.5. Discussion

Chapter 4
Characterizing the mode of action of Brevibacillus laterosporus B4 for control of bacterial brown strip of rice caused by A. avenae subsp. avenae RS-1
4.1. Abstract
4.2. Introduction
4.3. Materials and methods
4.3.1. Bacterial strains and chemicals used
4.3.2. In vitro inhibition assessment
4.3.3. Characterization of the nature of antibacterial metabolites
4.3.4. Pot experiment
4.3.5. Bactericidal mode of action
4.3.5.1. Biofilm formation assay and transmission electron microscopy
4.3.5.2. Recovery of pathogen and validation
4.3.5.3. RNA isolation and real-time quantitative PCR
4.3.6. Statistical analysis
4.4. Results
4.4.1. In vitro trials
4.4.2. Biological control of bacterial brown strip disease in rice
4.4.3. Mechanism of biological control
4.4.3.1. Molecular nature of the antibacterial metabolites
4.4.3.2. Inhibition of biofilm formation
4.4.3.3. Disruption of cell integrity in A. avenae subsp. avenae
4.4.3.4. Differential expression of virulence-related genes in response to biological control .
4.5. Discussion

Chapter 5
Alleviation of cold and drought tolerance in rice by using consortium of chemical inducers and rhizobacterial strains
5.1. Abstract
5.2. Introduction
5.3. Materials and Methods
5.3.1. Bacterial strains and chemical inducers used
5.3.2. Preliminary screening
5.3.3. Compatibility trials
5.3.4. Synergistic effect on plant growth under cold/drought stresses
5.3.4.1. Measure of MDA, proline and chlorophyll contents
5.3.4.2. Enzyme assays
5.3.4.3. Analysis of gene expression by RT-qPCR
5.3.5. Statistical analysis
5.4. Results
5.4.1. Primary trials
5.4.2. Compatibility tests
5.4.3. Co-inoculation improved growth and stress tolerance in rice plants
5.4.3.1. Change in lipid peroxidation, electrolyte leakage and proline content
5.4.3.2. Bacterization enhanced leaf chlorophyll under drought and cold stresses
5.4.3.3. Enzyme activities
5.4.3.4. Co-inoculation treatment induced changes in plant gene expression
5.4.4. Data analysis
5.5. Discussion

General conclusions

References

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Acknowledgment

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My first and foremost profound thanks and gratitudk go to Almighty AdOh who gave me strength and the volition, guide and hep me to complete this work. I believe that without his gracious andmercy I would not be able to accomplish this Dissertation.

I wouldTike to express the deepest appreciation and sincerest gratitude to Professor Xie Guan-m for his scholarly and continuous guidance, encouragement, valuable advices and suggestions throughout the course of this study.

I greatly appreciate the valuable suggestions and assistance of associated Professor Li Bin and Dr Zhu Bo during my research work.

I wish to express my heartiest thanks to all my dear friends Imran Khan, Sayed Ali, Naqeeb Zaib, Suleman Khan, all my Pakistani friends at Zhejiang University particuOrly Dr. Mumtaz Ali Saand, Essa Ali, Sayed Hussain, Shams Ali Baig, Zia UdOh, Niaz Mohammad for their kind help and nice company during my stay in China.

Words are lacking to express my gratitude to my wonderful ab mates A6dufWareth Almoneafy, Sied AhmedEfsheikh, Yu shang, Tian Wen Xiao, Yang ChunCan, Zhang Guoqing, Shi Yu and Zhou qi Cui for their kind a^ssistan^ce.

My great profound thanks and sincere gratitude to my afectionate and loving Parents, and my uncles Naseer Uddn, Maidaan Ustad, Sultan Khan and my grandma (May Allah bless his soul rest in peace) who always support and encourage me in all of my Ife.

I wish to pay my all of my love to my fa 仕 hfulpartner Dr. Zarqa Nawaz Mirza who stands and shares with me every happiness and hardest moments and encourages me to achieve this success, to Hiba, Naqeeb Uiah, Muhee6, Najee6, Raqee6, Ikram, Safia, Palowsha, Masooma, and Kashan Hussain Khan who always carry and bring all happiness to my life.

Finally my great feeling and exclusive love go to my all brothers and sisters May Allah 61ess all of them and 6ring all happiness to their life. May Alah assist everybody who one day tries to hep me even if he coud not.

List of Tables

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List of Tables

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Abstract

Plant growth-promoting rhizobacteria (PGPR) are free-living, soil-borne bacteria that colonize the rhizosphere and, when applied to crops, enhance the growth of plants, protect from disease and induce systematic resistance against abiotic stress. The present study was undertaken to estimate the potential use of rhizobacterial strains to control the bacterial brown stripe (BBS) caused by A. avenae subsp. a^venae and brown sheath rot caused by P. fuscovaginae respectively, under in vitro and in vi^vo conditions, and study their potential mechanisms. In addition, a consortium of these two PGPRs and different chemical inducers were used to induce systematic resistance in rice plants against abiotic cold and drought stress.

Initially, about 120 rhizobacterial isolates were isolated from rice rhizosphere and examined for their antagonistic activities against A. avenae subsp. avenae and 尸 fuscovaginae, the causal agents for bacterial brown stripe (BBS) and brown sheath rot of rice. Among them, two isolates Bk7 and B4 showed highest potential of antagonistic activity in in vitro assay against P. fuscovaginae and A. avenae subsp. avenae respectively, and further screened for their biocontrol efficacy under greenhouse conditions against individual pathogen. Strain Bk7 and its metabolites significantly suppressed the growth of P. fuscovaginae with 93 % efficacy in laboratory, and exhibited biocontrol efficacy of 76.6 %, by reducing the disease incidence of brown sheath rot to 16.9 % in glasshouse, followed by B4. Moreover, Bk7 significantly (p > 0.05) increased plant height by 46.4 % and fresh weight by 84.3 %, followed by B4, which increased the plant heights and fresh weights by 28.3 and 51.7 % respectively.

Similarly, the application of isolate B4 and its culture filtrates (70 %; v/v) exhibited inhibitory effects against A. avenae subsp. avenae i^ vitro. In greenhouse, 71.9 % reduction of disease incidence of BBS was recorded in the rice plants treated with isolate B4, and considerably increased the plant heights and fresh weights by 28.3 and 51.7 % respectively. Moreover, B4 suppressed the biofilm formation capability of A. avenae subsp. avenae, severely disrupted cell membrane integrity that caused the leakage of intracellular substances, and down-regulated the expression of virulence-related genes (coding for ABC

transporter, protein F, OmpA/MotB domain-containing protein, TonB-dependent receptors) which are responsible for biofilm formation, motility, niche adaptation, membrane functionality and virulence of A. avenae subsp. avenae. Rhizosphere bacterial strain Bk7 was identified as B. amyloliquefaciens strain Bk7, and B4 was identified as Bre^vibacillus laterosporus B4, based on the analysis of 16S rDNA internal transcribed spacer sequences and a fatty acid methyl ester analysis.

