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Review of Literature

REVIEW OF LITERATURE

The global increase in human population and environmental pollution has the
ill-fated consequence that worldwide food production may soon become scarce.
Therefore, in twenty first century, an environmentally safe and sustainable crop
production to feed the increasing population will be one of the great challenges.
Moreover, severe yield losses of economically important crops due to various plant
pathogens and pests are also of major concern around the world. In spite of good
agronomic and horticultural practices, producers often rely heavily on chemical
fertilizers and pesticides. Over the past 100 years, such chemical inputs to agriculture
have significantly contributed to the remarkable improvements in crop production and
quality (Junaid et al., 2013). However, excessive and indiscriminate use of
agrochemicals has resulted in long list of harmful effects on environment and human
health (Gunnell et al., 2007; Leach and Mumford, 2008), and led to considerable
changes in consumer’s attitude towards the approach of using various agrochemicals in
agriculture. “Furthermore, emerging, re-emerging, and endemic plant pathogens
continue to challenge our ability to safeguard plant growth and health worldwide”
(Miller et al., 2009). Therefore, the health awareness of the people together with the
development of resistant pathogens due to the continuous misuse of the chemicals
contributes to the restricted use of chemicals in crop protection. Under such
circumstances, there is a great need for sound and ecologically friendly approaches in
agriculture for enhanced agricultural production and to cater the increasing world’s
population. The use of microbes for biological control of plant pathogens and for
enhancement of plant growth has been emerged as promising alternative to reduce the
use of chemical pesticides and fertilizers (Compant et al., 2005; Glick, 2012; Saraf et
al., 2014). Different microbes belonging to different groups of organisms such as
bacteria, fungi, actinobacteria have been studied as potential biocontrol and growth
promoting agents (Whipps, 2001; Raiijmakers et al., 2002; Beneduzi et al., 2012;
Santoyo et al., 2012). Among these antagonistic and plant growth promoting
microorganisms, actinobacteria have received widespread attention due to their ability
to produce vast array of secondary metabolites (El-Tarabily, 2008; Khamna et al., 2009;
Palaniyandi et al., 2013b; Sharma, 2014b).

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2.1 ACTINOBACTERIA

Actinobacteria formerly referred to as actinomycetes is a large and most widely


distributed bacterial phylum in nature. They are primarily soil inhabitants but are also
present in a wide variety of aquatic and marine habitats (Asquith et al., 2013) and play an
important role in natural geochemical cycles. These bacteria also inhabit different plant
tissues as endophytes. This diverse group of high G+C, Gram-positive bacteria exhibits
varied morphological, physiological and metabolic properties that allow them to prosper in
a wide range of environments. The taxonomy of the actinobacteria is complex and the
phylum actinobacteria is classified into six orders, 10 suborders, 42 families, 110 genera
and more than 1,000 species (Stackenbrandt et al., 1997). Mankind’s interest in
actinobacteria, originated in the early nineteenth century, primarily due to their metabolic
versatility in the production of chemically diverse and biologically active secondary
metabolites (Berdy, 2005; Nett et al., 2009). The most remarkable among these metabolites
are the antibiotics (Lechevalier and Lechevalier, 1967) because of which these bacteria tend
to occupy a coveted position in the pharmaceutical industry (Selvakumar et al., 2014).
From total 23,000 bioactive secondary metabolites, over 10,000 of compounds are
produced by actinobacteria, which constitutes 45% of all discovered bioactive microbial
metabolites Among actinobacteria, Streptomyces spp. have gained significant importance
because of their maximum contribution in number of bioactive metabolites (with diverse
biological activities) produced by actinobacteria. From total, around 76% (7600) of the
compounds are produced by streptomycetes (Berdy, 2012).

In addition to pharmaceutical industry, they have long gained significance in the


agroindustry as unparalleled sources of antifungal compounds (effective against fungal
phytopathogens), cell wall degrading enzymes, plant growth regulators, antiparasitic
agents and insecticidal agents which are conducive in crop production (Tanaka and
Omura, 1993; Xiao et al., 2002; Jayakumar, 2009; Dhanasekaran et al., 2012). Therefore,
these bacteria constitute the potential to be used as biocontrol agents by suppressing the
growth and development of a wide range of plant pathogens, and as plant growth
promoting agents (Behal, 2000; Doumbou et al., 2002; Ouhdouch et al., 2001; Chung et
al., 2005; Gadelhak et al., 2005; Glick, 2012). Thus, actinobacteria have several utilities
using different mechanisms in the agroindustry (Figs. 2.1 and 2.2), which are described
in greater detail in the following sections.

