REVIEW OF LITERATURE T - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/51033/9/09_chapter...

Use of Antagonistic Soil Actinomycetes for the Management of Root Pathogenic Fungi of Dalbergia sissoo 12

REVIEW OF LITERATURE

he objective of this study was to review the published research works on the

management of plant pathogens by application of various biological control

agents and to understand the mechanisms of biological control.

2.1 Biological Control:

Biological control can be defined as “the directed, accurate management of common

components of ecosystems to protect plants against pathogens” (Kennedy and Smith

1995). Harley (1985) defines the biological control as the study and utilization of

parasites, predators, and pathogens to regulate populations of pests. It 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. Microbes that contribute to disease control are most likely those that could

be as classified competitive saprophytes, facultative plant symbionts and facultative

hyperparasites (Pal and Gardener, 2006). Biological control of plant diseases is slow,

gives few quick profits, but can be long lasting, inexpensive and harmless to life.

Biocontrol systems do not eliminate neither pathogen nor disease but bring them into

natural balance (Dhingra and Sinclair, 1995). Odum (1953) proposed that the

interaction of two populations be defined by the outcomes for each. The types of

interactions were referred to as mutualism, protocooperation, commensalism,

neutralism, competition, amensalism, parasitism, predation, etc.

2.2 Biological Control Agents:

The term applies to the use of organism antagonists to suppress diseases as well as the

use of host specific pathogens to control weed populations. The organism that

suppresses the pathogen is referred to as the biological control agent (BCA) (Pal and

Gardener, 2006). Microorganisms that grow in the rhizosphere are ideal as biocontrol

agents, since the rhizosphere provides the front-line defence for roots against attack

by pathogens. Pathogens encounter antagonism from rhizosphere microorganisms

before and during primary infection and also during secondary spread in the roots

(Suprapta, 2012). Microorganisms with potential to provide effective and useful

biological control of plant diseases already exist on or within plants or soils as part of

a vast and largely untapped natural, biological and genetical resource. Quantative

T


assessment indicates that these microbes comprise wide range of organisms like

yeasts, amoebae, mycorrhizae, viruses, fungi, bacteria, actinomycetes and blue green

algae (Joshi, 2005). The biocontrol agent is introduced systematically into the

population of the pathogen and in successful cases may have the ability to progress to

epidemic proportions thus controlling the pathogenic agent. The essential prerequisite

of a successful biocontrol agents are:-

1. Active in an environment conductive for the pathogens

2. Compatible with other agents

3. Survive agricultural introduction

4. Colonize the appropriate substrate

5. Effectively suppress the pathogen

6. Long time survival in the substrate

7. Simple and cheap multiplication, packing distribution and application

8. No health hazards

9. Economical

A multitude of organisms have been implicated as agents responsible for biocontrol of

several plants diseases. A review of biological control agents is given below:-

2.2.1 Protozoans:

So far, very little information is available on soil amoeba as biological control agent

in comparison to bacteria, fungi, viruses and actinomycetes. Mycophagy is the

phenomenon of feeding on fungi by amoebae. Anderson and Patrick (1978) reported

that certain free living, soil inhibiting, amoeboid organisms perforated and feed on

mycelium and chlamydospores of Thielaviopsis basicola and conidia of Cochliobolus

sativus. Duczek (1983) reported Cashia mycophaga fed by engulfing and completely

digesting fungal hyphae without causing perforations. Some other species viz.

Arachnula impatiens, Leptomyxa reticulate, Vampyrella lateritia and Thecamoeba

granifera sub sp. minor were also reported to cause large perforations (Old, 1977; Old

and Patrick, 1979; Pussard, et al., 1980). Duczek and Wildermuth (1991) measured

the populations of mycophagous amoebae which feeds on hyphae of Bipolaris

sorokiniana and found that over 90% of the mycophagous amoebae occurred in the 0–

15 cm layer of the soil profile. Adl (2003) discussed the mechanisms of fungivory, as

the amoebae attach to the hyphae and dissolve the chitin wall, then inserted the


pseudopodium through these perforations, ingest cytoplasm by phagocytosis and

pinocytosis and digest in food vacuoles.

2.2.2 Viruses:

Mycoviruses are viruses that infect fungi and have the potential to control fungal

diseases of crops when associated with hypovirulence (Yu et al., 2010). The term

hypovirulence is mostly used with mycoviruses. Hypovirulence is thought to play a

role in counterbalancing plant diseases in nature (Yu et al., 2010). Hypovirulence has

been described as a form of induced resistance, antibiosis and hyperparasitism. Choi

and Nuss (1992) defined “hypovirulence as simply a pathogen phenotype where

virulence is reduced”. The viruses always recognised as pathogenic organisms,

however, they have also been identified as one of the potential biocontrol agents for

fungal pathogens (Fulbright et al., 1988). Use of viruses for the control of plant

pathogens and insect pests is comparatively a recent phenomenon although

possibilities were apparent for a fairly long time. Viruses are found ubiquitously in

major groups of filamentous fungi, and an increasing number of novel mycoviruses

are being reported (Aoki et al., 2009). Chiba et al. (2009) reported the use of

Rosellinia necatrix-mycoviruses for the management of Rosellinia necatrix, an

ascomycete causing white root rot, a devastating disease worldwide, particularly in

fruit trees in Japan. Yu et al. (2010) successfully isolated Sclerotinia sclerotiorum

hypovirulence-associated DNA virus 1 (SsHADV-1), for the management of S.

sclerotiorum.

2.2.3 Blue Green Algae:

Cyanobacteria (blue green algae, BGA), which constitute the largest, most diverse,

and most widely distributed group of photosynthetic prokaryotes, along with

eukaryotic algae, make up most of the world’s biomass (Cannell, 1993) but they

received very little attention as potential biocontrol agent of plant diseases. Kulik

(1995) use the BGA extract to inhibit the growth of Chaetomium globosum,

Cunninghamella blakesleeana, Aspergillus oryzae, Rhizoctonia solani, Fusarium sp.,

Pythium sp., and Sclerotinia sclerotiiorum. The filtrates of Phormidium fragile and

Nostoc muscorum have the potential to inhibit the growth of the sugar beet pathogens

Fusarium verticillioides, Rhizoctonia solani and Sclerotium rolfsii (Rizk, 2006). The

water, ethyl acetate, chloroform, methanol and diethyl ether extracts of three blue


green algal species (Anabaena flos aquae (Linnaeus) Bory; Anabaena variabilis

(Kützing) and Oscillatoria angustissima West and West) were examined against

Candida albicans, C. tropicalis (yeast), Aspergillus niger, A. flavus, and were found

to show varying degree of inhibition (Khairy and El-Kassas, 2010). Nostoc

entophytum and N. muscurum showed antagonistic activity against the causal agent of

soybean root rot Rhizoctonia solani (Osman et al., 2011). Three strains of BGA (C4,

C8 and C12) were found effective against phytopathogenic fungi Fusarium solani, F.

oxysporum, F. oxysporum lycopersici, F. moniliforme, Pythium debaryanum and

Rhizoctonia solani (Dukare et al., 2013).