The study of bioactivity mechanisms revealed that both strains viz. B. amyloliquefaciens strain Bk7 and Brev. laterosporus B4 were able to Indole acetic acid, Siderophores and ammonia. In contrast, only strain Bk7 was able to solubilize tri-calcium phosphate in Pikovskaya medium and showed the ability to form biofilm. The supernatants from culture filterates of B4 and Bk7 were found to be of protein nature, as evidenced by ammonium sulfate precipitation and subsequent treatment with protease. PCR assay confirmed the presence of five lipopeptide biosynthetic gene markers (srfAA,fe^D, bm;yB, bac^ and ituC) in the genome of strain Bk7, and four (srfAA, fenD, bmyB and bacA) in B4. Moreover, Real-time qPCR of these genes demonstrated that surfactin, iturin and bacylisin coding genes from Bk7 were highly expressed in response to P.fuscovaginae exposure in vitro, which could suggest the potential antibacterial related mechanism is associated to their ability to secret the corresponding lipopeptide in surrounding niche.

Another study was carried out to assess the synergistic effect of B4 and BK7 (BB), salycilic acid and P-aminobutyric acid (SB) individually or in comination (BBSB) for cold and drought stress tolerance in rice plants. After withholding water for 20 days, rice seeds treated with BBSB showed 100% survival rates; improved seedling height (35.4 cm), shoot numbers (6.12); and showed minimum symptoms of chlorosis (19%), wilting (4%), necrosis (6%) and rolling in rice leaves. Likewise, BB inoculation resulted into dark green leaves with reduced overall necrosis (23%), wilting (13%), leaf rolling (5%) and chlorosis; and improved the growth of rice seedlings subjected to chilling stress. The results exhibited that BBSB and BB lead to generate a divergent signals in rice plants that mobilized from roots to leaves, which maintained high superoxide dismutase and catalase activity, proline and cholorohyll content, and reduced MDA content and electrolyte leakage to protect integrity of plant cells and reduce oxidative stress. Besides, the expression of OslMYB3R-

2, OsDREB1A, OsNAC6 and OsGolS1 was remarkably up-regulated by BB under cold, while the expression of OsDIL, OsWRKY11, OsGADPH, OsAP37, OsDREB1 A and OsNAC6 was up-regulated by BBSB treatment under drought stress, suggesting that these genes play important role in abiotic stress tolerance in rice plants. The results indicated that BB and BBSB conferred induced systematic tolerance to chilling and drought in rice plants, by maintaining the cell integrity and photosynthetic activity, enhancing antioxidant enzyme activities and up-regulating the expression of abiotic stress-related genes under stress conditions.

Key Words: Rhizobacteria, Biological control, Abiotic stress tolerance, Acidovorex avenae subsp. avenae, Pseudomonas fuscovaginae, Rice, Bioactivities related mechanisms, Induced systemic resistance.

堠择诅，主葸(pgpr)i—卖仓由习居土镶的的如葸，劣被后用子农〇物的，能诅逬 毡物的/主长、场土毡物矣导系蜣抵逆佟。本邛究的€的K年伐PGPR葸株在輿 (本和•:€(本舍(斗下对抵由A. avenae subsp. avenae和P.fuscovagi+nae名）起的木福如葸沒

穩舍矣（BBS )和木#如葸佟吋筠穩廇矣的增能还其，主场机机理。同的，踩意 PGPR葸株和不同化学褚导物《含褚导木#毡株的秒，主物佟份湛和子軍胪淦的系蜣 抵佟。

从木餡堠择土镶中分輿出120种堠择如葸，刺盍他扪的木餡如葸佟穩舍矣葸和木# 如葸佟吋筠穩廇矣葸的尨抵佟。其中，BK7和B4兩株葸在輿(本舍饵下的冱兩种矣 屈如葸罢云畏矣尨抵读佟。在湛重舍饵下BK7还其代射卢物能 . g薯抑别93 %吋筠 穩廇矣葸的代长，滅少16.9 %发矣專，达76.6 %的，主场敛果，B4葸的场敛次之。 同的，BK7葸罢薯（P>0.05)代力。毡物株矣46.4%和鏵童84.3%;其次IB4的毡 物株矣和鏵童分到代28.3%和51.7%。

B4葸还其(喜养漆液（70 %; v/v)表®出# A. avenae subsp. avenae矣雇如葸的抑别 敛果。在湛重中这B4葸还(喜晷潙液处理的木餡毡株滅少3木#如葸佟穩舍矣发矣 專71.9%，0代力。3禮物株矣28.3%和童军童51.7%。碎究.2云，B4葸抑别A.avenae subsp. avenae的/主物赌衫成能力，户童破坏3如胞赌的宅整沒，导绞如胞而物廣的 珍漏，衿下例3资音’主物赌的衫成、能功沒、’主卷遠后、赌功能和A. avenae subsp. avenae的绞矣力的沒矣沒相菓的I因的表达（ABC科运/f■全，/f■全F,OmpA/MotB 杳仓，TonB旅箱佟爰(本杳仓的偽砝）。晷子16S rDNA的而科杀向隊S存列的分 科和鴻标酸中基錄的分科，棍絲如葸葸株BK7被磬生寿B. amyloliguefciens strain Bk7 和 B4 梭餐复泠 Brevibacillus laterosporus B4。

约B. amyloliquefaciens Bk7和Brev. laterosporus B4的生物这性的奶哈机辦碎贫炎

现，兩种葸株都能够卢，主钊哚乙酸，锬裁(本和氨。相吐之下，S荀葸株BK7I能 够在Pikovskaya介廣该解病酸三耗和與充衫成’主物赌的能力。从B4和BK7 (喜养I

液中馭得的署全肩，通过滅酸德:•冗波和署全絲居读处理，这PCR检刺在B4公必衮

3 4个鴻赋’主物含成I因絲祕物存在（sfAA，fenD，bmyB and bacA)和在BK7公必衮 3 5个鴻赋’主物含成I因絲祕物存在（sfAA, fe^D，bmyB, bacA and ituC )。此夕卜， 因的qPCR必衮，BK7的surfactin, iturin和bacylisin偽接I因在暴露子(本夕卜

的能够的木#吋筠如葸佟穩廇矣葸矣敛表达，冱^能表明增在的抵葸相菓机别和他 扪在洵围济繞分滅鴻赋荀菓。

另一項碎究中逬#拜f古3 B4和BK7 (BB )，木移酸和#独的P-aminobutyric acid (SB)亵《含（BBSB)在份湛和子軍胪淦射爰佟舍饵下的木#毡株公的#同敛后。这 过20天断木处理居，用BBSB处理木餡种子表现出100%的存读專；接矣3幼餐的