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Fig. 2.1: Different applications of actinobacteria (Sharma, 2014b)

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Plant growth ACC deaminase


Antibiotics Biocontrol
promotion
s

Cell wall degrading enzymes


Stress alleviation

Hyperparasitism Plant hormones


m
Phosphate solubilization
Promotion of symbiosis Siderophores
Competition
Suppression

Pathogenic fungus Pathogenic bacteria Actinobacteria Mycorrhiza N2 fixing bacteria

Fig. 2.2: Mechanisms exhibited by plant-associated actinobacteria in biocontrol


and plant growth promotion (PGP) (Palaniyandi et al., 2013b)

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2.2 ACTINOBACTERIA AS BIOCONTROL AGENTS

2.2.1 Fungal Phytopathogens

The growing demand for low-input agriculture has resulted in an incessant


attention towards microbes which are able to enhance plant nutrition and health along
with improved soil quality and thus a solution for environmental problems which have
beset us since inception of mankind. Actinobacteria, one of the major constituents of the
microbial biomass present in soil, serve as a research target with regard to sustainable
agriculture. The genus Streptomyces is well known as one of the major sources of
bioactive natural products including biocontrol and plant growth promoting metabolites
(Behal, 2000; Terkina et al., 2006; Palaniyandi et al., 2013b; Selvakumar et al., 2014).

In 1890, Gasperini reported actinobacteria for the first time as potential killers of
fungi and bacteria. Later in 1932, Tims studied an actinobacterium which was
antagonistic towards Pythium, a fungal pathogen of sugarcane. Afterwards, Waksman
(1937) studied actinobacteria in detail for their antagonistic effect upon other
microorganisms. Since then there are numerous reports on the potential of
actinobacteria, mainly Streptomyces spp. as potent biocontrol tools in the control of
various phytopathogens, especially phytopathogenic fungi (Tahvonen and Avikainen,
1987; El-Abyad et al., 1993; Yuan and Crawford, 1995; Trejo-Estrada et al., 1998ab;
Chamberlain and Crawford, 1999; Berg et al., 2001; Doumbou et al., 2002; Berdy,
2005; Zucchi et al., 2008; Errakhi et al., 2009; Boukaew et al., 2011; Kim et al., 2015).

A major example of Streptomyces as biocontrol agent is S. griseoviridis strain


K61, isolated from light coloured Sphagnum peat. It has been reported to antagonize
variety of plant pathogens including Alternaria brassicicola, Botrytis cinerea, Fusarium
avenaceum, Fusarium culmorum, Fusarium oxysporum f.sp. dianthi, Pythium
debaryanum, Phomopsis sclerotioides, Rhizoctonia solani and Sclerotinia sclerotiorum
(Tahvonen, 1982ab; Tahvonen and Avikainen, 1987). Afterwards in 1992, Mohammadi
and Lahdenpera used the Streptomyces strain K61 in root dipping or growth nutrient
treatment of greenhouse cucumbers, eut flowers, potted plants and various other
vegetables. In 1995, Yuan and Crawford characterized another actinobacterium, S.
lydicus WYEC108 as potentially potent biocontrol agent for reducing Pythium seed and

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root rots. El-Tarabily et al. (1997) isolated and screened streptomycete and non
streptomycete actinobacteria for biocontrol of cavity spot disease of carrots caused by
Pythium coloratum and two isolates, Streptomyces janthinus and Streptomyces albidum
were the most effective in suppressing the disease in inoculated plants. In the same year,
Elson et al. (1997) reported Streptomyces sp. for biological control of
Helminthosporium solani causing silver scurf of potato tubers. Marten et al. (2001)
reported the use of RhizovitR obtained from Streptomyces rimosus to control a wide
range of fungi including Pythium spp., Phytophthora spp., R. solani, A. brassicicola,
and Botrytis sp. Later on, Liu et al. (2004) reported a high antagonistic activity of S.
rimosus against Fusarium solani, Fusarium oxysporum f. sp. cucumarinum, Verticillium
dahliae, R. solani, Fulvia fulva, B. cinerea, Alternaria alternata, S.
sclerotiorum and Bipolaris maydis. Dhanasekaran et al. (2005) demonstrated biocontrol
potential of Streptomyces isolates which significantly reduced the R. solani damping off
in tomato plants. Streptomyces species J-2 and B-11, isolated from rhizosphere soil of
sugarbeet, significantly reduced the incidence of root rot in sugar beet after the
treatment of Sclerotium rolfsii infested soil with their biomass and culture filtrates
(Errakhi et al., 2007).