2.2.4 Fungi:

Fungi have received maximum attention as antagonists possibly because of the case in

handling and identification compared to other microorganisms. The mycoparasitic

activity of these organisms is attributed to a combination of successful nutrient

competition, rapid growth on various organic substrates, the production of cell wall-

degrading enzymes and potent broad spectrum antibiotics (Brunner et al., 2005; Joshi,

2005).

The genus Penicillium includes many ubiquitous species which are able to

colonize very diverse natural environment because of their capacity to adapt to

extreme environmental conditions and utilize almost any kind of organic substrate

(Nicoletti and De Stefano, 2012). Fang and Tsao (1995) evaluated Penicillium

funiculosum against Phytophthora root rots of Rhododendron spp. and Citrus sinensis

and found that 3 strains of the P. funiculosum was effective against Phytophthora

citrophthora in Rhododendron spp. and Phytophthora parasitica on C. sinensis.

Anthracnose disease of the grape causing fungi Colletotrichum gloeosporioides was

reported to be control by the use of Penicillium chrysogenum KMITL44 (Soytong et

al., 2005). Sartaj et al. (2011) studied the antagonistic effect of Penicillium sp.

EU0013 on tomato growth and Fusarium oxysporum f. sp. lycopersici and suggested

that Penicillium sp. EU0013 was capable of enhancing growth and protecting tomato

plants against fusarial wilt. Nicoletti and De Stefano (2012) found P. restrictum

antagonistic to Rhizoctonia solani.

Fungi from the genus Trichoderma were found antagonistic to the plant

pathogenic fungi such as Rhizoctonia solani, Sclerotium rolfsii, Pythium


aphanidermatum, Fusarium oxysporum and F. culmorum (Chet and Inbar, 1994).

Harsh and Ojha (2000) have reported control of fusarial wilt of Moringa

pterygosperma seedlings using Trichoderma virens. Harsh and Kapse (1999) have

successfully developed the biocontrol formulation against fungal decay of stored

bamboo using T. harzianum and T. pseudokoningii and reported their viability for

more than two years at room temperature in tropical climatic conditions when stored

in dry powder form using baggasse. Gajera et al. (2011) studied the effect of three

Trichoderma strains (T. virens, T. viride and T. harzianum) against the collar rot

disease-causing fungus Aspergillus niger and observed that T. viride inhibited

maximum (86.2%) growth of test fungus, followed by T. harzianum (80.4%) and

least by T. virens (61.20%). Similar findings were also reported by other workers

(Prabhu and Urs, 1998; Raju and Murthy, 2000; Rao and Sitaramaih, 2000; Goel et

al., 2008). Dar et al. (2011) used antagonistic and mycorrhizal fungi against root rot

pathogen of blue pine (Pinus wallichiana), F. oxysporum, R. solani and reported

effectiveness in descending order of T. viride, T. harzianum, mycorrhizal fungi

Pisolithus tictorius and Laccaria laccata. Hamid et al. (2012) tried four biocontrol

agents against F. solani f. sp. pisi (Jones) and found that T. harzianum exhibited

highest inhibition parentage (78.60) followed by T. viride (75.72), Gliocladium virens

(69.52) and Pseudomonas fluorescence (68.37).

Naik and Sen (1994) reported that A. niger were found effective against a

spectrum of nine pathogenic isolates of Fusarium sp. A. terreus strain showed in vitro

antagonistic activity against the plant pathogen Sclerotinia sclerotiorum (Lib.) de

Bary (Melo et al., 2006). Dutt et al. (2009) reported that in presence of A. terreus, the

infection of Rhizoctonia solani was reduced in rice seedlings.

Mycorrhizal fungi are also strong contenders for providing biological control

through competition for space by virtue of their ecologically obligate association with

roots. Ectomycorrhiza have been shown to prevent pathogens from reaching the root

surface as the fungus forms a continuous sheath which acts as a physical barrier to

infection (Joshi, 2005). Harsh et al. (1994) found that post emergence damping-off of

Moringa pterygosperma caused by Fusarium acuminatum was successfully controlled

by padding with Vesicular-Arbuscular Mycorrhizal (VAM) inoculation around the

seeds. Ectomycorrhizal fungi like Paxillus involutus effectively controlled root rot

caused by F. oxysporum and F. moniliforme in red pine (Pal and Gardener, 2006).


Inoculation of sand pine with Pisolithus tinctorius, another ectomycorrhizal fungus,

controlled disease caused by Phytophthora cinnamomi (Pal and Gardener, 2006).

Inoculation of apple-tree seedlings with the VAM fungi Glomus fasciculatum and G.

macrocarpum suppressed apple replant disease caused by phytotoxic myxomycetes

(Catska, 1994). Mechanisms for the protective activity of mycorrhizal fungi include

improvement of plant nutrition, root damage compensation, competition for

photosynthates or colonization/infection sites, production of anatomical or

morphological changes in the root system, changes in mycorrhizosphere microbial

populations, and activation of plant defence mechanisms (Azcón-Aguilar and Barea,

1996).

Endophytic fungi are taxonomically and biologically diverse but all share the

character of colonizing internal plant tissues without causing apparent harm to their

host (Wilson, 1995). Mejia et al. (2008) have shown that endophytic fungi can limit

pathogen damage in Theobroma cacao by the use of endophytic fungi viz.

Colletotrichum gloeosporioides, Clonostachys rosea and Botryosphaeria ribis against

Moniliophthora roreri (frosty pod rot), Phytophthora palmivora (black pod rot) and

Moniliophthora perniciosa (witches broom). Kusari et al. (2013) isolated the

endophytic fungi from rhizosphere of the medicinal plant, Cannabis sativa L. and

evaluated their antagonistic activity against Botrytis cinerea and Trichothecium

roseum and revealed that most dominant species was Penicillium copticola.

Dolatabadi et al. (2012) studied the effect of endophytic fungi Piriformospora indica

and Sebacina vermifera from Lentil (Lens culinaris Medic.) on Fusarium oxysporum

f. sp. lentis. F. oxysporum f. sp. cubense race 4 was also reported to be inhibited by

the fungal metabolites of endophytic fungi (Ting et al., 2010).