矣度（35.4cm)，发身敖（6.12);_里现3畏份的黄化（19%)，羞舅（4%)， 钚死（6% )在仗和木#卷吋。同釋，BB M种0果寿■:冢铼含吋子滅少整（本f不死 (23%)、羞舅（13%)、卷吋(5%)和黄化，接矣木#幼餐爰份湛胪淦下的，主长。0果屎 奇，BBSB和BB能够在木#公卢4 一个不同的戌考，值其从堠到吋子都保抻鉸矣的 起氧化物歧化鷂和过氧化氡鷂读佟、碱氨酸和吋铼音含蜃，衿續份MDA含蜃和电 解廣滲潙保护毡物如胞的宅整佟,滅少氧化后激。此外，在份湛舍饵下这BB处理的 OsMYB3R-2, OsDREBlA, OsNAC6 知 OsGolSl 猞表达气耳缺渔条 A 7 这 BBSB 让理的 OsDIL, OsWRKYll, OsGADPH, OsAP37, OsDREBlA 和 OsNAC6表达公调，冱表明冱#晷因在木餡毡株的秒，主物胪淦射爰佟中发撺童袭〇 用。0果表明，BB和BBSB遏过僱抻如胞的宅整佟和光含读佟，代錄抵氧化鷂读 佟，衿公调3，主物胪淦相菓晷因在胪淦舍饵下的表达桌褚导木餡毡株的份湛和子 軍的系说抵佟。

菓键请：木福棍择如葸，’主物场始，射秒’主物妙淦，木福如葸佟穩舍矣葸，木福如 葸佟吋筠穩廇矣葸，，主物读佟机别，褚导系蜣抵佟。

List of Abbreviations

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Introduction

Chapter 1 Introduction

Plant diseases control is necessary in order to maintain the quality and abundance of food, feed, and fiber produced by growers around the world. Numerous approaches may be employed to prevent, alleviate or control plant diseases. Beyond good agronomic and horticultural practices, agronomists often depend heavily on chemical fertilizers and pesticides. In past, such efforts to agriculture have added significantly to the remarkable improvements in crop yield and quality over the past 100 years. However, the environmental pollution triggered by excessive use and misuse of agrochemicals, as well as fear-mongering by some opponents of pesticides, has led to considerable changes in people’s attitudes towards the use of pesticides in agriculture. Today, there are strict rules on chemical pesticide use, and there is political pressure to remove the most hazardous chemicals from the market. Additionally, the spread of plant diseases in natural ecosystems may preclude successful application of chemicals, because of the scale to which such applications might have to be applied. Consequently, some pest management researchers have focused their efforts on developing alternative inputs to synthetic chemicals for controlling pests and diseases. Among these alternatives are those referred to as biological controls.

The terms “biological control” and its abbreviated synonym “biocontrol” refers to the purposeful utilization of introduced or resident living organisms, other than disease resistant host plants, to suppress the activities and populations of one or more plant pathogens[2]. The rich diversity of the microbial world provides a seemingly endless resource for this purpose. In addition, biological control likely to be stronger than synthetic chemicals in disease control. The durability of the biocontrol over synthetic chemicals is contributed by the complexity of the organismal interactions, the involvement of numerous mechanisms of disease suppression by a single microorganism, and the adaptation of most biocontrol agents to the environment in which they are used[3].

Biological control is a strategy that was proposed half a century ago. Indeed, a symposium held in Berkeley in 1965 was entitled: ‘Ecology of soil-borne plant pathogens; prelude to biological control’[4]. Organisms and procedures involved in biological control include: (i) avirulent or hypo-virulent individuals or populations within the pathogenic species, (2) antagonistic microorganisms, and (3) manipulation of the host plant to resist the pathogen more effectively. Biological control may be accomplished through several approaches including mass introduction of antagonists, plant breeding, and specific cultural practices aimed at modifying the microbial balance. Forty years later, biological control of plant diseases is still in its infancy. Despite its application and potential, biological control has not made the transition from research plots to farmers' fields very efficiently. Quite a few biocontrol agents (BCAs) are currently on the market [5, 6]. Apart from that, the success with biocontrol agents is often unpredictable and too variable for large-scale use, due to lack of knowledge of the biological control system and difficulty in obtaining a successful formulation. Simultaneously there is a demand from society for healthy foods with less chemical residues, and a great concern for preservation of the environment. In order to understand the failure of biocontrol and determine the mechanisms, it is helpful to appreciate the different ways that organisms interact with pathogen, the plant, the microbial community and the environment. Unluckily, very few systems have been studied in detail, and those which are studied in detail, it is unlikely to have sufficient insight to avoid failures and reduce variability. Numerous BCA(s) have been fruitful in research schemes, but scaling up production, providing effective formulation, and producing a stable and inexpensive product has affected them to languish as research marvels that do not reach the marketplace[3]. One of the solutions to overcome inconsistent performance of BCA(s) is to combine two or more beneficial microbes in a biocontrol preparation or combine them with some agrochemicals compounds. In comparison to single BCA, the mixtures of biocontrol agents have the potential for additional broad colonization of the rhizosphere, a more consistent expression of beneficial traits under a wider range of soil conditions, and of being antagonistic to a larger number of plant pests or pathogens [7, 8]. The major advantages of biocontrol agents applied in combination include: (i) multiple modes of action against the target pathogen; (ii) ability to affect more than one stage of the life cycle of the target organism; (iii) activity of microbes during different times in the growing season; (iv) increased consistency in performance over a wider range of soil conditions, stemming from the different environmental niches of the applied microbes; (v) potential to select organisms that affect more than one plant pathogen or pest, thus increasing the spectrum of uses for the product and the alleviation of abiotic stress such as temperature, drought, salt and other conditions [9, 10].

Nowadays more attention is paid to develop useful strategies for eco-friendly and sustainable agricultural practices, mainly depending on application and using of beneficial microorganism particularly Plant growth promotion rhizobacteria (PGPRs) as biofertilizers or biopesticides. [11-13]. PGPRs are defined as a wide range of soil bacteria that colonize the roots of plants and have the ability to enhance plant growth by increasing seed emergence, plant weight, and crop yields[14]. Several mechanisms are employed to achieve the above cited beneficial effects by PGPRs such as Nitrogen fixation, siderophore production, phosphate solubilization, biofilm formation, enhancing the availability of nutrients via root colonization, plant-growth hormone production, production of inhibitory allelochemicals, and induction of systemic resistance (ISR) in host plants to a broad spectrum of pathogens. Moreover, the PGPRs can also be applied for mitigation, environmental restoration, re-vegetation, promotion of soil aggregation under harsh environmental conditions and abiotic stress resistance in plants [10, 15]. A wide range of bacterial groups are considered as PGRs including: Ac~^-netobacter, Agrobacterium, Arthobacter, Azotobacter, Azospirillum, Burkholderia, Bradyrhizobium, Rhizobium, Franhia, Serratia, Thio-Bacillus, Pseudomonads, and Bacillus[16].