Nguyen et al. (2012) reported that application of culture suspension of S. griseus


H7602 protected pepper plants against Phytophthora capsici by reducing 47.35% of
root mortality. Treatment of cucumber seedlings with the strain Streptomyces 16R3B
reduced 71% damping-off caused by Pythium aphanidermatum under greenhouse
conditions (Costa et al., 2013). Bubici et al. (2013) demonstrated the biocontrol
potential of Streptomyces spp. where three of the four studied streptomycete isolates
significantly reduced corky root up to 64.9%. Also the foliar symptoms of Verticillium
wilt were reduced up to 48.3% by all the four species. These isolates effectively reduced
corky root severity in tomato in naturally infested field trials but none of the isolates
were effective in controlling Verticillium wilt of eggplant. Jung et al. (2013) also
demonstrated that treatment of wheat seeds with Streptomyces sp.BN1 prior to
Fusarium graminearum infection significantly decreased Fusarium head blight severity.
Later Choudhary et al. (2014 and 2015) documented the antagonism of Streptomyces
exfoliates strain MT9 and Streptomyces violascens MT7 towards several fruit rotting

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fungi. Cell suspension of S. violascens MT7 resulted in 100% disease reduction


(Rhizoctonia stolonifer soft-rot) on papaya fruits as compared to 0 % and 75.11%
disease reduction in the untreated control and carbendazim treated papaya fruits,
respectively. The development of Geotrichum candidum causing sour-rot on Citrus
reticulata Blanco (oranges) was also completely inhibited by treatment with cell
suspension of S. violascens MT7 (Choudhary et al., 2015). Kim et al. (2014a) reported
protection of pepper and cherry tomato plants from anthracnose of Colletotrichum
gloeosporioides by Streptomyces sp. A1022. All these reports showed the importance of
Streptomyces species in the biocontrol of diverse range of fungal phytopathogens.
Recently, Kobayashi et al. (2015) described the biocontrol potential of another
streptomycete, Streptomyces WoRs-501, which suppressed potato scab caused by
Streptomyces turgidiscabies and Streptomyces scabies in field pot trials. Culture broth
of Streptomyces hygroscopicus strain BS062 significantly controlled postharvest root
rot of ginseng, and strawberry gray mold disease caused by B. cinerea up to 73.9% and
58%, respectively (Kim et al., 2015).

These antagonistic actinobacteria exhibit several mechanisms which might


explain their ability to act as biocontrol tools. These properties include antibiosis
(production of antibiotics), cell wall degrading enzymes, root colonization, parasitism
(direct), and induction of systemic resistance in host plants (indirect). Antagonistic
microbes exhibiting multitude of mechanisms are more fruitful in controlling plant
diseases and therefore, are the preferred candidates for development of biocontrol
agents (Doumbou et al., 2002; Palaniyandi et al., 2013b). The different mechanisms of
plant disease control are as follows:

2.2.1.1 Production of antifungal compounds

Plant-beneficial antagonistic microbes produce several compounds to inhibit the


growth of plant pathogens and thus, effectively suppress the disease development.
According to statistics, approximately 60 % of agriculturally useful antibiotics are
produced by different Streptomyces species and they still remain the most fruitful
source for different types of bioactive metabolites, including agroactive type. A number
of studies document the plant disease suppressing ability of actinobacterial strains by

