Review

The mechachanism and mode of action in microbial biocontrol agents against plant disease.

Birhanu Gizaw*

Microbial Biodiversity Directorate, Ethiopian Biodiversity Institute, P. O. Box 30726 Addis Ababa, Ethiopia.

Abstract

Pathogenic microorganisms are the main constraints affecting the production and productivity of crops both in terms of quality and quantity up to 20–40% yield loss. Existing plant disease management relies predominantly on toxic pesticides. Continuous use of chemicals for plant disease control will have adverse effect on the environmental, human and animal health, biodiversity loss. Researchers are focusing on potential biological control microbes as viable optional for the management of pests and plant pathogens.The purposeful utilization of living organisms whether introduced or indigenous, other than the disease resistant host plants, to suppress the activities or populations of one or more plant pathogens is referred to as bio control. Biological control involves the use of beneficial organisms, their genes, and/or products, such as metabolites, that reduce the negative effects of plant pathogens and promote positive responses by the plant. In this direction, a number of commercial products have been registered both at national and inter-national levels based on different fungal and bacterial antagonists.Understanding mode of action in microbial antagonistic activities are very impotant to screen and formulate new potential biocontrol agent. The modes of mechanism included direct inhibition of spore germination and mycelial growth, competition with pathogen for space and nutrients, biofilm formation, sidrophore production, induction of host resistance and iron depletion, where the iron, methionine competition was considered as the key action.

Key word;-Biocontrol, biofilm, competation, Methionin, spore, Sidrophore

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1.Introduction

It is a persistent issue worldwide that an enormous number of plant pathogens varying from the smallest

viroid to more complex pathogens such as 700 viruses, 2000bacteria, 10000 fungi spp., oomycetes and

nematodes species cause many important plant diseases and are responsible for major crop losses at least

20–40% yield(Savary etal.,2012). Recently new fungal and fungal-like plant pathogen occurance have

increased by more than 7 fold since 2000 (Fisher et al.,2012). Every year plant diseases cause an

estimated 40 billion dollars losses worldwide either directly or indirectly (Roberts et al.,2006). To address

food security agricultural yields must increase to match the growing human population in the near future.

There is now a strong push to develop low-input and more sustainable agricultural practices that optional

alternatives to chemicals for controlling pests and diseases, a major factor of heavy losses in agricultural

production. Based on the adverse effects of some chemicals on human health, the environment and living

organisms researchers are focusing on potential biological control microbes as viable optional for the

management of pests and plant pathogens (Sharifah Farhana te al.,2018). The terms “biological control”

and its abbreviated synonym “biocontrol” have been used in different fields of biology most notably

entomology and plant pathology. In entomology, it has been used to describe the use of live predatory

insects, entomopathogenic nematodes, or microbial pathogens to suppress populations of different pest

insects. In plant pathology, the term applies to the use of microbial antagonists to suppress diseases as

well as the use of host specific pathogens to control weed populations . In both fields, the organism that

suppresses the pest or pathogen is referred to as the biological control agent(BCA). DeBach (1964)

defined biological control as "the action of parasites, predators, or pathogens in maintaining another

organism's population density at a longer average than would occur in their absence." Smith (1919) first

used term "biological control" to signify the use of natural enemies whether introduced or otherwise

manipulated to control insect pests. DeBach (1964) further refined the term and distinguished "natural

control" from "biological control" National Research Council took into account modern biotechnological

developments and referred to biological control as “the use of natural or modified organisms, genes, or

gene products, to reduce the effects of undesirable organisms and to favor desirable organisms such as

crops, beneficial insects, and microorganisms”, but this definition spurred much subsequent debate and it

was frequently considered too broad by many scientists who worked in the field (US Congress, 1995).

The history of Biological Control may be divided into 3 periods: 1. The preliminary efforts when living

agents were released rather haphazardly with no scientific approach. Little precise information exists on

successes during this time. Roughly 200 A.D. to 1887 A.D., 2. The intermediate period of more

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discrimminating biocontrol which started with the introduction of the Vedalia beetle, Rodolia cardinalis

Mulsant, for control of the cottony cushion scale in 1888. Period extended from 1888to ca. 1955; and 3.

The modern period characterized by more careful planning and more precise evaluation of natural

enemies. Period from 1956 to the present. Biological control was discovered by trial and error and then

practiced in agriculture long before the term itself came into use (Baker and Cook, 1974). The era of

modern biological control, involving the deliberate transfer and introduction of natural enemies of insect

pests, was launched 100 years ago with the highly successful introduction of the vadalia beetle from

Australia to California in 1888 to control the cottony cushion scale of citrus. the German plant pathologist

C. F. von Tubuef wrote a somewhat speculative article entitled "Biologische Bekampfung von

Pilzkrankheiten der Pflanzen." This is apparently the first reference in the scientific literature to the term

"Biologische Bekampfung" or "biological control" (Baker, 1987). More broadly, the term biological

control also has been applied to the use of the natural products extracted or fermented from various

sources. These formulations may be very simple mixtures of natural ingredients with specific activities or

complex mixtures with multiple effects on the host as well as the target pest or pathogen. biocontrol differ

depending on the target of suppression; number, type and source of biological agents; and the degree and

timing of human intervention. Most broadly, biological control is the suppression of damaging activities

of one organism by one or more other organisms, often referred to as natural enemies. There is increasing

evidence that biological control occurs naturally on plant surfaces (e.g. in the rhizosphere, phylloplane

and fruit surfaces) by the activity of epiphytic microflora. The most convincing indirect evidence for this

control comes from examples where the use of an agricultural chemical to control a particular leaf disease

results in the occurrence of another disease problem previously regarded as unimportant. The increasing

use of chemicals as pesticides to eliminate Plant pathogen has provided effective solutions in agriculture.