Amongst these bacterial genera, genus of Bacillus and Brevibacillus have several advantages that make them good candidates for use as BCA(s). In recent years, the PGPRs from these two genera attracted considerable attention because of their advantages over other PGPR strains in inoculant formulations, stable maintenance in rhizosphere soil, greater potentials in sustainable agriculture, and the production of several different types of insecticidal and antimicrobial compounds as well as inducement growth and defense responses in the host plants. Though, the success of PGPRs strains from Bacillus and Brevibacillus groups is highly contributed by the production of a vast array of secondary metabolites with potential antimicrobial activity. However, the spore forming capability of these species allow them to resist to the adverse environmental conditions, and permit easy formulation and storage of the commercial products[17]. Today the Bacillus-based products represent the most important class of microbial products for phytosanitary use commercially available[18]. Improvements in plant growth and productivity by the applications of Bacillus spp. are mediated by three different ecological mechanisms: promotion of host plant nutrition and growth, antagonism against plant pathogens and insect pests, and stimulation of plant host defense mechanisms.

Rice (Oryza sativa L.) has been cultivated as a major crop for more than 7000 years, and it currently sustains more than half the world population. Although Asia remains the main centre of production and consumption of rice, the importance of rice is increasing rapidly in Africa and Latin America, and exports of rice from the USA and Australia are of major importance to world rice trade. Rice is the staple food for more than 08 billion people in China, where 30% of the world’s rice is grown[19]. It is believed that the importance of this crop will increase in the future with the increasing world population. The high-yielding rice variety has resulted in an increase in rice production but requires large amounts of chemical fertilizers, leading to health hazards and environmental pollution. Rice is highly vulnerable at all stages of growth to pathogens that affect the quality and quantity of its yield. Of the 80 biotic and abiotic diseases described in rice, bacterial brown stripe caused by Acidovorax avenae subsp. avenae, brown sheath rot caused by Pseudomonas fuscovaginae, and cold and drought stresses are among the most destructive. Compared to salt and other stresses, drought, and cold stresses are more pervasive and economically damaging, by causing yield loss of about 17% and 15%, respectively[20]. A. avenae subsp. avenae and P fuscovaginae, which cause disease in many other plants with economic importance, including corn, oats, sugarcane, millet, and foxtail[21], are reported in many countries including Asia, Africa, America, and Europe [22, 23]. Due to some biotic- and abiotic factors, breeding for host resistance is not successful for all host species. Although there are several studies conducted on the biological suppression of rice diseases and numerous BCA strains have already been identified, their efficiency under conditions of abiotic stress has not been adequately elucidated. The abiotic environment has however been recognized as the main criterion determining the efficiency of antagonistic bacteria in soil[24]. Furthermore, the ability of these pathogens to infect a wide range of host plants in different families, beside their capabilities to survive in infested soil or plant debris for long period, the successful and effective management for this disease should mainly depend on the using sustainable agricultural techniques which offer endless and renewable solution for this problem.

Objectives:

The present study was undertaken with the aim to estimate the potential use of plant growth promoting rhizobacteria of Bacillus spp. and Brevibacillus spp. to control the bacterial brown stripe (BBS) caused by A. avenae subsp. a^venae and brown sheath rot caused by P. fuscovaginae under in vitro and in vivo conditions and reveal their potential mechanisms. In addition, a consortium of the two PGPRs and different chemical inducers were used to induce systematic resistance in rice plants against abiotic cold and drought stress.

The main objectives for this study were as follow:

1. To isolate and characterize the rhizobacteria associated with rice crops by biochemical and molecular analysis.
2. To evaluate the biocontrol efficacy of the obtained bacterial strains against BBS caused by A. avenae subsp. avenae and bacterial brown sheath rot caused by P. fuscovaginae, and their effects on plant growth parameters, and elicited tolerance against drought/cold stress in rice plants in vitro and in vivo.
3. To test and compare the effect of a mixture of two selected biocontrol agents, and chemical elicitors on survival rates and growth yield of rice plants under cold and drought stress conditions.
4. To explore the mechanisms associated with the biocontrol efficacy, plant growth promotion and induced resistance against cold/drought stress.

Chapter 2

Literature review

2.1. Current Status and Challenges of Rice Production in China

Rice is the primary food for over 65% of the Chinese people and is the subsistence crop for most resource-poor rice farmers and consumers in rural areas of China[25]. China positions first in total annual rice production (about 204 million tons in 2012) and produced 29% of the world’s rice (Figure 1)[26]. About 35% of the planting area used for grain crops, is occupied by rice in China, which accounts for 41% of total grain production according to data of 2006[27]. During the past fifty years, rice production in China has boosted by 3-folds, mainly due to increased grain yield rather than increased planting area. This upsurge has come from the development of high-yielding varieties and improved crop management practices such as nitrogen fertilization and irrigation. However, yield unproductivity of rice has been detected in the past ten years in China. As its population rises, China will need to produce about 20% more rice by 2030 in order to meet its domestic needs if rice consumption per capita stays at the current level[28]. The major problems challenging rice production in China are narrow genetic background, extra use of fertilizers and pesticides, breakdown of irrigation infrastructure, diseases, oversimplified crop management, and a weak extension system. In spite of these challenges, good research approaches which can drive increased rice production in China comprise, the development of new rice varieties with high yield potential, improvement of resistances to major diseases and insects, and to major abiotic stresses such as drought and heat, and the establishment of integrated crop management[28].

As in rice, yield potential is limited by some biotic and abiotic factors, some of these potential threats to rice production and yield, and their biological management strategies are reviewed in detail in the following sections.

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Figure 1. a. Average rice production of top 5 producers from the year 2010-2013. b. Total rice production in China and the world from 1961 to 2010.

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Figure 2. Rice fields in China

2.3.1. Bacterial Brown sheath rot of rice

In 1976, a new disease of rice namely sheath brown rot was noticed and regarded as the most important bacterial disease of rice in Hokkaido, Japan[23]. Compared to the established and renowned rice diseases, sheath brown rot caused by P. fusco^vag^-nae can be considered new but getting widespread and serious all over the world[29]. The disease has been reported in all continents of the world except Antarctica, and is favored by high elevation, low temperature and high humidity. At first, P. marginalis (P. fluorescens biovar II) was stated to be the causal agent of the disease. However, after more detailed studies, the bacterium was re-identified as a new species and was given a new name of P. fuscovaginae[23]. Since then, a number of reports on the new disease from other parts of the world explicitly Central Africa[30], Latin America [31, 32], Burundi[33], Mexico[34], Brazil, Cameroon, Madagascar, Reunion Island and Rwanda[35], the Philippines[36]and China[37]also were published. Sheath brown rot can occur in areas of high altitude (1200-1700 m above sea level), low temperature (20-22°C), and high humidity, in both temperate and tropical conditions. The disease has been reported to cause appreciable losses. For example, yield loss of as high as 72.2% were reported in Indonesia, total yield loss (almost 100%) in Madagascar, 46.0% in Perak[29].