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producing antibiotics. Various antifungal antibiotics produced by streptomycetes,


reported to suppress fungal plant diseases, are listed in Table 2.1. Streptomycin and
cycloheximide produced by S. griseus are the first antibiotics reported to control
bacterial and fungal diseases of plants (Leben and Keitt, 1954). Blasticidin S, a
nucleoside antibiotic discovered from Streptomyces griseochromogenes was the first
microbial fungicide commercially available for plant protection and has been used for
the control of Magnaporthe grisea (Herb) Barr. causing rice blast (Takeuchi et al.,
1957). However, some limitations related to blasticidin S encouraged further screening
of other biofungicides that eventually yielded kasugamycin, polyoxin, validamycin and
mildiomycin and all are used commercially in agriculture. Kasugamycin obtained from
the metabolites of Streptomyces kasugaensis and Streptomyces kasugaspinus showed in
vitro activities against yeast and some plant pathogenic fungi, including M. grisea
(Umezawa et al., 1965). In 1965, Ishiyama et al. showed that kasugamycin significantly
suppressed the development of mycelial growth of M. grisea on rice plants during in
vivo studies. In addition, Hamada et al. (1965) studied the use of ksasugamycin for the
protective and curative control of scab in pears and apples as well as leaf spot in celery
and sugarbeet. In the same year, Suzuki et al. (1965) isolated polyoxins from the culture
broth of Streptomyces cacaoi var. asoensis and used them commercially for controlling
grey mold diseases caused by B. cinerea Pers and black spot of Japanese pear caused by
Alternaria kikuchiana S. Tanaka (Isono et al., 1965). Later, Iwasa et al. (1971) reported
validamycin A from Streptomyces hygroscopicus var. limoneus, which was found to be
effective in controlling rice sheath blight caused by R. solani. Because of biodegrability
in soil, validamycin A was considered to be an ideal environmentally safe microbial
fungicide.

Streptomycetes which produced gelandamycins are promising biocontrol agents


(BCA) of several plant diseases. Geldanamycin produced in soil by S. hygroscopicus
var. geldanus accounts for the control of rhizoctonia root rot by antagonizing R. solani
(Rothrock and Gottlieb, 1984). The production of multiple antibiotics by the BCAs is
desirable because it leads to inhibition of diverse plant pathogens. For example,
Streptomyces violaceusniger YCED9 isolated from rhizosphere soil is a good model as
biological control agent as it produces three antimicrobial compounds which include

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nigericin, geldanamycin, and guanidylfungin A which inhibit Fusarium, Phytopthora


and Pythium species (Crawford et al., 1993; Trejo-Estrada et al., 1998b). Hwang et al.
(1996) isolated a manumycin-type antibiotic from Streptomyces flaveus which
displayed broad antifungal spectrum against several plant pathogenic fungi, including P.
capsici Leonian, Cladosporium cucumerinum Ellis & Arthur, M. grisea and Alternaria
mali Roberts. During screening for antifungal antibiotics for controlling plant diseases,
Kim et al. (1999) isolated streptimidone from Micromonospora coerulea, which
showed strong antifungal activity against P. capsici and completely suppressed the
development of Phytophthora blight in pepper plants comparable to the control efficacy
of the commercial fungicide metalaxyl. In 2000, Igarashi et al. reported new microbial
metabolite, fistupyrone from endophytic Streptomyces sp. TP-A0569 which inhibited
the in vivo infection of the seedlings of Chinese cabbage by A. brassicicola. 4-Phenyl-3-
butenoic acid and phenyl acetic acid discovered from several isolates of Streptomyces
spp. also showed potent control efficacy against Phytophthora blight in pepper plants
(Hwang et al., 2001; Lee et al., 2005; Kim et al., 2006). Another compound, 2-
methylheptyl isonicotinate from Streptomyces sp. 201, showed marked inhibitory
activity towards prevalent soil-borne phytopathogens, including Fusarium moniliforme,
F. oxysporum, R. solani, Fusarium semitectum, Fusarium solani and showed protective
effect to control Fusarium wilt in cauliflower (Bordoloi et al., 2002).