Among soil microorganisms, yeasts have received little attention as biocontrol

agents of soil-borne fungal plant pathogens in comparison to bacterial, actinomycetes,

and filamentous fungal antagonists. The ability of certain taxa of yeasts to multiply

rapidly, to produce antibiotics and cell wall-degrading enzymes, to induce resistance

of host tissues, and as promoters of plant growth indicates the potential to exploit

them as biocontrol agents and plant growth promoters (El-Tarabily and

Sivasithamparam, 2006b). Guo-Zheng et al. (2003) investigated three antagonistic

yeasts against Penicillium expansum and found that Trichosporon pullulans was most

effective to blue mold rot in the three yeasts. The yeasts Trichosporon pullulans


(Lindner.) Diddens et Lodder, Cryptococcus laurentii (Kuffer.) Skinner and

Rhodotorula glutinis (Fresenius) Harrison were sprayed onto sweet cherry (Prunus

avivum L. cv. Hongdeng) fruit in two orchards prior to harvest. C. laurentii was the

most effective antagonist of the three yeasts for control of postharvest decay of sweet

cherry over different storage conditions (Shi-Ping et al., 2004).

2.2.5 Bacteria:

Bacteria are important antagonists in biological control because of their fast growth,

ability to utilize different forms of nutrients under varying conditions, parasitism,

competition for nutrients and colonization potential, siderophore production,

production of different antibiotics, enzymes and other secondary metabolites,

degradation of pathogen virulence factors and induction of resistance in host plants

(Lee et al., 2013). The ability of bacteria to parasitize and degrade spores of fungal

plant pathogens is well established (El-Tarabily et al., 1997). Most species of Bacillus

are distributed globally and the widespread occurrence of subspecies of Bacillus

thuringiensis, B. subtilis and B. cereus with their ability to suppress the plant

pathogens have been widely recognized (Kumar et al., 2011). B. thuringiensis is

essentially used for insect pest control but the use of this bacterium as antagonistic

against plant pathogenic fungi was reported by Reyes-Ramírez et al. (2004). Sixty

strains of B. thuringiensis were tested against damping-off, root and stem rot of

Capsicum annuum L. caused by Rhizoctonia solani and suggested that three B.

thuringiensis strains have an excellent potential to be used as bio-control agents.

Fourteen B. thuringiensis isolates were tested for in vivo antifungal activity against

tomato late blight, wheat leaf rust, tomato gray mold, and barley powdery mildew

causing pathogens, 12 isolates exhibited strong control activity against barley

powdery mildew (Choi et al., 2007). Mohammad et al. (2013) screened the potential

of B. thuringiensis and B. kurstaki against R. solani and found that isolate B.t. D-1 and

the B.t. kurstaki HD-203 were found to be inhibiting R. solani from 49% to 64%.

The use of several other species of Bacillus is also reported as biological

control agents by other workers. Antifungal activity of B. coagulans against three

pathogenic species of Fusarium was examined, the result showed that B. coagulans

was more effective against F. culmorum (Czaczyk et al., 2002). Yuan et al. (2012)

found that volatile compounds produced by B. amyloliquefaciens completely inhibited


the growth of F. oxysporum f. sp. cubense. Zongzheng et al. (2009) studied the effect

of B. subtilis SY1 on eight different phytopathogens and also as PGPR, the result

confirmed that B. subtilis SY1 was more effective against Alternaria solani, F.

oxysporum f. sp. melongenae and F. oxysporum f. sp. lycopersici and also played an

important role in enhancing stress tolerance of the host plant. The culture filtrate and

the n-butanol extract of B. subtilis ZZ120 showed strong growth inhibition activity in

vitro against Fusarium graminearum, Alternaria alternata, R. solani, Cryphonectria

parasitica and Glomerella glycines (Li et al., 2012).

Pseudomonas spp. are aerobic, gram-negative bacteria, ubiquitous in soils, and

are well adapted to growing in the rhizosphere (Weller, 2007) have been applied for

biocontrol, promoting plant growth and bioremediation. The 2, 4-

diacetylphloroglucinol (DAPG) producing strains were major groups in biocontrol

microorganisms, because of their easy colonization, good competition and broad

antimicrobial spectrum (Gao et al., 2012). The biocontrol effect of P. fluorescens, P.

aeruginosa and Bacillus subtilis were evaluated on plant pathogenic fungi viz.

Fusarium oxysporum, Aspergillus niger and Alternaria alternata and P. fluorescens

has shown the highest growth promoting effect, followed by P. aeruginosa and least

was of B. subtilis (Khanuchiya et al., 2012). Pseudomonas chlororaphis and B.

amyloliquefaciens also have biocontrol effect evaluated in vitro against Sclerotinia

sclerotiorum (Fernando et al., 2007). Five strains of Pseudomonas were tested for

their antifungal activity against phytopathogens viz. Alternaria cajani, Curvularia

lunata, Fusarium sp. and Helminthosporium sp. and all of them were tested positive

(Anbuselvi et al., 2010).

The antifungal compounds extracted from P. fluorescens at 5% were found

inhibitory to the growth of Rhizoctonia solani (42.79%), Phytophthora parasitica

(28.57%), P. palmivora (25.98%) and Fusarium solani (20.45%) (Koche et al., 2013).

Isolates of Pseudomonas were evaluated for antifungal activity against five fungal

plant pathogens, i.e. F. oxysporum, Aspergillus niger, A. flavus, Alternaria alternata

and Erysiphe cruciferarum. Out of the five fungal pathogens studied, F. oxysporum

showed maximum extent of inhibition (51.76%) followed by A. niger (50.14%) and

least by Erysiphe cruciferarum (22.27%) (Singh et al., 2011). Antifungal activity of

P. fluorescens was studied by Goud and Muralikrishnan (2009) against Pythium


ultimim, Macrophomina phaseolina and Pyricularia oryzae, all three pathogenic fungi

were inhibited by P. fluorescens with inhibitory activities ranging from 50% to 80%.

Twelve isolates of P. fluorescens were evaluated by Alemu and Alemu (2013) for

their antagonistic activity against chocolate spot disease (Botrytis fabae) of faba bean.

P. fluorescens 10 (Pf 10) showed high antagonistic activity against B. fabae (88.1%).

Minaxi and Saxena (2010) examined the antagonistic effect of Pseudomonas

aeruginosa strain RM-3 against the phytopathogenic fungi and found that maximum

growth inhibition was found in M. phaseolina in plate assay (68%), whereas it was

93% in Dreschlera graminae in dual liquid assay.

2.2.6 Actinomycetes:

Actinomycetes are common soil inhabitants with an unprecedented ability to produce

novel microbial products exhibiting antibacterial, antifungal, antiviral as well as

antitumor properties. About 60% of the world's antibiotics were secreted by

actinomycetes (Liu, 2002; Liu and Jiang, 2004). Actinomycetes are an enormous

reservoir for antibiotics, bioactive metabolites and many are excellent biocontrol

agents for use in protecting plants against phytopathogens (González-Franco and

Hernández, 2009).