However, due to the fact that the excessive use of these chemicals such as thiabendazole and o-

phenylphenol causes environmental pollution, and as plant pathogenic agents are quickly become resistant

to chemical pesticides The one of the major disadvantage of chemical pesticides is that many of them are

not able to breakdown into simple and safer constituents and remained intact over a long timeperiod

polluting soil environmental(Gilden 2010). Synthetic pesticides are also non-targeted in nature as they

affect the broad spectrum of microbe including plant beneicial microbe and the whole biodiversity

loss.Considering the high price of pesticides and their accumulation in plants or soil which has harmful

effects on humans, extensive researches are being conducted in the world to replace this method with

more recent methods to confront fungicidal resistant pathogens. Since the late 1900s, scientists have made

great efforts to use natural antagonisms of terricolous microorganisms to protect plants. Biological control

depends on knowledge of biological interactions at the ecosystem, organism, cellular, and molecular

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levels and often is more complicated to manage compared with physical and chemical methods. (Baker

and Cook, 1974). Recently, the use of biological control agents, especially bacteria has attracted a lot of

attention due to the ability of some species to suppress different plant diseases and the possibility of

combining with other control methods (Arrebola et al.,2010 ). Therefore, various sources of antibiotic

production are screened, among which bacillus especially is an important alternative to extract antibiotics

and their industrial production. One reason for its growing popularity is its record of safety during the past

100 years considered as the era of modem biological control (Waage and Greathead, 1988). For the

management of plant diseases, including plant parasitic nematodes (Cook et al.,1983). All biological

control interactions between plants and microbes occur naturally at a macroscopic and microscopic level

in the form of mutualism (Bronstien, 1994) protocooperation (James etal.,1995), commensalism(Yoon et

al.,1977]), neutralism (Halimann 2001), competition (TrenbathB,1976), amensalism (Arthur 1989),

parasitism (Price, 1977), and predation (Price et al., 1980, Pal, 2006). Pathogen populations thus can be

limited by antagonistic microorganisms in very different ways. The nature of the mode(s) of action does

not only determine how a pathogen population is affected by the antagonist. Also the characteristics of the

microbial biocontrol agent depend on the exploited mode of action. Possible risks for humans or the

environment, risks for resistance development against the biocontrol agent, its pathogen specificity and its

dependency on environmental conditions and crop physiology may differ between different modes of

action. Preferences for certain modes of action for an envisaged application of a biocontrol agent will also

have impact on the screening methods used to select new antagonists (Köhl et al.,2011). The objective of

this paper is to review the modes of action and antagonisms in biocontrol microorganisms for disease

management.

2.Direct antagonism

2.1. Mycoparasitism / Hyperparasitism

Hyper-parasitism is the most considered and the most direct form of antagonism (Pal et al., 2006). This

kind of interaction is often observed between fungi. For bacteria, hyperparasitism rarely has been

reported. Bdellovibrio bacteriovorus is a predatory bacterium which has the unusual property to use

cytoplasm of other Gram-negative bacteria as nutrients (McNeely et al.,2017). Hyperparasites invade and

kill mycelium, spores, and resting structures of fungal pathogens and cells of bacterial pathogens

(Ghorbanpour et al.,2018). There are four major classes of hyperparasites: obligate bacterial pathogens,

hypoviruses, facultative parasites, and predators. (Tjamos et al., 2010). Hyper-parasitism involves tropic

growth of bio control agent towards the target organism, coiling, final attack and dissolution of target

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pathogens cell wall or membrane by the activity of enzymes (Tewari, 1996). The ability of any fungi to

attack the other fungal species and utilizing their nutrients is called Mycoparasitism (Atanasova et

al.,2013). It is a complex mechanism that generally involves the production of a cell wall lytic enzyme

that degrades the pathogen fungus cells wall such as cellulases, chitinases, β-1,3-glucanases, proteinases,

lipases and in case of hyperparasites of oomycota, cellulase. (Rabea et al., 2003, Horbach et al., 2011). It

is one of the main mechanisms involved in Trichoderma (Sharma, 1996). Trichoderma harzianum

exhibits excellent mycoparasitic activity against Rhizoctonia solsni hyphae (Altomare et al., 1999). The

ATP (adenosine triphosphate)-binding cassette (ABC) transporter proteins of Trichoderma work both in

the process of nutrient uptake and myco- parasitism also (Locher, 2004). Other mechanisms of parasitism

are associated with fungi such as Verticillium chlamydosporium and Paecilomyces lilacinus, which can

infect the egg masses and cysts of the cereal cyst and root knot nematodes. For example, oospores of

Phytophthora and Pythium spp. are frequently found to be infected by Olpidiopsis gracilis, whilst

sclerotia of R. solani are infected by the obligate sclerotial mycoparasite Verticillium biguttatum.(

Pankhurst et al.,2005). Generally mycoparasitism can be described in four sequential steps (Chet, 1987):

The first stage is chemotropic growth. The biocontrol fungi grow tropistically toward the target fungi that

produce chemical stimuli, a volatile or water- soluble substance produced by the host fungus serves as a

chemo attractant for parasites. The next step is recognition. Lectins of hosts(pathogens) and carbohydrate

receptors on the surface of the biocontrol fungus may be involved in this specific interaction. The third

step is attachment and cell wall degradation. Mycoparasites can usually either coil around host hyphae or

grow alongside it and produce cell wall degrading enzymes such as chitinases and b-1,3-glucanase to

attack the target fungus, The final step is penetration. The biocontrol agent produces appressoria-like

structures to penetrate the target fungus cell wall, and kill their hosts by cell wall degrading enzymes,

often in combination with antimicrobial secondary metabolites (Chet, 1987, Chet et al., 1998, Inbar and

Chet 1994, Di Pietro, et al, 1992, Harman et al.,2004). There are several fungal parasites of plant

pathogens, including those that attack sclerotia (i.e. Coniothyrium minitans) while others attack living

hyphae (i.e. Pythium oligandrum) and, a single fungal pathogen can be attacked by multiple

hyperparasites. For example, Acremonium alternatum, Acrodontium crateriforme, Ampelomyces

quisqualis, Cladosporium oxysporum, and Gliocladium virens are just a few of the fungi that have the

capacity to parasitize powdery mildew pathogens (Heydari and Pessarakli, 2010). There are 30

hyperparasitic species against Rhizoctonia solani belonging to 16 genera have been reported

byJeffries(1995). Approximately 30 fungal species which show hyperparasitism against rust pathogens,

including Cladosporium uredinicola against Puccinia violae (Traquair et al.,1984) and Alternaria

alternata against Puccinia striiformis f. sp. tritici (Zheng et al.,2017). The most studied mycoparasites are

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belonging to the genera Trichoderma and Clonostachys Other hyperparasites attack plant-pathogenic

nematodes during different stages of their life cycles (i.e. Paecilomyces lilacinus and Dactylella

oviparasitica). The molecular level induction of mycoparasitism was first reported in 1994 (Carsolio et

al., 1999), based on the study of regulation of an endochitinase-encoding gene (ech42). Ech42 was

expressed during the mycoparasitic interaction between T. harzianum and Rhizoctonia solani. Another

study showed that in the P1 mutant strain of T. atroviride, the expression of exochitinase nagI or

endochitinase ech42 gene was needed to induce mycoparasitism in treatments containing purified

colloidal chitin from the fungal cell walls (Vinale et al., 2008). Production and regulation of lytic enzymes

such as chitinases, glucanases, and proteases by Trichoderma spp also play key roles in the

mycoparasitism/biocontrol process (Mukherjee et al., 2008). In high glucose conditions, glucose is

metabolised preferentially through the repression of genes required for utilization of other carbon sources.