Shih et al. (2003) demonstrated biocontrol potential of Streptomyces padanus, as


treatment of cabbage seeds with its culture filtrate reduced the disease incidence
(damping-off of cabbage caused by R. solani AG-4). They further reported
fungichromin as an active ingredient responsible for antagonism of S. padanus against
R. solani. Another endophytic streptomycete, Streptomyces galbus R-5, also produced
fungichromin along with actinomycin X2, and treatment of tissue cultured
rhododendron seedlings with the strain offered protection against Pestalotia disease
(Hasegawa et al., 2006). Park et al. (2006) isolated antibiotic staurosporine from
Streptomyces roseoflavus strain LS-A24 which inhibited the mycelial growth of several
fungi and inhibited the development of Phytophthora blight on pepper plants
comparable to commercial fungicide, metalxayl. In 2014, Li et al. also reported
staurosporine from an endophytic Streptomyces sp. strain CNS-42 and revealed its

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involvement in biocontrol of F. oxysporum f. sp. cucumerinum. Park et al. (2008)


isolated valinomycin (a peptide antibiotic) from culture extract of Streptomyces sp.
strain M10 which controlled the development of Botrytis blight caused by B. cinerea in
cucumber plants similar to that of commercial fungicide, vinclozolin. Culture filtrate of
Streptomyces malaysiensis strain MJM1968 resulted in more than 80 % suppression in
fungal population after 14 days of treatment in soil. The compound responsible for
antagonism was found to be azalomycin which exhibited antifungal activity against A.
mali, F. oxysporum, C. gloeosporioides, R. solani, Fusarium chlamydosporum,
Cladosporium cladosporioides, and Pestalotia spp. (Cheng et al., 2010). Palaniyandi et
al. (2011) reported the use of antifungal antibiotics from actinobacteria for the control
of foliar disease of anthracnose in yam plants. Secondary metabolites of
Propionicimonas sp. ENT-18, a rare actinobacterium inhibited sclerotia formation in S.
sclerotiorum (Zucchi et al., 2010). Streptomyces lividans inhibit the growth of V.
dahliae by the producing prodiginines. During co-cultivation with V. dahliae,
Streptomyces lividans produced high amount of undecylprodigiosin and smaller
amounts of streptorubin B. Undecylprodigiosin inhibited V. dahliae by affecting
microsclerotia formation (Meschke et al., 2012).

Strevertenes A and B from Streptomyces psammoticus and Filipin III from


Streptomyces miharaensis showed inhibitory effects against the mycelial growth of A.
mali, Cylindrocarpon destructans, Colletotrichum orbiculare, Fusarium oxysporum
f.sp. lycopersici, and also inhibited the development of Fusarium wilt in tomato plants
caused by F. oxysporum f.sp. lycopersici comparable to commercial fungicide benomyl
under greenhouse conditions (Kim et al., 2011; Kim et al., 2012). Taechowisan et al.
(2012) purified two antifungal compounds, 3-methylcarbazole and 1-methoxy-3-
methylcarbazole, from endophytic Streptomyces sp. LJK109 which showed activity
against various phytopathogenic fungi. Later, Xiong et al. (2013) demonstrated
antagonistic activity of a novel polyene macrolide antibiotic, antifungalmycin 702
(produced by S. padanus JAU4234) against M. grisea, a rice blast fungus.

Nguyen et al. (2015) isolated 1H-pyrrole-2-carboxylic acid (PCA) from S.


griseus H7602 which showed strong antifungal activity against P. capsici under in vitro
conditions. Two more antifungal compounds i.e. guanidylfungin A and methyl
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guanidylfungin exhibiting activity against various plant pathogenic fungi and bacteria
were recovered from Streptomyces sp. A3265 (Minh et al., 2015). In another report,
Bafilomycins B1 and C1 isolated from the fermentation culture of Streptomyces
cavourensis NA4 exhibited significant broad spectrum antifungal activity against
different phytopathogenic fungi (Pan et al., 2015). An antifungal metabolite, 3-methyl-
3,5-amino-4-vinyl-2-pyrone from Streptomyces sp. N2 possess broad-spectrum
inhibitory activity towards plant pathogenic fungi such as Fusarium oxysporum f. sp.
niveum, R. solani, Fusarium oxysporum f. sp. vasinfectum, Pyricularia grisea,
Penicillium italicum and C. gloeosporioides. Antifungal compound also showed
protective effect against C. gloeosporioides to prevent anthracnose on grape fruits (Xu
et al., 2015). Streptomyces sp. strain Di944 was reported to control root rot and
damping-off of tomato seedlings caused by R. solani when applied as a seed treatment
or added to peat-based potting medium (Sabaratnam and Traquair, 1996, 2002). Later
this antagonist was identified as Streptomyces griseocarneus (Traquair et al., 2013).
Recently, in 2015, Sabaratnam and Traquair reported the presence of an antifungal
compound, designated as rhizostreptin, which is a pentaene macrolide complex, in the
cell-free culture filtrates of S. griseocarneus Di944 and demonstrated its involvement in
suppression of Rhizoctonia damping-off of tomato plants. More recently, Palaniyandi et
al. (2016) demonstrated that foliar application of culture filtrate extract of an
azalomycin-producing S. malaysiensis on Yam plants, significiantly reduced the disease
severity (yam anthracnose caused by C. gloeosporioides) to 26% as compared to
untreated plants where disease severity was >92%.