Crawford et al. (1993) isolated 267 actinomycete strains from four

rhizosphere-associated and four non-rhizosphere-associated British soils and tested

their antagonistic behaviour against fungi viz. Pythium ultimum, Phanerochaete

chrysosporium, Coriolus versicolor, Postia placenta, and Gloeophyllum trabeum.

Five isolates were very strong antagonists of the fungi, four were strong antagonists,

and ten others were weakly antagonistic. Yuan and Crawford (1995) showed that

Streptomyces lydicus WYEC108 was capable not only of destroying germinating

oospores of P. ultimum but also of damaging the cell walls of the fungal hyphae and

concluded that S. lydicus WYEC108 is potentially a potent biocontrol agent for use in

controlling seed and root rot.

Lim et al. (2000) selected 32 actinomycetes isolates, which showed the

inhibitory activity against mycelial growth of plant pathogenic fungi like Alternaria

mali, Colletotrichum gloeosporioides, Fusarium oxysporum, Magnaporthe grisea,

Phytophthora capsici and Rhizoctonia solani. Lee and Hwang (2002) isolated 1510

actinomycetes from 14 different sites in the western part of Korea. All the isolates


were classified as genus Streptomyces. The isolates showed strong antifungal activity

against A. mali, C. gloeosporioides, Fusarium oxysporum f. sp. cucumerinum and

Rhizoctonia solani. Dadwal and Jamaluddin (2003) isolated Streptomyces spp. from

soil and screened in vitro antagonistic activity against pathogens of forest species i.e.

Alternaria alternata, Curvularia lunata, Sarocladium oryzae, Fusairum oxysporum,

Ganoderma lucidum and Macrophomina phaseolina.

Rifaat (2003) analyzed taxonomic analysis of 114 actinomycete strains

isolated from water of the river Nile and its bottom sediments showed that most of the

water isolates belonging the genus Streptomyces and Micromonospora. Out of 68

Streptomyces strains obtained, 11 exhibited significant antifungal activity against

Aspergillus niger and Trichoderma viride proved to be the most susceptible to the

active substance present in the fermentation broths of Streptomyces strains. Aghighi et

al. (2004) investigated antagonistic activity of 110 isolates of soil actinomycetes

against plant fungal-pathogens from which 14 isolates were found active against

Alternaria solani, A. alternata, Fusarium solani, Phytophthora megasperma,

Verticillium dahliae and Saccharomyces cerevisiae.

Bafti et al. (2005) assayed antagonistic activity of 178 isolates of

actinomycetes collected from soils of Kerman province, south-east of Iran against

Fusarium oxysporum f. sp. melonis, the causal agent of fusarial wilt of cucurbits,

through agar disc and well diffusion methods. One strain Streptomyces olivaceus

(strain 115) showed anti fusarial activity. Antifungal activity was fungistatic type on

pathogen mycelia. It is prominent that amending greenhouse soil mixed with S.

olivaceus (strain 115) will reduce crop losses by the pathogen. Kathiresan et al.

(2005) isolated 160 isolates of actinomycetes from marine habitats and each isolate

was tested against four phyto-pathogenic fungi i.e. Rhizoctonia solani, Pyricularia

oryzae, Helminthosporium oryzae and Colletotrichum falcatum. Fifty one percent

isolates were found effective against P. oryzae and H. oryzae, 31% isolates were

found effective against R. solani and 12.5% were against C. falcatum. Among 160

isolates 16% were found effective against all the tested pathogens.

El-Tarabily and Sivasithamparam (2006a) reported that the non-streptomycete

actinomycetes may also be used as biocontrol agents against soil-borne fungal plant

pathogens or as a plant growth promoter. Muititu et al. (2008) successfully used two


actinomycetes antagonists for the control of late blight of tomatoes in greenhouse

caused by Phytophthora infestans. Prapagdee et al. (2008) assessed actinomycete

isolates from rhizosphere soils for in vitro antagonism against Colletotrichum

gloeosporioides and Sclerotium rolfsii. A potent antagonist against both plant

pathogenic fungi, designated SRA14, was selected and identified as Streptomyces

hygroscopicus. Potential antagonistic effects of actinomycetes were investigated on

the litter decomposing fungi: Chrysosporium pannorum, Cladosporium

cladosporioides, C. herbarum, Mortierella alpina, M. ramanniana var. angulispora,

M. ramanniana var. ramanniana, M. vinacea, Mucor hiemalis var. 1, M. hiemalis var.

2, M. racemosus, Paecilomyces carneus, Penicillium chrysogenum, P. montanense,

Trichoderma polysporum, Trichoderma sp. 2 and Zygorrhynchus moelleri. The results

showed a varying degree of inhibition from 41% to 100 % (Jayasinghe and Parkinson,

2008). Lee et al. (2008) investigated endophytic actinomycetes as biocontrol agents

against Chinese cabbage clubroot caused by Plasmodiophora brassicae, the strain

A004, A011 and A018 showed 58%, 33% and 42% inhibition respectively.

Abdelghani et al. (2009) investigated 51 actinomycetes isolated from different

soil samples of Palestine. A novel strain of Streptomyces albovinaceus (isolate 10/2)

was found to be maximum antibiotic producer and which has shown both broad

spectrum antibacterial and antifungal activities. Atta (2009) isolated 45 Streptomyces

strains which were screened for their antifungal properties. Among the 45

Streptomyces, the broad spectrum Streptomycs olivaceiscleroticus, AZ-SH514 was

selected for physico-chemical characteristics of the purified antifungal agent. The

compound was tested against human and plant pathogenic fungi like Aspergillus

niger, A. fumigatus, A. flavus, Fusarium oxysporum, Botrytis fabae, Penicillium

chrysogenum, Rhizoctonia solani and Alternaria alternata and was found promising.

Baniasadi (2009) assayed antifungal bioactivity of 50 isolates of actinomycetes

collected from soils of Kerman province of Iran, against Sclerotinia sclerotiorum, the

causal agent of stem rot in sunflower, through agar disc method and dual culture

bioassays. The results indicated that isolate No. 363 was a proper candidate for field

biocontrol studies. Jorjandi et al. (2009) investigated antagonistic activity of 50

isolates of soil actinomycetes. Active isolates were exposed to chloroform for

detection of antibiotics. From the tested isolates, 13 showed anti gray mold activities.

Exposure of active isolates to chloroform revealed that Streptomyces isolates number

347, 263 and 350 retained their antifungal activities. Minimum Inhibitory


Concentration (MIC) value and solubility of active crude extract in organic solvents

were determined for Streptomyces isolate no. 347 which showed a unique and stable

property of inhibiting Botrytis allii munn.