Analysis of the promoter sequence of mycoparasitism-related genes showed that control by carbon

catabolite repression occurred through binding of the CRE1 protein (Cortés et al. 1998; Kubicek &

Penttilä 1998; Mach et al. 1999; de las Mercedes et al. 2001; Donzelli et al. 2001). A Trichoderma

catabolite repressor gene (cre1) was cloned from Trichoderma spp. and molecular studies confirmed its

role in catabolite repression of the mycoparasitism-related gene ech42 (Ilmén et al. 1996; Lorito et al.

1996; Cziferszky et al. 2002). Nitrogen catabolite repression is another regulatory mechanism by which

genes required for utilisation of poor nitrogen sources are repressed in the presence of primary nitrogen

sources such as glutamine or ammonia. In T. atroviride, the response of the protein aseprb1 is also

controlled by nitrogen catabolite repression (Olmedo-Monfil et al. 2002). Repression is thought to be

mediated through interaction of regulatory proteins with GATA motifs within the promoter region has

been identified in other mycoparasitic genes from T. atroviride, T. harzianum, and T. hamatum (Cortés et

al. 1998; Donzelli et al. 2001; Steyaert 2002), Promoter analysis of a chitinase gene (ech42) and

proteinase gene (prb1) implicated in mycoparasitism has led to the prediction of a global induction

pathway for mycoparasitism-related genes (Cortés et al. 1998). The molecular level induction of

mycoparasitism was first reported in 1994 (Carsolio et al., 1999), based on the study of regulation of an

endochitinase-encoding gene (ech42). Generally In hyperparasitism, the pathogen is directly attacked by a

specific BCA that kills it or it spropagules. Where as in mycoparasitism, two mechanisms operate among

involved species of fungi. This may be hyphal of interfungus interaction i.e., fungus-fungus interaction,

several events take place which lead to predation viz., coiling, penetration, branching and sporulation,

resting body production, barrier formation and lyses. The bio-nematicide Bacillus firmusalso colonizes the

rhizosphere of the plant where it parasitizes the eggs and larvae of nematodes especially of the

rootknotnematodes.

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Mycoprasitic coiling of Trichoderma atrovide around Botrytus cinerea hyphae.Arrow indicate site of penetration

3.Indirect Antagonism

3.1. Competition for nutrion and space

Competition is diffcult to study mechanistically: it is likely more important in natural environments,

where resources are limited and competitors plentiful. In community ecology, niche and nutrient

competition have been intensely studied as determinants of species diversity. Germination and growth of

plant pathogens depend on nutrient uptake. From the microbial perspective, soils and living plant surfaces

are frequently nutrient limited environment. So to colonize the phytosphere, a microbe must effectively

compete for the available nutrients (Pal et al., 2006). Both the biocontrol agents and the pathogens

compete with one another for the nutrients and space to get established in the environment. This process

of competition is considered to be an indirect interaction between the pathogen and the biocontrol agent

whereby the pathogens are excluded by the depletion of food base and by physical occupation of site

(Lorito et al., 1994). Competition for carbohydrates in the carbohydrate rich wound environment in yeast

seen and competition for the limited amounts of nitrogen sources such as amino acids play the key roles

in the antagonistic interactions . Yeast can consume a broad range of carbohydrates such as disaccharides

and monosaccharides but also various nitrogen sources (Spadaro et al.,2010). Spadaro and Droby (2016)

reviewed competition processes between antagonistic Pichia guilliermondii and pathogenic Penicillium

digitatum, P. expansum, B. cinerea, or Colletotrichum spp. in wounds of different fruits and

Aureobasidium pullulans and P. expansum in apple wounds.The competition for nutrients is concerned

biocontrol agents compete for the rare but essential micronutrients, such as iron and manganese especially

in highly oxidized and aerated soils. In these soils iron is present in ferric form, which is insoluble in

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water and where the concentration may be as low as 10 -8 M, too low to sport the microbial growth

(Lindsay, 1979). Iron is required in several metabolic processes including tricarboxylic acid cycle,

electron transport chain, oxidative phosphorylation, and photosynthesis (Messenger and Barclay 1983;

Fardeau et al. 2011). It also regulates the biosynthesis of porphyrins, vitamins, antibiotics, toxins,

cytochromes,siderophores, pigments, and aromatic compounds, and nucleicacid synthesis (Messenger and

Barclay 1983). Recently it has also been observed that iron plays an important role in the microbial

biofilm formation as it regulates the surface motility of microorganism (Glick et al. 2010; Cai et al. 2010).

Competition for micro nutrients exists because biocontrol agents have more efficient utilizing uptake

system for the substances than the pathogens (Nelson, 1990). This property can be attributed to the

production of iron binding ligands called siderophores as in Erwinia caratovora (Kloepper et al. 1980).

Siderophores are low molecular-weight chelating agents with a high affinity for ferric iron. Siderophores

are produced by microorganisms under restricted iron conditions (Haas, 2014). (Fig2). Till to date more

than 500 different siderophores were reported, of which 270 were well characterized (Boukhalfa et al.

2003), while the rest remain uncharacterized and their functions are yet to be determined (Ali and Vidhale

2013). Siderophores exhibiting novel structures with two types of ligands and modified aminoacids, not

found elsewhere in nature with variation from one species to another. They exhibit requisite (I)

hydrophilic properties for chelating iron in extracellular aqueous environment (II) lipophilic properties for

entering through the lipoprotenaceous membrane receptors of the cell and (III) hydro-lipo-phile properties

depending upon the aqueous or fatty environment under which they are destined to function. Depending

on the oxygen ligands for Fe (III) coordination, siderophores can be classified into three main categories,

namely (1) hydroxamate(C=O, N-(OH &aminoacids) and (2) catecholates(derivates if 2-3 dihydroxy

benzoic acid) groups and carboxylates, (Ali & Vidhale 2013, Winkelmann and Drechsel (1997) have

classified bacterial siderophores in to 5 types namely (i)catecholate (ii) hydroxamates (iii) peptide

siderophores (iv)mycobactin and related siderophores (v) citrate hydroxamate siderophores. Table 1.