Table 2.1: Antibiotics produced by actinobacteria to control fungal phytopathogens


Antagonist Disease Causative Antibiotic Reference
pathogen
S. cacaoi var. Rice sheath R. solani Kùhn Polyoxin B and D Isono et al.,
asoensis blight 1965
S. Broad range Alternaria, Blasticidin S Kono et al.,
griseochromogenes of plant Botrytis, 1968
2A-327 diseases Corticium,
Gloeosporium,
Glomerella,
Macrosporium,
Ophiobolus,
Penicillium and
Piricularia

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Antagonist Disease Causative Antibiotic Reference


pathogen
S. hygroscopicus var. Sheath blight Pellicularia sasaki Validamycin Iwasa et al.,
limoneus No. T-7545 of rice (Jinggangmycin) 1971
Damping off R. solani
of cucumber
Streptomyces Powdery Several crops Mildiomycin Iwasa et al.,
rimofaciens Niida mildew 1978
S. kasugaensis Rice blast P. oryzae Kasugamycin Yamaguchi,
disease 1982
Micromonospora sp. Rice root R. solani Dapiramicin Shomura et
SF-1917 disease al., 1983
S. hygroscopicus var. Root rot of R. solani Geldanamycin Rothrock
geldanus Pea and Gottlieb,
1984
S. griseus Asparagus Fusarium spp. Faeriefungin Smith et al.,
root diseases 1990
S. hygroscopicus Powdery Erysiphe Gopalamycin Nair et al.,
mildew of cichoracearum, 1994
Cucumber Erysiphe graminis
Brown rust tritici,
of wheat F. culmorum,
Powdery Plasmopara
mildew of viticola,
wheat F. culmorum,
Culm rot of P. viticola and
wheat Helmenthosporium
Downy teres
mildew
grape
Gray mold of
pepper
Rice blast
Net blotch of
barley
S. violaceoniger Phytophthora Phytophthora Tubercidin Hwang and
blight of infestans Kim, 1995
pepper
S. flaveus strain A-11 P. capsici, Manumycin-type Hwang et
M. grisea, C. Antibiotic, SW-B al., 1996
cucumerinum,
and A. mali
S. violaceusniger Grass R. solani Nigericin and Trejo-
YCED9 seedling Guanidylfungin A Estrada et
disease al., 1998b
Streptomyces Phytophthora P. capsici Phenylacetic Acid Hwang et
humidus blight of al., 2001
Pepper
S. melanosporofacies Potato scab S. scabiei Geldanamycin Agbessi et
al., 2003

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Antagonist Disease Causative Antibiotic Reference


pathogen
S. padanus Damping-off R. solani Fungichromin Shih et al.,
of cabbage 2003
S. aureofaciens Anthracnose Colletotrichum 5,7-dimethoxy-4- Taechowisan
CMUAc130 of banana musae pmethoxylphenylcoumarin, et al., 2005
Wilt of F. oxysporum 5,7-dimethoxy-4-
wheat phenylcoumarin
Micromonospora sp. Rice blast P. oryza 2,3- dihydroxybenzoic Ismet et al.,
M39 acid, phenylacetic 2004
acid, cervinomycin A1 and
A2
S. malaysiensis Blotch of Stagonospora Malayamycin Li et al.,
wheat nodorum 2008
Streptomyces sp. Powdery Sphaerotheca Neopeptin A and B Kim et al.,
KNF2047 mildew fusca 2007
of cucumber
S. cavourensis subsp. Anthracnose C. 2-Furancarboxaldehyde Lee et al.,
cavourensis SY224 of pepper gloeosporioides 2012
S. griseus H7602 Phytophthora P. capsici 1H-pyrrole-2-carboxylic Nguyen et
blight of acid al., 2015
Pepper
S. cavourensis NA4 Fusarium F. oxysporum Bafilomycins B1 and C1 Pan et al.,
wilt 2015
Streptomyces sp. N2 Fruit C. gloeosporioides 3- Xu et al.,
anthracnose methyl-3,5-amino-4-vinyl- 2015
2-pyrone