Khamna et al. (2009) isolated actinomycetes from the rhizosphere of 16

medicinal plants and assessed for in vitro antagonistic activity against 6 plant

pathogenic fungi; Alternaria brassicola, A. porri, Cladosporium gloeosporioides,

Fusarium oxysporum, Penicillium digitatum and Sclerotium rolfsii. Streptomyces

spectabilis CMU-PA101 isolated from rhizosphere of pandanus palm (Pandanus

amaryllifolius) was very effective in producing bioactive metabolite against 6 plant

pathogenic fungi. Bharti et al. (2010) reported the isolation of 316 actinomycetes

from 69 soil samples from different localities of Garhwal region of Uttarakhand,

India. The growth pattern, mycelial coloration was documented. Among 316 isolates,

98 (31.01%) isolates exhibited antifungal activity against one or more human

pathogens. Out of 98 active isolates, 19, 67, 42, 37, 18 and 25 showed activity against

Trichophyton rubrum, Microsporum gypseum, M. canis, Aspergillus flavus, A.

fumigatus and Candida albicans, respectively, while seven isolates showed activity

against all the fungal pathogens.

Kavitha et al. (2010) isolated 4 different actinomycete strains (A1, A2, A3 and

A4) from the laterite soil samples of Guntur region. Growth pattern and antifungal

profiles of the strains were evaluated against the test fungi such as Aspergillus flavus,

A. niger, Candida albicans and Fusarium oxysporum. Based on the cultural,

morphological and physiological characteristics, the strains A1, A2 and A4 were

identified as the species of Streptomyces while the strain A3 was assigned to

Nocardia. Among the 4 tested strains, Streptomyces sp. A1 showed strong antifungal

activity which may provide a potent source for antifungal metabolites. Sharma and

Parihar (2010) isolated actinomycetes from the soil, extracting the antifungal

compounds from these isolated actinomycetes and then tested the extract against the

growth of Aspergillus niger, A. flavus, Fusarium oxysporum, Alternaria alternata and

Rhizopus stolonifer, and found that nearly all the extracts were effective against the

test fungi. Thenmozhi and Kannabiran (2010) isolated 8 strains of actinomycetes from

the marine sediments collected at the Puducherry coast, India. All the eight strains

were primarily screened for antifungal activity against A. fumigatus, A. niger and A.

flavus. The metabolites were extracted using ethyl acetate, lyophilized and screened


for antifungal activity against the three Aspergillus species by agar well diffusion

method and a potential strain was further identified and designated as Streptomyces

sp.VITSTK7.

Intra et al. (2011) isolated 304 actinomycetes and tested for their inhibitory

activity against Colletotrichum gloeosporioides and C. capsici which cause

anthracnose disease as well as the non-pathogenic Saccharomyces cerevisiae. Most

isolates (222 out of 304, 73.0%) were active against at least one indicator fungus or

yeast. Fifty four (17.8%) isolates were active against anthracnose fungi and 17 (5.6%)

could inhibit the growth of all three fungi. Streptomyces sp. A1022 had a strong

antagonism and broad spectrum against tested phytopathogens including Alternaria

longipes, Cercospora canescens, Colletotrichum gloeosporioides, Diaporthe citri,

Magnaporthe grisea, and Sclerotinia sclerotiorum (Lee et al., 2011).

A total of 137 actinomycetes cultures, isolated from 25 different herbal

vermicomposts, were characterized for their antagonistic potential against Fusarium

oxysporum f. sp. ciceri by dual-culture assay by Gopalakrishnan et al. (2011). Five

isolates (CAI-24, CAI-121, CAI-127, KAI-32 and KAI-90) were characterized for

antagonistic potential against Fusarium oxysporum f. sp. ciceri, Macrophomina

phaseolina, Rhizoctonia bataticola, (three strains viz. RB-6, RB-24 and RB-115). In

the dual-culture assay, three of the isolates, CAI-24, KAI-32 and KAI-90, also

inhibited all three strains of R. bataticola in chickpea, while two of them (KAI-32 and

KAI-90 have good antagonistic potential as these inhibited all the tested pathogens.

Ganeshan et al. (2011) studied antagonistic activity of halophilic actinomycetes

against several bacterial and fungal species and revealed that all the strains have

excellent inhibitory activity.

A potent Actinomycete isolate 9p was studied in dual plate assay by Srividya

et al. (2012), and the strain exhibited antagonistic activity in the following order

against Collectotrichum gleosporioides (21.4%); Alternaria brassicae (33.33%);

Rhizoctonia solani (35.7%) and Phytophthora capsici (36.6%). Antifungal

metabolites produced by Streptomyces pactum strain S131 was evaluated against

Aspergillus niger, A. flavus, Candida albicans, Fusarium oxysporum, Fusarium

moniliforme, Rhizoctonia solani and Pythium sp. which was most effective in case of

A. niger (Saleh et al., 2013).


Crude extract of antifungal compounds isolated from Streptomyces spp. were

assayed for the antifungal activity and were found effective against test pathogens

namely Candida albicans, Aspergillus flavus and A. fumigatus (Sweetline and Usha,

2013). Saxena et al. (2013) isolated one actinomycetes GS 22 which is found

potential antagonist to C. albicans and A. niger. Actinomycetes from marine

sediments were investigated for their antagonistic potential against some bacterial

pathogens and fungal phytopathogens Rhizoctonia solani, Macrophomina phaseolina,

Fusarium udum and F. oxysporum f. sp. lycopersici. One isolate Streptomyces sp.

LCJ85 was found to be more efficient in the production of secondary metabolites

(Mohanraj and Sekar, 2013). Khucharoenphaisan et al. (2013) investigated

actinomycetes isolate R 58 against Colletotrichum gloeosporioides and revealed that

the secondary metabolite produced by the strain is thermostable. Actinomycete

isolate, KSA-818 was found to be active against unicellular and filamentous fungi viz.

Saccharomyces cerevisiae, Candida albicans, Aspergillus niger, A. fumigatus, A.

flavus, A. terreus, Fusarium solani, F. oxysporum, F. moniliforme, Alternaria

alternata, Botrytis cinerea, Penicillium chrysogenum and Rhizoctonia solani (Atta

and Reyad, 2013).