Fungal siderophores have been classified into five families (i) ferrichromes (ii) coprogens (iii)

rhodotoluric acid (iv) fusarinines (v) rhizoferrins.The majority of fungal siderophores belong to the

hydroxamate class. Exceptions are the carboxylate-type siderophore rhizzoferrin produced by various

Mucorales and the catecholate pistillarin produced by the marine species Penicillium bilaii .(Thieken et

al., 1992, Capon et al., 2007), whiles prokaryotic organisms produce both hydroxamates and

catecholates. Many siderophores produced by bacteria and fungi are strong enough to remove iron from

host-binding proteins. In case of gram-negative bacterial membranes, an outer membrane receptor, a

periplasmic binding protein, and a cytoplasmic membrane protein belonging to ATP-binding cassette

transporter (ABC-transporter) are involved in the transport of siderophore iron (Fe (Ahmed and 7

Holmstrom 2014). Once siderophores bound to ferric iron moves to cytosol, the ferric iron gets reduced to

ferrous form and the ferrous form of iron becomes free from the siderophores. After release of iron,

siderophores either get degraded or recycled by excretion through efflux pump system. For example, A.

fumigatus secretes two main hydroxamate siderophores, triacetylfusarinine C and fusarinine C, which

have higher affinity for iron than transferrin and are capable of obtaining iron directly from the protein

(Hissen etal., 2004).Siderophores chealate the Fe (II) ions and the membrane bind protein receptors

specifically recognize and take up the Siderophore-Fe-complex (Mukhopadhyay and Mukherjee, 1998).

This results in making iron unavailable to the pathogen, which produce less siderophores with lower

binding power. The result is less pathogen infection and biological control. Iron competition is the mode

of action of several fungal antagonists. For example, Trichoderma asperellum producing iron-binding

siderophores controls Fusarium wilt (Segarra et al.,2010). P. putida produce the pseudofactin siderophore

that have ability to abolish the Fusarium oxysporum and Rhizoctonia solani from rhizosphere by lowering

iron availability in soil (Beneduzi et al., 2012). The yeast Metschnikowia pulcherrima transforms

pulcherriminic acid and iron ions to the red pigment pulcherrimin. This process leads to iron depletion in

media inhibiting development of B. cinerea, A. alternata, and P. expansum (Saravanakumar et al.,2008).

Recently, it was shown that Saccharomycopsis schoenii lacks several components of the sulfur

assimilation pathway and thus likely acquires methionine from its prey (Junker et al. 2019). Among

yeasts, the inability to take up sulfur is specifc to Saccharomycopsis, but some plant pathogenic fungi and

Trichoderma species show a similar phenomenon, which may indicate that methionine is an important

target for such organisms and highly competed over (Junker et al.2019).

Type of siderophore Class

1 Caecholate Enterobacterine

2 Hydroxamate Aferrioxamines

Ferrichrom

Aerobacline

3 Carboxylate Rhizoferrin

4 Mixed Lysine derivatives Myobactine

Ornithine derivatives pyoverdine

Histamine derivatives Anguibctine

Table .1. Sidrophore class

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Fig.2. Sidrophor Iron chelation

Biofilm formation may also be considered a specific and highly successful strategy to compete for space.

Bioflms are microbial communities that live and grow on surfaces and can be comprised of a single

species or represent multi-species consortia (Costa-Orlandi et al. 2017). A biofilm comprises any

syntrophic consortium of microorganisms in which cells stick to each other and often also to a

surface.These adherent cells become embedded within a slimy extracellular matrix that is composed of

extracellular polymeric substances (EPS).The cells within the biofilm produce the EPS components,

which are typically a polymeric conglomeration of extracellular polysaccharides, proteins, lipids and

DNA. Because they have three-dimensional structure and represent a community lifestyle for

microorganisms, they have been metaphorically described as "cities for microbes".(Hall-Stoodley et

al.,2004, .Aggarwal et al.,2016, Watnick et al.,2005, López et al.,2010). Microbes form a biofilm in

response to various different factors, which may include cellular recognition of specific or non-specific

attachment sites on a surface, nutritional cues, or in some cases, by exposure of planktonic cells to sub-

inhibitory concentrations of antibiotics. A cell that switches to the biofilm mode of growth undergoes

a phenotypic shift in behavior in which large suites of genes are differentially regulated. A biofilm may

also be considered a hydrogel, which is a complex polymer that contains many times its dry weight in

water. Biofilms are not just bacterial slime layers but biological systems; the bacteria organize themselves

into a coordinated functional community. (O'Toole et al.,1998, Karatan et al., 2009, Hoffman et al.,2005,

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An D, Parsek ,2007). The formation of a biofilm begins with the attachment of free-floating

microorganisms to a surface. The first colonist bacteria or yeast of a biofilm may adhere to the surface

initially by the weak van der Waals forces and hydrophobic effects they can anchor themselves more

permanently using cell adhesion structures such as pili. During surface colonization bacteria cells are able

to communicate using quorum sensing (QS) products such as N-acyl homoserine lactone (AHL). Once

colonization has begun, the biofilm grows by a combination of cell division and recruitment.

Polysaccharide matrices typically enclose bacterial biofilms. In addition to the polysaccharides, these

matrices may also contain material from the surrounding environment, including but not limited to

minerals, soil particles, and blood components, such as erythrocytes and fibrinThe final stage of biofilm

formation is known as dispersion, and is the stage in which the biofilm is established and may only

change in shape and size.The development of a biofilm may allow for an aggregate cell colony (or

colonies) to be increasingly resistant to antibiotics. Cell-cell communication or quorum sensing has been

shown to be involved in the formation of biofilm in several bacterial species. The process of biofilm

development is summarized by five major stages of biofilm development , Initial attachment, Irreversible

attachment, Maturation I, Maturation II, Dispersion (O'Toole et al., 1998, Briandet et al.,2001, Takahashi

et al.,2010, Donlan 2002, Watnick, etal., 2000)

In biocontrol yeasts, bioflm formation, mainly in the phyllo- and carposphere (i.e., in wounds), is now

considered an important mode of action and has been widely studied. Besides P. fermentans,bioflm

formation has also been implicated in the protective and biocontrol activities of A. pullulans, Kloeckera

apiculata, S. cerevisiae, Pichia kudriavzevii, W. anomalus, and M. pulcherrima (Chi et al. 2015; Klein

and Kupper 2018; Ortu et al. 2005; Pu et al. 2014; Wachowska et al. 2016). In a S. cerevisiae for strain,

bioflm cells were far more effcient than planktonic cells in colonising the inner surface of apple wounds,

thereby controlling the development of blue mould caused by P. expansum (Ortu et al. 2005; Scherm et al.

2003)(Fig.3).

Fig3.Colonisation  a of the inner surface of an apple wound by the  Saccharomyceslcerevisiael or strain M25.