2.1.1.1. The pathogen (P. fuscovaginae) and its disease symptoms on rice Kingdom: Bacteria

Phylum: Proteobacteria

Class: Gamma Proteobacteria

Order: Pseudomonadales

Family: Pseudomonadaceae

Genus: Pseudomonas Species: P.fuscovaginae

P. fuscovaginae nom. rev. (fus. co. va. gi' nae, L. adj. fuscus fuscous; L. fem. n. vagina vagina, sheath; M. L. fem. n. fuscovaginae of a fuscous vagina). Gram-negative, non-spore-forming, rodshaped cells with round ends, 0.5 to 0.8 by 2.0 to 3.5 ^m. Cells occur singly or in pairs and are motile by means of one to four polar flagella. After 4 to 5 days at 28°C on nutrient agar moderate growth consisting of white to light brown, smooth, glistening, raised, translucent, circular, butyrous colonies 3 to 5 mm in diameter is produced. A green fluorescent, diffusible pigment is produced on King's medium B. No slime is produced on nutrient agar containing 5% sucrose. P. fuscovaginae belongs to the authentic rRNA group I of pseudomonads pathogenic toward several crop species, such as rice, maize, sorghum, and wheat [34, 38, 39].

Bacterial brown sheath rot of rice is characterized by longitudinal brown to reddish brown necrosis 2-5 mm wide ranging the total length of the leaf sheath and blade[23]. The disease symptoms could be detected as early as at seedling stages and the infected dead seedlings. Affected sheaths enfolding the panicle may show widespread water soaking and necrosis with ill-defined margins. But, if the infection happens at advanced growth stages, the field of infected rice plants turn into yellowish. Lower parts of the leaf sheath usually turn light or dark brown. Glumes become discolor before emerging from such panicles. Grain on affected tillers maybe completely discolored, sterile and empty[40]. At the later stages of infection, the whole leaf sheath becomes necrotic (Figure 3).

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Figure 3. Typical symptoms of brown sheath rot diseases on rice seedlings caused by P. fuscovaginae.

2.1.2. Bacterial brown stripe of rice

Bacterial brown stripe (BBS), caused by A. avenae subsp. avenae , was first reported on rice in Japan[41]. This disease was firsts seen lowland and upland nurseries and in greenhouse pots. The disease is frequently detected in rice-growing countries. Since then the disease has been reported to cause severe losses in rice in Philippines, China, Korea, and Iran, as well as in Africa, Latin America and Portugal in Europe[42]. In China, the causal agent has often been referred to as P. syringae pv. panici or P. panici. However, neither epithet has been validly published[43]. The pathogen causes disease in many plants with economic importance, including rice, corn, oats, sugarcane, millet, and foxtail[44]. Since A. avenae subsp. avenae is seed born pathogen in rice, therefore, infected seeds are important sources of primary inocula and a means of dissemination of the pathogen to new areas[45]. Seed transmission of the BBS pathogen A. avenae subsp. avenae , was first demonstrated in South Korea, and a petri dish method for its detection was established [46, 47]. Losses are associated to the inhibition of seed germination and to seedling damage in nursery boxes adapted to mechanize transplanters. Infected seed may act as an important source of dissemination of the bacterium from one geographical area to another. During 1998-1999, nursery paddies of Iran were surveyed and samples showing water-soaked stripes on the leaves and sheaths were collected. It was reported that BBS of rice appears to be widespread among various local rice cultivars[48]. The pathogen was detected in 60% of seed samples tested, indicating that A. avenae subsp. avenae is widely distributed in Tanzania[49]. Maximum infection (75%) was found in cv. Karuna from India. There were no marked differences in percentage of seedling infection recorded in petri dishes and germination stands. The bacterial stripe developed in 5 to 30 % of the seedlings from seed samples harvested in 1976.

2.1.2.1. The pathogen (A. avenae subsp. avenae) and its symptoms on rice

Kingdom: Bacteria

Phylum: Proteobacteria

Class: Betaproteobacteria

Order: Burkholderiales

Family: Comamonadaceae

Genus: Aci^ovorax

Species: Acidovorax avenae

A. avenae subsp. avenae is gram-negative, non-spore-forming, non-capsulated rod, 1.2- 3.0 ^m x 0.4-0.6 ^m, with 1-2 polar flagella[50]. The bacterium is positive for oxidase, nitrate reduction and starch hydrolysis. A. avenae subsp. avenae could be isolated from discolored seeds as well as from healthy looking seeds of rice. The pathogen significantly affects seed germination and seed vigor when 10[10]cfu/mL and 10[8]cfu/mL of the bacterium were inoculated on rice seeds. The bacterium could have transmitted from seeds to seedlings with 0.2%-5.0% of infection. The symptoms of the brown stripes starts on the bottom of stems of 5 days old seedling, after emergence and frequently extend into the sheaths, then spreading along the leaf midrib and throughout the entire seedling at the one-leaf stage[51]. According to Xie et al.[50]The symptoms of seedlings induced by naturally infected seeds can be described as the appearance of the water-soaked strip on the basal part of the leaf sheath. They soon become dark brown and could be measured up to 10 cm in length and 1 mm in width, sometimes merging to form wider lesion. In some cases, young leaves are attacked leading to bud rot, which eventually die (Figure 4.)[50]. Although this worldwide rice disease has been proved economically important, very little is known about the molecular basis of A. avenae subsp. avenae in rice plants. Recently, the genome of A. avenae subsp. avenae RS-1 has been released by Xie et al.[22]2011), which made a great contribution to understand this pathogen and control of BBS. Liu et al.[44]characterized the association of A. avenae subsp. avenae Type IV pili (TFP) with BBS in rice, and indicated that the pilP gene and TFP in A. avenae subsp. avenae play a key role in plant pathogenicity, twitching motility, and biofilm formation. Earlier, Bahar et al.[52]and Tammam et al.[53]reported that TFP play an important role in DNA uptake, attachment to surfaces, twitching motility, and biofilm formation in numerous bacterial genera[54]. More, recently, Ibrahim et al.[55]obtained differential expression of in vivo and in vitro protein profile of outer membrane of A. avenae subsp. avenae. It was observed that the major proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo, indicating that these proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existence of Type VI secretion system core components, which can eventually help to explore the molecular mechanism of the pathogen and control disease.

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Figure 4. Typical symptoms of BBS on rice seedlings caused by A. avenae subsp. avenae.