Some other workers also used actinomycetes as a biocontrol agent against

other microorganisms along with fungi. Saadoun and Al-Momani (1997) have

assessed the antibacterial effect of the actinomycetes from Jordan soil and found that

strains were active against Agrobacterium tumefaciens. Jiménez-Esquilín and Roane

(2005) investigated 122 actinomycete isolates and screened against 9 fungal species

and 6 bacterial species for the production of antimicrobial compounds. Four

rhizosphere isolates, Streptomyces amakusaensis, S. coeruleorubidus, S. hawaiiensis

and S. scabies, showed broad-spectrum antifungal activity against three or more

fungal species in dual culture assays. Nedialkova and Naidenova (2005) studied 40

actinomycete strains, isolated from Antarctica soil and were tested for antagonistic

activity against 7 Gram-positive, 7 Gram negative bacteria and yeasts and 16 phyto-

pathogenic fungi. During the initial screening, 60 % of the strains showed inhibition

potential against test microorganisms. Ten of them had a broader spectrum of

antimicrobial activity. Jeffrey (2008) isolated 62 actinomycetes from soil samples

collected from Agriculture Research Center Semongok, Sarawak. All the isolates

were then subjected to antimicrobial testing using selected phytopathogens as test


strains and it was observed that 3, 25, 35 and 37 of the isolates showed antagonistic

reaction with Fusarium palmivora, Bacilus subtilis, Pantoae dipersa and Ralstonia

solanacearum, respectively. Six of the most promising isolates were selected and

identified using their 16S rRNA sequence. All six isolates were identified as

Streptomyces spp. Oskay (2009) investigated 16 antibiotic-producing Streptomyces

spp. isolated from the north Cyprus soils for their ability to inhibit the growth of six

unicellular and filamentous fungi and six bacteria including both human and plant

pathogens such as Fusarium oxysporum, Aspergillus niger, Alternaria alternata,

Trichoderma hamatum, Cladosporium oxysporum, Penicillium spp. and

Staphulococcus aureus, Klebsiella pneumoniae, Kocuria rhizophila, Escherichia coli,

Salmonella typhimurium and methicillin-resistant Staphylococcus aureus MRSA in

vitro. One promising strain, designed as KEH23 exhibited strong antifungal activity.

Atta et al. (2011) isolated 28 actinomycete strains from soil sample collected from a

farm in Jabbar district, Al-Khurmah governorate, KSA. One of the actinomycete

cultures, symbol 143 was found to produce a wide spectrum antifungal agent against

Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumonia, Pseudomonas

aeruginosa, Saccharomyces cerevisiae, Candida albicans, Aspergillus niger, A.

flavus, Fusarium oxysporum and Penicillum chrysogenum. The nucleotide sequence

of the 16s RNA gene (1.5 Kb) of the most potent strain evidenced 77% similarity with

Streptomyces albidoflavus and named S. albidoflavus143.

The effect of secondary metabolites produced by actinomycetes on plant

pathogens are also studied by other workers. Igarashi et al. (2003) investigated the

effect of endophytic actinomycetes on plant growth. Crop seeds were bacterized with

the spores of endophytic actinomycetes and grown in a green house. Of the tested

microorganisms, Streptomyces hygroscopicus S-17 induced the significant growth

promotion of tomato ca 2 times in height and ca 8 times in fresh weight compared to

the control. One of the secondary metabolites, pteridic acid A showed growth

promotion in the root formation test of kidney bean hypocotyls and the tobacco BY-2

cell culture. El-Mehalawy et al. (2005) investigated the factors affecting the

antifungal production of four actinomycetes species i.e. Streptomyces lydicus, S.

ederensis, S. erumpens and S. antimycoticus like carbon source, nitrogen source,

temperature and pH. The chemical analysis of the culture filtrates of the species

revealed the presence of 13 chemical compounds in the culture filtrate of S. lydicus,


12 chemical compounds in the culture filtrate of each of S. ederensis and S. erumpens

and 11 chemical compounds in the culture filtrate of S. antimycoticus and tested for

their efficacy against four human pathogenic yeast and mold fungi species;

Aspergillus niger, Candida albicans, C. parapsilosis, Saccharomyces cerevisiae and

four plant pathogenic fungal species viz. Alternaria solani, Cephalosporium maydis,

Fusarium oxysporum f. sp. lycopersicis and Penicillium digitatum. Rugthaworn et al.

(2007) tested 200 of actinomycete isolates from soil samples in Sakaerat Biosphere

Reserve and Suwanvajokkasikit Field Corps Research Station for the ability of

chitinase production and growth inhibition of three phyto-pathogenic fungi; Fusarium

sporotrichiodes, Rhizoctonia solani, Sclerotinia rolfsii and were found very effective.

Nonoh et al. (2010) studied the effect of acetonitrile-methanol extracts of 361

actinobacterial isolates obtained from Aberdares, Arabuko Sokoke, Lake Bogoria, Mt

Kenya, Kakamega, Ruma, Shimba Hills and Imenti Forest National Parks in Kenya,

on the plant pathogenic fungi; Fusarium oxysporum, Fusarium sp. and

Colletotrichum kahawae. Five isolates that were antagonistic against all test fungi

were investigated further and were also found to have antibacterial activity against S.

aureus and E. coli.

2.3 Mechanisms of Biological Control:

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 (Table 2.1).

Table 2.1 Different mechanisms of biological control (Pal and Gardener, 2006).

S. No. Type Mechanism Examples

1 Direct

antagonism

Hyperparasitism/predation Lytic/some nonlytic mycoviruses

Rosellinia necatrix-mycoviruses

Cashia mycophaga

Vampyrella lateritia

Trichoderma virens

2 Mixed-path

antagonism

Antibiotics 2,4-diacetylphloroglucinol

Phenazines


candicidin B

Cycloheximide

Lytic enzymes Chitinases

Glucanases

Proteases

Unregulated waste

products

Ammonia

Carbon dioxide

Hydrogen cyanide

Physical/chemical

interference

Blockage of soil pores

Germination signals consumption

Molecular cross-talk confused

3 Indirect

antagonism

Competition Exudates/leachates consumption

Siderophore scavenging

Physical niche occupation

Induction of host

resistance

Contact with fungal cell walls

Systemic acquired resistance

(SAR) and induced systemic

resistance (ISR)

Phytohormone-mediated induction

2.3.1 Direct Antagonism:

Direct antagonism results from physical contact or a high-degree of selectivity for the

pathogen by the mechanism(s) expressed by the Biological Control Agents (BCA).

Hyperparasitism by obligate parasites of a plant pathogen would be considered the

most direct type of antagonism. In hyperparasitism, the pathogen is directly attacked

by a specific BCA that kills it or its propagules. There are four major classes of

hyperparasites: obligate pathogens, hypoviruses, facultative parasites, and predation

(Pal and Gardener, 2006). Obligate pathogens are usually bacteria. Bacillus

thuringiensis is essentially used for insect pest control as obligate parasite (Reyes-

Ramírez et al., 2004). A classical example of hypovirulence is the Rosellinia

necatrix-mycoviruses that infects Rosellinia necatrix, a fungus causing white wood

rot, which causes hypovirulence, a reduction in disease-producing capacity of the

pathogen (Chiba et al., 2009). Parasitism occurs when the antagonist feeds upon or


within the pathogen, resulting in a direct destruction or lysis of propagules and

structure (Bull et al., 1998). The activities of various parasites, those parasitize plant

pathogens; result in biocontrol (Lo et al., 1997). Acremonium alternatum,

Acrodontium crateriforme, Ampelomyces quisqualis, Cladosporium oxysporum, and

Gliocladium virens are just a few examples of the fungi that have the capacity to

parasitize powdery mildew pathogens (Kiss, 2003; Heydari and Pessarakli, 2010).