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 b Penicillliumlexpansum germ tubes grow onto the yeast cells, but contact with the apple tissue is prevented by a thick yeast cell layer. The presence of an extracellular matrix is likely to assure an elective protection of the apple tissue (Ortu et al. unpublished)

3.2. Induction of host resistance

Plants actively respond to a variety of environmental stimuli, including gravity, light, temperature,

physical stress, water and nutrient availability. Plants also respond to a variety of chemical stimuli

produced by soil- and plant-associated microbes. Such stimuli can either induce or condition plant host

defenses through biochemical changes that enhance resistance against subsequent infection by a variety of

pathogens. Induction of host defenses can be local and/or systemic in nature, depending on the type,

source, and amount of stimuli. Plants are central players in a complex food web in which numerous

members profusely take advantage of the plant’s resources. Besides microbial pathogens and insect

herbivores, plants also nurture a vast community of commensal and mutualistic microbes that provide the

plant with essential services, such as enhanced mineral uptake, nitrogen fixation, growth promotion, and

protection from pathogens(Shoresh et al.,2010). These plant microbiota are predominantly hosted by the

root system, which deposits up to 40% of the plant’s photosynthetically fixed carbon into the rhizosphere,

rendering this small zone around the roots one of the most energy-rich habitats on Earth (Bais 2006).

Several genera of the rhizosphere microbiota, which are referred to as plant growth–promoting

rhizobacteria (PGPR) and fungi (PGPF), can enhance plant growth and improve health (Lugtenberg

2009, Shoresh et al.,2010). Evidence showed PGPR strains can promote plant health through stimulation

of the plant immune system (Alstrom, 1991 Van Peer 1991). The term induced resistance is a generic

term for the induced state of resistance in plants triggered by biological or chemical inducers, which

protects nonexposed plant parts against future attack by pathogenic microbes and herbivorous insects

(Kuc, 1982) Generally, induced resistance confers an enhanced level of protection against a broad

spectrum of attackers (Walters 2013). In the 1960s, Ross coined the term SAR for the phenomenon in

which uninfected systemic plant parts become more resistant in response to a localized infection

elsewhere in the plant (Ross, 1961). There are different Systemic Acquired Resistance like Pathogen-

Induced Systemic Acquired Resistance Signaling, Herbivore-Induced Resistance Signaling, hormonal

regulation of induced systemic resistance by beneficial microbes. Using Microbial Biocontrol agents

(MBCAs) for disease control through induction of resistance or priming relies on a complex sequence of

events where the MBCA initially has to establish on the host, followed by the release of specific inducers

which are recognized by specific receptors by the plant and there after triggering pathways within the host

plant resulting in the onset of defense reactions or priming to make the plant ready for later challenges by 11

pathogens. The first part of this sequence of events depends on the MBCA, the second part on the genetics

and physiological status of the plant. (Romanazzi et al.,2016). In the current concept of the plant immune

system, pattern-recognition receptors (PRRs) have evolved to recognize common microbial compounds,

such as bacterial flagellin or fungal chitin, called pathogen- or microbe-associated molecular patterns

(PAMPs or MAMPs) (Boller et al ,2009, Zipfel, 2009). Plants also respond to endogenous plant-derived

signals that arise from damage caused by enemy invasion, called damage-associated molecular patterns

(DAMPs) (Boller et al ,2009,). Pattern recognition is translated into a first line of defense called PAMP-

triggered immunity (PTI), which keeps most potential invaders in check (Fig 5). Successful pathogens

have evolved to minimize host immune stimulation and utilize virulence effector molecules to bypass this

first line of defense, by either suppressing PTI signaling or preventing detection by the host (Bardoel et

al.,2011, De Jongeet al., 2010, Dodds 2010, Pel MJC et al., 2013). In turn, plants acquired a second line

of defense in which resistance (R) NB-LRR (nucleotide-binding–leucinerich repeat) receptor proteins

mediate recognition of attacker-specific effector molecules, resulting in effector-triggered immunity (ETI)

(Dodds et al.,2010). ETI is a manifestation of gene-for-gene resistance (Flors HH. 1971), which is often

accompanied by a programmed cell death at the site of infection that prevents further ingress of biotrophic

pathogens that thrive on living host tissue. The onset of PTI and ETI often triggers an induced resistance

in tissues distal from the site of infection and involves one or more long-distance signals that propagate an

enhanced defensive capacity in still undamaged plant parts (Dempsey et al., 2012 , Shah et al.,

2013).Systemic acquired resistance (SAR), is mediated by salicylic acid (SA), a compound which is

frequently produced following pathogen infection and typically leads to the expression of pathogenesis-

related (PR) proteins. These PR proteins include a variety of enzymes some of which may act directly to

lyse invading cells, reinforce cell wall boundaries to resist infections, or induce localized cell death. A

second phenotype, first referred to as induced systemic resistance (ISR), is mediated by jasmonic acid

(JA) and/or ethylene, which are produced following applications of some nonpathogenic rhizobacteria.

Interestingly, the SA- and JA- dependent defense pathways can be mutually antagonistic, and some

bacterial pathogens take advantage of this to overcome the SAR. For example, pathogenic strains of

Pseudomonassyringae produce coronatine, which is similar to JA, to overcome the SA-mediated pathway

(He et al. 2004).Recently, epigenetic regulation of pathogen- and β-aminobutyric acid (BABA)-induced

priming for SA-dependent defenses and herbivore-induced priming for JA-dependent defenses was shown

to be inherited by the next generation via chromatin remodeling or DNA methylation (Luna et al.,2012,

Rasmann et al.,2012). Hence, plants seem to have the capacity to memorize a stressful situation and

subsequently immunize not only themselves but also their offspring against future attacks (Pastor et

al.,2013). Generally This induced resistance is of two types representing two distinct pathway responses:

12

systemic acquired resistance (SAR) and induced systemic resistance (ISR). Typically, SAR is induced by

pathogens while ISR is salicylic acid in dependent and is induced by non-pathogenic bacteria (Van Loon

et al., 1998). For example, S.cerevisiae, Rhodosporidiumlpaludigenum, Candidalsaitoana, C.loleophila

and Metschnikowia species induce an innate immune response and eventually cause resistance against

phyllosphere pathogens in fruits (De Miccolis Angelini etal. 2019; Droby etfal. 2002; Hadwiger etfal.

2015; Hershkovitz etfal. 2012; Sun et al. 2018).

4.Mixed-path antagonism

4.1. Antibiotics

The term antibiotic was coined from the word „antibiosis‟ which literally means “against life”. In the

past, antibiotics were considered to be organic compounds produced by one microorganism which are

toxic to other microorganisms (Russell, 2004). It is secondry antimicrobial metabolites belonging to

heterogeneous groups of organic, low-molecular weight compounds produced by microorganisms that are

deleterious to the growth or metabolic activities of other microorganisms (Thomashow et al.,1997).