2.1.3. Abiotic stress

Abiotic stresses (such as high salinity, drought and flood, high and low temperatures) are major factors that reduce the agricultural productivity of rice[1]. The predominantly rice-growing areas in Asia (〜130 million hectares) are often susceptible to severe abiotic stresses, the most common being drought. Drought has become the most important restraint to realizing the yield potential of rice across all agro-climatic zones. In certain years, abiotic stresses cause crop losses by as much as 50%[56]and drought alone may cause yield losses of as much as 15%[57]. Drought spells across Asia have become more frequent and severe, leading to irregular and insufficient irrigation of the crop and depletion of groundwater resources leading to 100% yield losses in certain areas[58]. Insufficient water availability leads to a host of bio-chemical, physiological, and metabolic changes in rice. These changes, include a host of biochemical pathways associated with signal perception, transduction, and regulation of gene expression in a temporal and spatial pattern.

Similarly, rice seedlings are particularly sensitive to chilling in early spring, which causes slow development, yellowing, withering, reduced tillering, and stunted growth[59]. During the last 4 decades of environment variation, increased minimum air temperatures during growing seasons have caused a substantial decrease in rice yields in China and the Philippines and are predicted to continue [60, 61]. A significant number stress-inducible genes are reported to be responsive to both water stress and low temperature. Some of these genes are induced only by water stress, and several genes respond only to low temperature[62]. These abiotic stress-related genes can be classified into three groups: genes encoding for signal transduction components; functional proteins; and transcription factors (Figure 5).

Oh et al.[63]stated that the expression of stress-related genes is largely regulated by specific transcription factors. The promoters of stress responsive genes typically have cis-regulatory elements such as DRE/CRT, ABRE, and MYCRS/MYBRS and are regulated by various upstream transcriptional factors[64]. Transcription factors such as OsDREB1A, OsAP37, OsMYB3R-2, and OsNAC6 are proteins that act together with other transcriptional regulators, including chromatin remodeling/modifying proteins, to employ or obstruct RNA polymerases to the DNA template[65]. When plants are exposed to abiotic stress, stress signals activate regulatory genes, which can be dependent or independent of hormones or second messengers. In turn, regulatory genes act on many functional genes. Expression of functional genes can lead to protection of cells, either directly or by causing a physiological response in the cells. Plants acquire abiotic stress tolerance via either mechanism (Figure 5). Major mechanisms of the regulatory networks underlying environmental stress adaptation, pathogen recognition, and defense include reactive oxygen species (ROS) signaling, plant hormones, changes in redox status, and inorganic ion fluxes, such as Ca[2]+ [66, 67].

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Figure 5. Schematic model for molecular responses to abiotic stress in plants[1].

2.2. Approaches to improve the health of rice plant

Biotic and abiotic stress tolerance is a very complex trait. It is challenging to develop significantly stress-tolerant rice cultivars due to the many factors that influence stress tolerance and because of difficulties in reproducing stress strength and duration in the field[1]. Further evidence for abiotic and biotic stress resistance crosstalk comes from studies of the effects of exogenous application of chemicals that sensitize plant defense responses, a phenomenon called priming[68]. Very less efforts are made in history to cure the above cited bacterial rice diseases. Recently, inhibitory effect and mode of action of chitosan solution against rice bacterial brown stripe pathogen A. avenae subsp. avenae strain RS-1 was examined[69]. They indicated that the chitosan at the 0.1, 0.2, and 0.4 mg/mL markedly inhibited the pathogen growth, mainly due to membrane disruption and lysis, reduction of biofilm formation, and altered the expression of ompA/motB gene. Our survey of literature could not find any report regarding the control brown sheath rot of rice disease. However, many chemical and biological agents have been used and reported, which to induce systemic resistance and increase tolerance to abiotic stresses in rice and other plants. For example, application in Arabidopsis thaliana of P-aminobutyric acid (BABA), a non-protein amino acid, results in enhanced resistance to a wide range of stresses including cold, heat, drought, and salinity stress, as well as enhanced resistance to biotrophic as well as necrotrophic fungi[70]. The broad spectrum protective effect of BABA against numerous plant diseases has been well-documented in the literature. It is evidenced that the protective effect of BABA is due to a potentiation of natural defense mechanisms against biotic and abiotic stresses[71]. On other hand, exogenous application of SA renders many crop plants more tolerant to an extensive array of abiotic stresses[72], and similar observations have also been reported after treatment with jasmonates[73]. Yang et al.[74]have stated that SA significantly modulated redox balance and protected rice plants from oxidative stress caused by aging as well as biotic and abiotic stress. Furthermore, treatment of rice with SA and jasmonic acid has been reported to rapidly induce the expression of a PtdIns-specific phospholipase C, which correlated with induced resistance against the blast fungus Magnaporthe grisea[75].

2.3. Plant growth-promoting rhizobacteria

Plant growth-promoting rhizobacteria (PGPR) are free-living, soil-borne bacteria, isolated from the rhizosphere, which, when applied to seeds or crops, enhance the growth of the plant or reduce the damage from soil-borne plant pathogens[76]. It has been estimated that more than 100 million tons of nitrogen, potash and phosphate-chemical fertilizers have been used annually in order to increase plant yield[77]. The potential negative effect of chemical fertilizers on the global environment and the cost associated with production has led to research with the objective of replacing chemical fertilizers with bacterial inoculants.

Bacterial inoculants which help in plant growth are generally considered to be of two types a) symbiotic and b) free-living [78, 79]. Beneficial free-living bacteria referred to as PGPR are found in the rhizosphere of the roots of many different plants[80]. Breakthrough research in the field of PGPR occurred in the mid-1970s with studies demonstrating the ability of Pseudomonas strains capable of controlling soil-borne pathogens to indirectly enhance plant growth and increase the yield of potato and radish plants [76, 81]. The effect of PGPR on agricultural crops has been investigated and published by various authors in in the last two decades with recent applications on trees [82, 83]. During 1983 and 1984 more than 4,000 bacterial strains were isolated from the rhizosphere of plants grown in the Canadian High Arctic and screened for the ability to fix nitrogen. Some strains demonstrated the ability to reduce acetylene and colonize roots of canola when grown at low temperatures[84]. Strains which exhibited the potential to be PGPRs were identified as P. putida, P. putida biovar B, P. f^^oresce^s, Arthobacter citreus and Serratia liquefaciens. The ability of these strains to be used as bacterial inoculants in agriculture was tested in greenhouse and field trials with different formulations and they increased the yield of canola in both types of trial. Salamone[85]reported the growth-promoting effect of P. fluorescens strain G20-18 on wheat and radish plants by production of cytokinin phytohormones. As the effect of PGPR on plants was demonstrated, the concept of PGPR began to gain importance and a large number of bacterial strains have been isolated, screened and evaluated for plant growth promotion [77, 86, 87].