Predation include the attack of pathogens by the predatory organisms e.g. Cashia

mycophaga fed by engulfing and completely digesting fungal hyphae without causing

perforations. Some other species viz. Arachnula impatiens, Leptomyxa reticulata,

Vampyrella lateritiae and Thecamoeba granifera subsp. minor were also reported to

cause large perforations (Old, 1977; Old and Patrick 1979; Pussard, et al., 1980).

There are several fungal parasites of plant pathogens, including those that attack

sclerotia (Coniothyrium minitans) while others attack living hyphae (Pythium

oligandrum) (Pal and Gardener, 2006).

2.3.2 Mixed-path Antagonism:

Mixed path antagonism includes antagonistic activity based on the ability of the BCA

to produce various kinds of enzymes, antibiotics or toxic metabolites inhibitory to

pathogens (Narayanasamy, 2013).

2.3.2.1 Antibiosis:

Antibiosis is the antagonism resulting from the production of secondary metabolites

by one microorganism toxic to other microorganisms (Alabouvette et al., 2006).

Antibiotics are microbial products that can, at low concentrations, poison or kill other

microorganisms. Most microbes produce and secrete one or more compounds with

antibiotic activity. 2,4-diacetylphloroglucinol (DAPG) producing strains of

Pseudomonas are major groups in biocontrol microorganisms (Gao et al., 2012).

Pseudomonas fluorescens produces siderophores, phenazines, 2,4-

diacetylphloroglucinol and cyanide (Koche et al., 2013). Streptomyces griseus

produced both candicidin B and cycloheximide. Bacillus amyloliquefaciens produce

both bacillomycin and fengycin (Czaczyk et al., 2002). 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 (Pal and Gardener, 2006).

Table 2.2 Different antibiotics with their source and target pathogens.

S. No. Antibiotics Source Target pathogen Reference

1. Amphotericin

B

Streptomyces

nodosus

Yeast, fungi Harald et al.

1974

2. Blasticidin S S.

griseochromogenes

Fungi Takeuchi et al.

1958

3. Candicidin B S. griseus Yeast, fungi Andrew et al.

1954

4. Candihexin S. viridoflavus Fungi Martin and

McDaniel, 1974

5. Nanaomycin S. rosa Fungi Omura et al.

1974

6. Purpuromycin Actinoplanes

ianthinogenes

Fungi and yeast Coronelli et al.

1974

7. Zorbonomycin S. bikiniensis Fungi and yeast Argoudelis et al.

1971

8. Variotin Paecilomyces varioti Fungi and yeast Tanaka and

Umezaw, 1962

9. 2,4-

diacetylphlorogl

ucinol

P. fluorescens F113 Pythium spp. Shanahan et al.

1992

10. Bacillomycin D B. subtilis AU195 A. flavus Moyne et al.

2001

11. Bacillomycin,

Fengycin

B. amyloliquefaciens

FZB42

F. oxysporum Koumoutsi et al.

2004

12. Zwittermycin A

B. cereus

UW85

Phytophthora

Medicaginis

Smith et al. 1993

13. Geldanamycin S. hygroscopicus

var. Geldanus

R. solani Deboer et al.

1970

14. Chaetomin Chaetomium

globosum

P. ultimum Di-Petro et al.

1992

15. Gliotoxin T. virens P. ultimum Anitha and

Murugesan, 2005


16. Pyoluteorin P. fluorescens Fungi Howell and

Stipanovic, 1980

17. Natamycin S. nataensis Fungi Struyk et al.

1957

18. Polyoxins S. cacaoi var.

Asoensis

Fungi Isono et al. 1969

19. Validamycin S. hygroscopicus Fungi Wu et al. 2012

20. Gliotoxin T. virens Rhizoctonia solani Wilhite et al.

2001

2.3.2.2 Enzymes:

Diverse microorganisms secrete and excrete other metabolites that can interfere with

pathogen growth and other activities. A variety of microorganisms produce and

release lytic enzymes that can hydrolyze a wide variety of polymeric compounds,

including chitin, proteins, cellulose, hemicellulose, and DNA. Expression and

secretion of these enzymes by different microbes can sometimes result in the

suppression of plant pathogen activities directly (Pal and Gardener, 2006), For

example, control of Sclerotium rolfsii by Serratia marcescens appeared to be

mediated by chitinase expression (Ordentlich et al., 1988) and β-1,3- glucanase

contributes significantly to biocontrol activities of Lysobacter enzymogenes strain C3

against Pythium (Palumbo et al., 2005). The potential use of endophytic

actinomycetes for controlling Pythium aphanidermatum in cucumber was evaluated

for Actinoplanes campanulatus, Micromonospora chalcea and Streptomyces spiralis,

all of which produced high levels of cell-wall degrading enzymes (β-1,3, β-1,4 and β-

1,6-glucanases) (El-Tarabily et al., 2009). Lysobacter and Myxobacteria are known to

produce abundant amounts of lytic enzymes, and some isolates have been shown to be

effective in suppressing fungal plant pathogens (Bull et al., 2002). Trichoderma spp.

towards pathogens such as Rhizoctonia solani has been studied (Chet and Baker,

1981). It involves specific recognition between the antagonist and its target pathogen

and several types of cell wall degrading enzymes to enable the parasite to enter the

hyphae of the pathogen. Several other researchers also reported same enzymes from

different strains (Haran et al., 1993; Rey et al., 2001; Viterbo et al., 2002; Brunner et

al., 2005). Bacillus thuringiensis var. israelensis produce chitinase enzyme which is

responsible for antagonistic activity against plant pathogenic fungi (Reyes-Ramírez et

al., 2004).


Table 2.3 Cell wall lytic enzymes identified in Trichoderma spp. and their

features (Steyaert et al., 2003).