Antibiotics produced by bacteria include volatile antibiotics (hydrogen cyanide,aldehydes, alcohols,

ketones) and nonvolatile antibiotics: polyketides(diacetylphloroglucinol; DAPG and mupirocin),

heterocyclic nitrogenous compounds (phenazinederivatives: pyocyanin, phenazine-1-carboxylic acid;

PCA, PCN, and hydroxyphenazines) andphenylpyrrole antibiotic (pyrrolnitrin) (de Souza et al. 2003,

Ahmad et al. 2008). Hydrogen cynid (HCN) effectively blocks the cytochrome oxidase pathways and is

highely toxic to all aerobic microorganism at picomolar concentration(Ramett et al., 2003).Supression of

black rot tobacco caused by Thielaviopsis basicola caused to be due to hydrogen cynid production(Howell

et al., 1988). In response to stressful conditions, bacteria secrete several types of antibiotics with varying

specificity and modes of action (Glick, 2012). Antibiotics produced by PGPR include kanosamine, 2,4-

diacetylphloroglucinol (2, 4-DAPG), Martínez-Viveros oligomycin A, butyrolactones,xanthobaccin

phenazine-1-carboxylic acid, pyrrolnitrin, zwittermycin A, viscosinamide (Viveros et al.,2010). The

bacterial strain of P. fluorescens BL915 involve in the production of antibiotic known as pyrrolnitrin have

ability to inhibit deterioration of Rhizoctonia solani. 2,4-DAPG is an extensively studied antibiotic

involved in the membrane destructionof Pythium spp.(De Souza et al.,2003). Pseudomonas spp. also

synthesizes phenazine that contains the antagonistic activity against Fusarium oxysporum (Beneduzi et

al.,2012). In Pseudomonas many antibiotic metabolites such as pyrrolnitrin have been studied

(Raaijmakers and Mazzola,2012). Many Bacillus ssp. Produced antibiotics like circulin, polymyxin and

colistin that are actively involved in the growth inhibition of pathogenic fungi as well as Gram-negative

13

and Gram-positive bacteria. In Bacillus, especially lipopeptides such as iturin, surfactin, and fengycin

have been investigated (Ongena and Jacques,2008).(Table.2) Six classes of antibiotic compoundsnamely

phenazines, phloroglucinols, pyoluteorin, pyrrolnitrin,cyclic lipopeptides (diffusible compounds) and

hydrogen cyanide(HCN; volatile compound) are better correlated with the biocontrol of root diseases

(Haas and Defago, 2005; Beneduzi and Ambrosini,Passaglia, 2012). Two antibiotics namely

biosurfactants and lipopeptide have gained attention due to their biocontrol potential againstwide-

spectrum phytopathogens including bacteria, fungi, oomycetes,protozoa, nematodes and plants (Al-Ajlani

et al., 2007; de Bruijn et al.,2007; Raaijmakers et al., 2010). Huge numbers of known antibiotics are

produced by actinomycetes (8700 different antibiotics), bacteria (2900) and fungi (4900) (Bérdy,2005).

Production of antimicrobial metabolites, mostly with broad-spectrum activity, has been reported for

biocontrol bacteria belonging to Agrobacterium, Bacillus, Pantoea, Pseudomonas, Serratia,

Stenotrophomonas, Streptomyces, and many other genera. Bacillus produces twelve major antibiotics

including bacillomicin, mycobacillin, fungistatin, iturin, fengycin, plipastatin, surfactin, bacilysin, etc.

(Stien, 2005; Al-Ajlani et al., 2007) whereas Pseudomonas spp. produce only six antibiotics

(Shanmugaiah et al., 2010). More recently, Pseudomonas putida WCS358r strains genetically engineered

to produce phenazine and DAPG displayed improved capacities to suppress plant diseases in field-grown

wheat (Glandorf et al. 2001). Also fungal antagonists can produce antimicrobial compounds. For

Trichoderma and closely related Clonostachys (former Gliocladium), 6-PAP, gliovirin, gliotoxin, viridin

and many more compounds with antimicrobial activity have been investigated (Ghorbanpour et al.,2018).

Antibiotics at low concentrations can be involved in signaling and microbial community interactions,

communication with plants, and regulation of biofilm formation. Raaijmakers and Mazzola(2012). A

wide range of functions of antimicrobial metabolites at low concentrations: there is evidence that

antimicrobials including lipopetides protect bacteria from grazing by bacteriovorus nematodes such as

Caenorhabditis elegans. Also volatile antibiotic compounds may play a role in long-distance interactions

amongst soil organisms including bacterial predators. Lipopeptides of Bacillus and Pseudomonas are

involved in the surface attachment of bacterial cells and biofilm formation by activating signaling

cascades finally resulting in the formation of extracellular matrices which protect microorganisms from

adverse environmental stresses. Several biocontrol strains are known to produce multiple antibiotics

which can suppress one or more pathogens. For example, Bacillus cereus strain UW85 is known to

produce both zwittermycin (Silo-Suh et al. 1994) and kanosamine (Milner et al. 1996). The ability to

produce multiple antibiotics probably helps to suppress diverse microbial competitors, some of which are

likely to be plant pathogens. The ability to produce multiple classes of antibiotics, that differentially

inhibit different pathogens, is likely to enhance biological control. More recently, Microbial genome

14

analysis revealed huge numbers of cryptic antibiotic gene clusters encoding still unknown antibiotics.

Antibiotics mode of action has different mechanism , The antimicrobial potency of most classes of

antibiotic are directed at some unique feature of the bacterial, structure or their metabolic processes. The

most common targets of antibiotics. The mechanism of antibiotic actions are as follows: (Talaro and

Chess, 2008; Madigan and Martinko, 2006; Wright, 2010)

Inhibition of cell wall synthesis Breakdown of cell membrane structure or function Inhibition of the structure and function of nucleic acids Inhibition of protein synthesis Blockage of key metabolic pathwaysAntibiotics play an important role in disease management, used as biocontrol agent and faced

challenge due to limitations because antibiotics are prepared under natural circumstances. Ecological

and other components that effect the antimicrobial action of antibiotics were examined to utilize the

potential of antibiotics that are produced by PGPR in crop protection.