Rhizosphere bacteria promote plant growth and yield either directly or indirectly [80, 88]. The direct mechanisms of plant growth promotion may involve the synthesis of substances by the bacterium or facilitation of the uptake of nutrients from the environment[77]. The indirect promotion of plant growth occurs when PGPR lessen or prevent the deleterious effects of plant pathogens on plants by production of inhibitory substances or by increasing the natural resistance of the host[89]. The direct growth promoting mechanisms are as follows i) nitrogen fixation ii) solubilization of phosphorus iii) sequestering of iron by production of siderophores iv) production of phytohormones such as auxins, cytokinins, gibberellins and v) lowering of ethylene concentration [77, 80, 88]. For example, strain GR12-2, P. putida, isolated from the rhizosphere of plants growing in the Canadian High Arctic, was found to promote growth of canola cv. Tobin by fixing nitrogen and enhancing the uptake of phosphate under gnotobitoic conditions[84], by synthesizing siderophores that can solubilize and sequester iron from the soil and supply it to the plants Glick[88], by production of the phytohormone IAA[90]and by lowering of ethylene concentration via production of the enzyme ACC deaminase[91]. The indirect mechanisms of plant growth promotion by PGPR include i) antibiotic production ii) depletion of iron from the rhizosphere iii) synthesis of antifungal metabolites iv) production of fungal cell wall lysing enzymes v) competition for sites on roots and vi) induced systemic resistance [77, 78, 88].

2.3.1. Mechanisms involved in PGPR bioactivity

Because biological control can result from many different types of interactions between organisms, researchers have focused on characterizing the mechanisms operating in different experimental situations. In all cases, pathogens are antagonized by the presence and activities of other organisms that they encounter. Direct antagonism results from physical contact and/or a high-degree of selectivity for the pathogen by the mechanism(s) expressed by the BCA(s). In contrast, indirect antagonisms result from activities that do not involve sensing or targeting a pathogen by the BCA(s). The different mechanisms of antagonism occur across a spectrum of directionality related to the amount of interspecies contact and specificity of the interactions are assembled by Pal et al.[2](Table 1).

Table 1. Types of interspecies antagonisms leading to biological control of plant pathogens.

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The list of indirect mechanisms used by PGPR is substantial. Some have been included here, with the most relevant being discussed in detail:

2.3.1.1. Antagonism against plant pathogen

2.3.1.1.1. Race for root colonization

Successful colonization and persistence in the plant rhizosphere are required for PGPR to exert their beneficial effect on plants[92]. Root colonization, which is a complex process, is under the influence of various parameters such as bacterial traits, root exudates, biotic and abiotic factors[93], is also considered to be a crucial step in the application of microorganisms for beneficial purposes such as biofertilization, phytostimulation, biocontrol and phytoremediation[94]. The method chosen for studying the traits associated with root colonization depends on the objective and different approaches and techniques have been used to quantify and identify inoculated strains on the host plant[94]. A mutant strain P. JJuorescens WCS365 with Tn5lacZ mutation colonized the roots to a lesser extent than the wild-type[95]. Dekkers et al.[96]showed that the gene encoding NADH dehydrogenase I plays an important role in root colonization. Another gene required for efficient colonization is the sss gene, encoding a site-specific recombinase of the lambda integrase family which helps in adapting cells to rhizosphere conditions[97]. Further, it was hypothesized that a two-component system involving genes coJR and coJS plays an important role in the root colonizing ability of P. fJuorescens strain WCS365. Miller et al.[98]has shown that the gene rpoS is essential for plant root colonization by P.putida in a competitive environment. Rainey[99]identified as many as twenty genes that were induced during root colonization using a novel promoter trapping technology. The root surface and surrounding rhizosphere are significant carbon sinks. Thus, along root surfaces there are various suitable nutrient-rich niches attracting a great diversity of microorganisms, including phytopathogens. Competition for these nutrients and niches are fundamental mechanisms by which PGPRs protect plants from phytopathogens. PGPR reach root surfaces by active motility facilitated by flagella and are guided by chemo-tactic responses[100]. ). Known chemical attractants present in root exudates include organic acids, amino acids, and specific sugars[101]. Some exudates can also be effective as antimicrobial agents and thus give ecological niche advantage to organisms that have adequate enzymatic machinery to detoxify those[102]. The quantity and composition of chemo-attractants and antimicrobials exuded by plant roots are under genetic and environmental control. This implies that PGPR competence highly depends either on their abilities to take advantage of a specific environment or on their abilities to adapt to changing conditions. As an example, AzospiriJJum chemotaxis is induced by sugars, amino acids, and organic acids, but the degree of chemotactic response to each of these compounds differs among strains[103]. PGPR may be uniquely equipped to sense chemo-attractants, e.g., rice exudates induce stronger chemotactic responses of endophytic bacteria than from non-PGPR present in the rice rhizosphere[104]. It has also been recently demonstrated that the high bacterial growth rate and ability to synthesize vitamin B1and exude NADH dehydrogenases contribute to plant colonization by PGPR[105]. Another determinant of root colonization ability by bacteria is type IV pili, better known for its involvement in the adhesion of animal and human pathogenic bacteria to eukaryotic cells. The type IV pili also play a role in plant colonization by endophytic bacteria such as Azoarcus sp.[106]. Bacterial traits required for effective root colonization are subject to phase variation, a regulatory process for DNA rearrangements orchestrated by site specific recombinase[97]. In certain PGPR, efficient root colonization is linked to their ability to secrete a site-specific recombinase[97].

2.3.1.1.2. Biofilm formation

Biofilms are build colonies of single or multispecies of microbial cells adherent to biotic or abiotic surfaces and/or in intimate contact with each other, encased in a self-produced matrix of extracellular polymeric substances (EPS). The colonization of plant surfaces by plant-associated microbial populations shows similarities to the formation of biofilms on abiotic surfaces with certain genetic determinants common to both processes[107]. As defined by Saleh-Lakha and Glick[108]these bacterial accumulations have the competence to interconnect chemically with one another through quorum sensing, functioning as a single unit. Consequently, PGPR when they are in biofilm mode should perform well in inhibiting competing organisms, nutrient uptake, quick responses, and adaptation to changing environmental conditions. However, the natural existence of PGPR in the soil has not been adequately investigated, and the knowledge of biofilmed mode of PGPR and their actions is vastly unexplored. Some reports have emphasized that the plant- associated biofilms have a greater aptitude to defend themselves from exterior stress and microbial competition that are characteristic of the rhizosphere, and also to produce advantageous effects in plant growth promotion [109, 110]. As a recent progress in biofertiliser research, biofertilisers have been produced from developed fungal - rhizobial biofilms in vitro[111], which are now known as BFBFs[112]. Seneviratne et al.[110]have recently observed the heavy colonization of FBBs/FRBs on root hairs of rice, tea (CameJJia sinensis), Anthurium (Anthurium andraeanum), and wheat (Triticum aestivum) (Figure 6.). These BFBFs exhibited increased biological N2- fixation, organic acid, mineral nutrient release in the soil and plant growth hormone production etc., compared to mono- or mixed cultures of the microbes without biofilm formation[112]. Moreover, the combined application of BFBFs and N2-fixers significantly increased soil organic C by ca. 20%, and reduced leaf transpiration by ca. 40%. It also supported plant growth, rhizoremediation and soil moisture conservation in comparison to the 100% chemical fertilization[113]. Molecular and genetic studies have determined that biofilms vary significantly from single microbes in planktonic mode of growth in vital features such as gene expression[114]and physiological functions[115]. More, Stoodley et al.[116]reported that BCA(s) show an elevated antimicrobial tolerance as a result of biofilm structure, physiological adaptation, and the adherent nature of microbial cells. These biofilm-forming bacterial species can produce a variety of antimicrobial metabolites which include broad spectrum lipopeptides of BaciJJus, such as surfactins that are potent biosurfactants and important for maintaining the aerial structure of biofilms[102]. Root colonization by B. subtiJis 6051 forms a stable, extensive biofilm and secretes surfactin, act together to protect plants against infection by other pathogenic bacteria (Bais, 2004). The biofilm-forming strains of B. thuringiensis suppress the quorum sensing dependent virulence of the plant pathogen Erwinia carotovora through a new form of microbial antagonism called signal interference[117]. Timmusk et al.[118]who reported that PaeniBaciJJuspoJymyxa forms biofilms around the root tip and behaves as a root-invading bacterium attributing a possible mechanism in biocontrol and drought tolerance-enhancing activities.