S. No. Group name Specific names Features

1. ß-glucanases Exo-ß-1,3- or ß-l,6-glucanases Cleave ß linkages from

the non-reducing ends of

the chain

Endo-ß-1,3- or ß-l,6-glucanases Cleave ß linkages

internally at random in ß-

glucans and their

oligomers

ß-1,3- or ß-l,6-glucosidases Cleave oligo- and

disaccharides

2. Cellulases ß-1,4-D-glucan

cellobiohydrolases

exocellulases

Cleave cellobiose units

from the ends of cellulose

and its oligomers

Endo-ß-l,4-glucanases Cleave ß linkages

internally at random

ß-1,4-glucosidases Cleave cellobiose units to

glucose

3. Chitinases Chitin ß-l,4-chitibiosidase

exochitinases

Cleaves ß linkages from

the non-reducing end of

chitin and its oligomers

Endochitinase Cleave ß linkages

internally at random in

chitin and its oligomers

ß-l,4-N-acetylhexosaminidase

exochitinases

Cleave chitin, its

oligomers and chitobiose

from the non-reducing

end

4. Proteinases Attack specific amino

acid residues within the

polypeptide chain


2.3.2.3 Other Products:

Other microbial by-products may also contribute to pathogen suppression. Hydrogen

cyanide (HCN) is the most potent volatile compound produced by many soil bacteria.

The HCN produced by fluorescent Pseudomonas have been well proved to have

exemplary antifungal activity against phytopathgens (Jayaprakashvel and

Mathivanam, 2011). HCN effectively blocks the cytochrome oxidase pathway and is

highly toxic to all aerobic microorganisms at picomolar concentrations (Pal and

Gardener, 2006). Kumar et al. (2005) assessed the HCN, phenazine-1-carboxamide

and indole-3-acetic acid production by Pseudomonas aeruginosa and found that the

strain P. aeruginosa strain PUPa3 antifungal activity is due to its ability of

phenazine-1-carboxamide and HCN production. Volatile and non-volatile compounds

and hydrolytic enzymes produced by Trichoderma species and antibiotics, Fe-

chelating siderophores and hydrogen cyanide produced by P. fluorescens inhibited the

conidial germination and mycelial growth of Fusarium oxysporum infecting Arachis

hypogaea (Rajeswari and Kannabiran, 2011). Inorganic volatiles such as ammonia,

produced by Enterobacter cloacae, appear to be one of many mechanisms that

bacteria use in the biocontrol of Pythium ultimum-induced damping-off of cotton

(Howell et al., 1988). The influence of carbon dioxide on antimicrobial activity as it

creates an anaerobic environment by replacing the existent molecular oxygen. The

antifungal activity of carbon di-oxide (CO2) is due to the inhibition of enzymatic

decarboxylations and to its accumulation in the membrane lipid bilayer resulting in

dysfunction in permeability (Lindgren and Dobrogosz, 1990). While it is clear that

biocontrol microbes can release many different compounds into their surrounding

environment, the types and amounts produced in natural systems in the presence and

absence of plant disease have not been well documented and this remains a frontier

for discovery (Pal and Gardener, 2006).

2.3.3 Indirect Antagonism:

2.3.3.1 Competition:

The process of competition is considered to be an indirect interaction whereby

pathogens are excluded by depletion of a food base or by physical occupation of site.

Generally, nutrient competition has been believed to have an important role in disease

suppression (Lo, 1998). Soils and living plant surfaces frequently compete in nutrient

limited environments. To successfully colonize the phytosphere, a microbe must


effectively compete for the available nutrients. On plant surfaces, host-supplied

nutrients include exudates, leachates and senesced tissues. Additionally, nutrients can

be obtained from waste products of other organisms and the soil. While difficult to

prove directly, much indirect evidence suggests that competition between pathogens

and non-pathogens for nutrient resources is important for limiting disease incidence

and severity (Pal and Gardener, 2006). Competition for nutrients is a general

phenomenon regulating population dynamics of microorganisms sharing the same

ecological niche and having the same physiological requirements when the trophic

resources are limited (Alabouvette et al., 2006). Competition for nutrients (e.g.

carbohydrates, nitrogen, and oxygen) and space is often suggested as a potential

mechanism of action in biological control systems (Spadaro et al., 2010).

Competition for minor elements also frequently occurs in soil. Competition for

iron is one of the modes of action by which fluorescent pseudomonads limit the

growth of pathogenic fungi and reduce disease incidence or severity (Loper and

Lindow, 1993). Iron is extremely limited in rhizosphere (10-8

M); in highly oxidised

and aerated soil it is present in ferric form which is insoluble in water. This very low

concentrationcan not support the growth of microorganisms. To survive in such an

environment, organisms produce iron binding ligands called siderophores, having

high ability to obtain iron from the environment (Loper, and Henkels 1999; Shahraki

et al., 2009). Mandeel and Baker (1991) studied interactions between pathogenic and

non-pathogenic F. oxysporum, postulated that the root surface had a finite number of

infection sites that could be protected by increasing the inoculum density of the non-

pathogenic strain.

2.3.3.2 Induction of Host Resistance:

Any plant reacts to stresses from biotic or abiotic origin by elicitation of defence

reactions. The plant reacts to: (i) physical stresses such as including gravity, light,

temperature, physical stress, water and nutrient availability, (ii) inoculation by

pathogenic or non-pathogenic organisms, (iii) chemical molecules from natural or

synthetic origins (Alabouvette et al., 2006)). Systemic acquired resistance (SAR) and

induced systemic resistance (ISR) are two forms of induced resistance wherein plant

defenses are preconditioned by prior infection or treatment that results in resistance

against subsequent challenge by a pathogen or parasite (Choudhary et al., 2007).


Induction of host defences can be local or systemic in nature, depending on the type,

source, and amount of stimuli (Pal and Gardener, 2006). A number of strains of root-

colonizing microbes have been identified as potential elicitors of plant host defences.

Some strains of Pseudomonas and Trichoderma are known to strongly induce plant

host defences (Harman et al., 2004; Haas and Defago, 2005). The existence of

salicylic acid-independent ISR pathway has been studied in Arabidopsis thaliana,

which is dependent on jasmonic acid (JA) and ethylene signaling. Specific

Pseudomonas strains induce systemic resistance in Arabidopsis, as evidenced by an

enhanced defensive capacity upon challenge inoculation. Salicylic acid (SA) and non-

expressor of pathogenesis-related genes1 (NPR1) are key players in systemic acquired

resistance. Trichoderma harzianum when inoculated on to roots or on to leaves of

grapes provides control of diseases caused by Botrytis cineria on leaves spatially

separated from the site of application of the biocontrol agent (Deshmukh et al., 2006).

Saksirirat et al. (2009) evaluated the efficacy of Trichoderma strains in inducing

resistance in tomato. Strains of Trichoderma were inoculated in soil and activity of

chitinolytic and glucanasese in the leaves was examined at 0, 5, 8, 11, 14 days after

inoculation. It was found that the activity of enzymes in the leaves of tomato

increased up to 14th

day. Combination of ISR and SAR can increase protection against

pathogens that are resisted through both pathways besides extended protection to a

broader spectrum of pathogens than ISR/SAR alone (Choudhary et al., 2007).

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