Some of antibiotics produced by BCAs

Antibiotic Source Target pathogen Disease Reference2, 4-diacetyl-phloroglucinol Pseudomonas fluorescens F113 Pythium spp. Damping off Shanahan et al. (1992)

Agrocin 84 Agrobacterium radiobacter Agrobacterium tumefaciens Crown gall Kerr (1980)Bacillomycin D Bacillus subtilis AU195 Aspergillus flavus Aflatoxin contamination Moyne et al. (2001)

Bacillomycin, fengycin Bacillus amyloliquefaciens FZB42

Fusariumoxysporum

Wilt Koumoutsi et al. (2004)

Xanthobaccin A Lysobacter sp. strain SB-K88 Aphanomycescochlioides

Damping off Islam et al. (2005)

Gliotoxin Trichodermavirens

Rhizoctonia solani Root rots Wilhite et al. (2001)

Herbicolin Pantoea agglomerans C9-1 Erwinia amylovora Fire blight Sandra et al. (2001)Iturin A B. subtilis QST713 Botrytis cinerea and R. solani Damping off Paulitz and Belanger (2001),

Kloepper et al. (2004)

Mycosubtilin B. subtilis BBG100 Pythiumaphanidermatum

Damping off Leclere et al. (2005)

Phenazines P. fluorescens 2-79 and 30-84 Gaeumannomyces graminis var. tritici

Take-all Thomashow et al. (1990)

Pyoluteorin, pyrrolnitrin

P. fluorescens Pf-5 Pythium ultimum and R. solani Damping off Howell and Stipanovic (1980)

Pyrrolnitrin, pseudane

Burkholderia cepacia R. solani and Pyricularia oryzae Damping off and rice blast

Homma et al. (1989)

Zwittermicin A Bacillus cereus UW85 Phytophthora medicaginis and P. aphanidermatum

Damping off Smith et al. (1993)

Table. 2

15

4.2.Hydrogen cyanide (HCN) production

Considerable numbers of free-living rhizospheric bacterial communities, mainly Pseudomonas sp.

(Ahmad et al., 2008; Muleta et al., 2007), are capable of generating HCN by oxidative decarboxylation

from direct precursors such as glycine, glutamate, or methionine. Other rhizobacterial genera reported to

produce HCN include Bacillus (Ahmad et al., 2008) and Chromobacterium (Muleta et al., 2007). HCN

secreted by Pseudomonas fluorescent strain CHAO has been demonstrated to stimulate root

hairvormation and suppress back root rot caused by Thielaviopsis basicola in tobacco plant (Voisard et

al., 1989). Cyanogenesis in bacteria accounts in part for the biocontrol capacity of the strains that suppress

fungal diseases of some economically important plants (Voisard et al., 1989).

4.3.Production of lytic enzymes

Extracellular hydrolytic enzymes such as chitinases, glucanases, proteases and lipases, achieve disease

suppression through lysis of pathogenic fungal cell walls (Maksimov et al., 2011). Except oomycetes, cell

walls of most phytopathogenic fungi are made up of chitin (C8H13O5N), an unbranched, longchain

polymer of glucose derivatives, composed of ß-1,4-linked units of the amino sugar N-acetyl D-

glucosamine (NAG) (Shaikh and Sayyed, 2015). Chitinase activity of PGPR has been well explored for

suppression of fungal phytopathogens (Kim et al., 2008). The role of lytic enzymes such as chitinase and

ß-1,3-glucanase in suppression of anthracnose pathogen Colletotrichum gloeosporioides Penz. have been

established (Vivekananthan et al., 2004) Pseudomonas stutzeri produces extracellular chitinase and ß-1,3-

glucanase, which lyse the pathogen Fusarium sp.. Cladosporium werneckii and B. cepacia can hydrolyze

fusaric acid (produced byFusarium), (Compant et al., 2010).Furthermore, chitinase produced by Serratia

plymuthica C48 was found to inhibit spore germination and germ tube elongation in Botrytis cinerea

(Frankowski et al., 2001). Lysis of fungal cell walls is a direct method of pathogen inhibition which

indirectly promotes plant growth. Antagonistic bacteria Serratia marcescens reduce mycelial network of

Sclerotium rolfsii by expressing chitinase (Ordentlich et al.,1988). Lysobacter is capable of producing

glucanase that is involved in the control of diseases caused by Bipolaris and Pythium sp. (Palumbo et al.,

2005). Hydrolytic enzymes directly contribute in the parasitization of phytopathogens and rescue plant

from biotic stresses [Haran et al., 1996]. The proteases reduced the activities of the pathogen enzymes

exo- and endopoly galacturonase , pectin methyl esterase, pectate lyase, chitinase, b- 1,3- glucanase and

cutinase, that are essential for the pathogen during host infection. The  secretion of  chitinolytic

enzymes is considered a desirable characteristic for biocontrol agents as it  allows degrading

fungal cell walls, Chitin degrading activity has been measured in biocontrol yeasts of 

16

the genera Aureobasidium,Candida,Debaryomyces,Metschnikowia, Meyerozyma, 

Pichia,Saccharomyces,Tilletiopsis,  and  Wickerhamomyces and in Saccharo mycopsis (Zajc et al. 2019).

4.5. Bacteriocin

Bacteriocins are proteinaceous toxins that are secreted by bacteria that lives in competitive microbial

environment. They destroy the neighboring bacterial species by damaging the bacterio-cinogenic cells

(Riley et al., 2002). Bacteriocins are very effective in reducing or inhibiting the growth of

phytopathogens (Beneduzi et al., 2012). Bacteriocins have narrow killing spectrum as compared to

conventional antibiotics and these have damaging effect on the bacteria that are closely relative of

bacteriocin producing bacteria (Riley et al., 2002). Colicins are most prominent bacteriocins synthesized

by Escherichia coli. Similarly, megacins is produced by B. megaterium; marcescins from Serratia

marcescens; cloacins from Enterobacter cloacae; and pyocins comes from P. pyogenes (Cascales et

al.,2007). Bacteriocins that are produced by Bacillus spp. remarkably gain importance due broad range of

inhibition of fungal, yeast, gram positive and gram negative species that may have some pathogenic effect

on animals and human beings (Abriouel et al.,2011).

5.Antagonisms in Rhizospher, phylospher and endophyt microbes

The rhizosphere concept was first introduced in 1904 by Dr Lorenz Hiltner as the soil compartment

influenced by the root (Smalla et al., 2006; Hartmann, 2008). The implication of root exudates as a

nutrient source for bacteria was then initiated, explaining why bacterial density was more impor tant than

in bulk soil. This phenomenon was called the ‘rhizosphere effect’ by Rovira (1956), another major

contributor in rhizosphere research (Burns, 2010). Rhizosphere has been broadly subdivided into the

following three zones (Pratibha et al., 2013). (1).Endorhizosphere (interior of the root): that consists of the

root tissue including the endodermis and cortical layers; (2). Rhizoplane (interior of the root): is the root

surface where soil particles and microbes adhere. It consists of epidermis, cortex and mucilaginous

polysaccharide layer; (3). Ectorhizosphere: that consists of soil immediately adjacent to the root. The

ability to secrete a vast array of compounds into the rhizosphere is one of the most remarkable metabolic

features of plant roots, with nearly 49% of all photosynthetically fixed carbon being transferred to the

rhizosphere through root exudates (Prakash and Karthikeyan, 2013). The phyllosphere is broadly defined

as the surfaces and internal parts of the aerialstructures of plants, including flowers, fruits,stems and

leaves. Specialized microbialcolonists, phytopathogens, spoilage organisms and periodic immigrants have