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Figure 6. Root hairs of rice (a), tea (b), and anthurium (c) colonized by microbial biofilms (BF), when fungal-bacterial biofilms (FBB) or fungal-rhizobial biofilms (FRB) were inoculated under axenic conditions. Darkness is due to cotton blue stain absorbed by the extra cellular polymeric substances (EPS) produced by the BF. Reprinted from Seneviratne et al.[110].

2.3.1.1.3. Siderophores production

Iron is an essential nutrient for plants. Iron deficiency is exhibited in severe metabolic alterations because of its role as a cofactor in a number of enzymes essential to important physiological processes such as respiration, photosynthesis and nitrogen fixation. Iron is quite abundant in soils but is frequently unavailable for plants or soil microorganisms since the predominant chemical species is Fe[3]+, the oxidized form that reacts to form insoluble oxides and hydroxides inaccessible to plants or microorganisms. Plants have developed two strategies for efficient iron absorption. The first consists of releasing organic compounds capable of chelating iron, thus rendering it soluble. The second strategy consists of absorbing the complex formed by the organic compound and Fe[3]+, where the iron is reduced inside the plant and readily absorbed.

Some rhizosphere bacteria are able to release iron-chelating molecules to the rhizosphere and hence serve the same function as the plants[119]. Siderophores are low molecular weight compounds, usually below 1 kDa, which contain functional groups capable of binding iron in a reversible way[120]. Although various bacterial siderophores differ in their abilities to sequester iron, in general, they deprive pathogenic fungi of this essential element since the fungal siderophores have lower affinity[121]. Some PGPR strains go one step further and draw iron from heterologous siderophores produced by cohabiting microorganisms[121]. Siderophore biosynthesis is generally tightly regulated by iron sensitive Fur proteins, the global regulators GacS and GacA, the sigma factors RpoS, PvdS, and Fp^vI and quorum sensing auto inducers such as N- acyl homoserine lactone[122]. However, some data demonstrate that none of these global regulators is involved in siderophore production. Neither GacS nor RpoS significantly affected the level of siderophores synthesized by Enterobacter cloacae CAL2 and UW4[123]. In addition, GrrA/GrrS, but not GacS/GacA, are involved in siderophore synthesis regulation in Serratia pJymuthicastrain IC1270, suggesting that gene evolution occurred in the siderophore producing bacteria[124]. A myriad of environmental factors can also modulate siderophores synthesis, including pH, the level of iron and the form of iron ions, the presence of other trace elements, and an adequate supply of carbon, nitrogen, and phosphorus[125]. Tn5 insertion mutants of strain 267 defective in siderophore production did not differ from the wild-type in promoting the growth of clover, suggesting that the siderophore production had no effect on stimulating nodulation. In contrast Gill et al.[126]revealed that mutants of Rhizobium meliotithat were unable to produce siderophores were able to nodulate the plants, but the efficiency of nitrogen fixation was less compared to the wild-type, signifying the importance of iron in nitrogen fixation. KJuyvera ascorbata, a siderophore-producing PGPR, was able to protect plants from heavy metal toxicity[127].

2.3.1.1.4. Antibiotic-mediated suppression

The basis of antibiosis as a biocontrol mechanism of BCA has become increasingly better understood over the past two decades. Antibiotics are microbial toxins that can, at low concentrations, poison or kill other microorganisms. Most microbes produce and secrete one or more compounds with antibiotic activity. In some instances, antibiotics produced by microorganisms have been shown to be particularly effective at suppressing plant pathogens and the diseases they cause. Interestingly, some antibiotics produced by PGPR are finding new uses as experimental pharmaceuticals[128], and this group of bacteria may offer an untapped resource for compounds to deal with the alarming ascent of multidrug-resistant human pathogenic bacteria. Antibiotics more recently discovered in biocontrol strains are d-gluconic acid[129]and 2-hexyl- 5-propyl resorcinol[130]. Volatiles other than hydrogen cyanide, such as 2,3-butanediol, or blends of volatiles produced by BaciJJus spp[131]can be involved in plant protection. Finally, lipopeptides (LPs) produced by B. subtiJis and by pseudomonads have been implied in biocontrol.

One of the most frequently used and well-studied organisms, the rhizobacterium B. subtiJis, has an average of 4 - 5% of its genome dedicated to antibiotic synthesis and has the capability to synthesize more than two dozen structurally diverse antimicrobial compounds[132]. Members of the BaciJJus genus are often considered microbial factories for the production of a vast array of biologically active molecules potentially inhibitory for phytopathogen growth, such as kanosamine or zwittermycin A from B. cereus[133]. Some examples of antibiotics reported to be involved in plant pathogen suppression are listed in Table 2. Among these antimicrobial compounds, LPs of the surfactin, iturin and fengycin (or plipastatin) families have well-acknowledged potential uses in biotechnology and biopharmaceutical applications due to their surfactant properties. Different groups of LPs can add an advantage to the producing BaciJJus strains in specific ecological niches[134]. Iturin production appears to be restricted to B. subtiJis[135]and B. am^yJoJiquefaciens[136]. Surfactin and related variants such as lichenysin have been isolated from B. coagulans[137], B. pumiJus and B. Jicheniformis[138], and fengycin production was identified in B. cereus[139]and B. thuringiensis[140]in addition to B. subtiJis[141]and B. am^yJoJiquefaciens[136]. Recent advances show that they can act not only as ‘ antagonists ’ or ‘ killers ’ by inhibiting phytopathogen growth but also as ‘ spreaders? by facilitating root colonization and as[6]immuno-stimulators' by reinforcing host resistance potential.

Table 2. Some of antibiotics produced by BCAs[2]

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