17

all been describedas residents of this physically diverse habitat. Microorganisms that can grow in the

rhizosphere are ideal for use as biocontrol agents, since the rhizosphere provides the front-line defense

for roots against attack by pathogens. Pathogens encounter antagonism from rhizosphere microorganisms

before and during primary infection and also during secondary spread on the root. In some soils described

as microbiologically suppressive to pathogens. microbial antagonism of the pathogen is especially great,

leading to substantial disease control(Schneider, R. W. 1982). The bacteria known as plant growth-

promoting rhizobacteria (PGPR) live in close vicinity to the plants (endosphereor rhizosphere) and play a

key role in the transformation of many organic and inorganic compounds making them available for plant

growth such as nitrogen, phosphorus, potassium, iron, and zinc( Olanrewaju et al., 2017). PGPR exert

beneficiale effects on plant growth and yield by the production of plant growthregulating substances, such

as indole-3-acetic acid (IAA), gibberellic acid, cytokinin and 1-aminocyclopropane-1-carboxylate (ACC)

deaminase(Glick, 2014). The ACC-deaminase producing PGPR facilitate plantgrowth by decreasing plant

ethylene levels and reducing stress caused by biotic (such as phytopathogenic bacteria/fungi) and abiotic

factors (such as heavy metals, salt, and drought) (Glick et al., 2007). Antifungal metabolites produced by

these rhizobacteria were identified as antibiotics (iturin, surfactins, fengycin, DAPG, Phenazine, etc.), cell

wall degradingenzymes (protease, chitinase, and cellulase), plant growth promotion enzymes and

hormones (indole-3-aceticacid, ACC-deaminase, phosphates, nitrogen fixation), N-acyl-homoserine

lactones and siderophores. (Saira Ali et al.,2020) Bacillus spp. have been tested on a wide variety of plant

species for abilityto control diseases. They are appealing candidates for biocontrol because they produce

endospores that are tolerant to heat and desiccation. The antagonistic effects by PGPR over various phyto

pathogens bolster the possibilities for their useas biocontrol agents (Sang et al., 2011; Lamsal et al.,2013).

Recent findings suggest that competition fornutrient, niche exclusion, induced systemic resistanceand

production of metabolites such as antibiotics,siderophores and hydrogen cyanide are the chief modes of

biocontrol activity in PGPR (Ambrosini andBeneduzi, 2012). The rhizosphere, influenced by root

secretions,can contain up to 1011microbial cells per gram root (Egamberdieva et al.,2008) and more than

30,000 prokaryotic species(Mende et al.,2011). Soil microorganisms (free-living,associative, and

symbiotic rhizobacteria) belonging to the genera like Acinetobacter,Burkholderia, Enterobacter,

Alcaligenes,Arthrobacter, Azospirillum, Azotobacter, Bacillus, Erwinia, Flavobacterium,

Rhizobium,Serratia, Xanthomonas, Proteus, andPseudomonas are the integral parts of rhizosphere biota

(Glick.,1995) and exhibit successful rhizosphere colonization. Diveres microbial genera are identified

from rhizospher of different plants, and horticulture, for example, The fungal genera Alternaria,

Clonostachys, Fusarium, Penicillium and Rhizoctonia are cited to be commonly encountered in both the

rhizosphere and the geocaulosphere of potato (Pieta & Patkowska, 2003; Fiers et al., 2010). Three genera,

18

known to mainly contain rhizobacteria species,were consistently identified in each microenvironment:

Agrobacterium, Bacillus and Pseudomonas. To a lesser extent, genera such as Arthrobacter, Comamonas,

Curtobacterium,Enterobacter, Paenibacillus, Pantoea, Serratia, Sphingobacterium,Stenotrophomonas,

Variovorax and Xanthomonas are frequently found in the vicinity of the potato rhizospher for instance.

6. Biontrol recent progress & future perspectives

Biological control has been used for centuries, but the first big wave of activity in the modern era

followed the spectacular success of the introduction in the late 1880s of the parasitic fly, Cryptochaetum

iceryae (Williston) (Diptera: Cryptochaetidae), and the vedalia beetle, Rodolia cardinalis (Mulsant)

(Coleoptera: Coccinellidae) to control cottony-cushion scale (Icerya purchasiMaskell) (Hemiptera:

Monophlebidae) in California citrus orchards (Caltagirone 1981). However, in the mid-1940s’ the growth

and success of the synthetic pesticide industry caused biocontrol use to almost disappear until the

publication of Rachael Carson’s ‘Silent Spring’ (Carson 1962), While biological control is still seen by

many as a preferable and sustainable alternative for chemical pest control, it should play a much more

important role in managing insects, weeds and diseases. In augmentative biological control, the situation

has changed in the last five years from a dip in uptake of biocontrol around the year 2000 (van Lenteren

2012) to much improved adoption (van Lenteren et al. 2017). This has come about by political

developments in Europe and Asia, and also in Latin America. Demands of retailers and consumers, and

actions by NGOs have helped to instigate this change. In emerging countries such as Brazil, biological

control research and implementation is gaining momentum for both augmentative and classical

biocontrol. (Parra 2014). China and India are also countries that have invested widely in biological control

research, training and adoption. China, for example, has recognised that pesticides and fertilizers have

created serious damage to ecosystems and created food security issues and, as a result, the use of agri-

chemicals will be capped from 2020. In the European Union, the Sustainable Use of pesticides Directive

2009 came into force starting in 2011 to encourage development and introduction of Integrated

PestManagement (IPM) and alternative techniques to reduce dependency on the use of pesticides.

initiatives and developments would seem to herald every prospect for increased funding for biological

control and IPM research in the future and the IOBC is set to play an important implementation and

facilitating role in the renaissance and development of biocontrol worldwide.

19

7. Conclusion

Microbial biological control agents protect plants from damage by diseases via different modes of

action.They may induce resistance or prime enhanced resistance against infections by a pathogen in plant

tissues without direct antagonistic interaction with the pathogen. Another indirect interaction with

pathogens is competition for nutrients and space. MBCAs may also interact directly with the pathogen by

hyperparasitism or antibiosis. Hyperparasites invade and kill mycelium, spores, and resting structures of

fungal pathogens and cells of bacterial pathogens. Production of antimicrobial secondary metabolites with

inhibiting effects against pathogens is another direct mode of action. Low amounts of in situ secreted

secondary metabolites support antagonists to gain a competitive advantage. In some cases, biocontrol

agents have been selected which secrete already efficient secondary metabolites into the growth media

during mass production that are applied together with or without living cells of antagonists in the

biological control product. The rhizosphere is the site of complex dynamic plant–microorganism

interactions and is of interest from multiple environmental perspectives for biocontrol study.

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