2.2.1.2 Cell wall degrading enzymes

Actinobacteria have also been reported to produce a variety of extracellular


hydrolytic enzymes (cellulase, chitinase, protease, phospholipase and glucanase) due to
which they are able to degrade various biopolymers in soil. The ability of actinobacteria
to produce extracellular enzymes has gained attention of researchers due to their
important role in biocontrol of plant diseases (Mahadevan and Crawfard, 1997; Trejo-
Esterada et al., 1998a; Mukherjee and Sen, 2006; Chater et al., 2010; Lee et al., 2012;
Palaniyandi et al., 2013a). In particular, chitinases and glucanases have been the most
studied enzymes for antagonism of plant pathogens (Valois et al. 1996; El-Tarabily et
al., 2000; Prapagdee et al., 2008). Therefore, chitinase and glucanase producing
actinobacteria are important biocontrol agents for fungi containing chitin and glucan in
their cell walls. Among actinobacteria, streptomycetes are among the dominant
chitinolytic microorganisms present in the soil (Chater et al., 2010).

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Table 2.2 represents the detailed list of hydrolytic enzymes producing


antagonistic Streptomyces spp. Taechowisan et al. (2003b) studied chitinase production
in endophytic Streptomyces aureofaciens CMU Ac 130 and its antagonism towards
fungal phytopathogens by lysing their cell walls. Streptomyces halstedii AJ-7 which
suppressed the P. capsici, causal agent of phytopthora blight in red peppers, was also
reported to produce extracellular chitinase enzyme of 55kDa size (Joo, 2005a, b).
Mukherjee and Sen (2006) purified and characterized chitinase enzyme from
Streptomyces venezuelae P10 and also reported its antifungal activity against
phytopathogens. S. violaceusniger XL-2 suppressed wood rotting fungi such as
Phanerochaete chrysosporium, Postia placenta, Coriolus versicolor, and Gloeophyllum
trabeum by producing chitinase enzyme (Shekhar et al., 2006). Later in 2014, Nagpure
et al. also reported that antifungal activity of S. violaceusniger MTCC 3959 against
brown and white rot fungi is due to the different mycolytic enzymes (chitinases,
glucanases and proteases) present in the cell free extract. In 2008, Quecine et al. also
reported the potential of chitinase producing endophytic streptomycetes as biocontrol
agents to control phytopathogenic fungi. Anitha and Rabeeth (2010) described the
antifungal activity of S. griseus against fungal phytopathogens. This strain produced
chitinase enzyme when grown in medium containing fungal cell walls as sole source of
carbon. Treatment with bioactive components (chitinase and glucanase) of S.
aureofaciens reduced the incidence of anthracnose disease (caused by C.
gloeosporioides) on mango trees and increased the fruit yield (Haggag et al., 2011). S.
cavourensis SY224 suppressed the anthracnose in pepper by producting chitinase and
glucanase enzymes (Lee et al., 2012). Purified chitinase obtained from Streptomyces
sporovirgulis inhibited growth of fungal phytopathogens such as A. alternata,
Penicillium purpurogenum and Penillium sp. (Brzezinska et al., 2013). Sajitha and
Florence (2013) reported antagonism of Streptomyces sp. against Lasiodiplodia
theobromae causing sapstain on rubberwood and attributed this to the production of
chitinase enzyme which degrade the fungal cell wall.

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Awad et al. (2014) isolated another active chitinase producer strain


Streptomyces glauciniger WICC-A03 which showed antagonism towards
phytopathogenic fungi. S. exfoliatus strain MT9 produced several cell wall lytic
enzymes and displayed strong and broad-spectrum antagonism by suppressing mycelial
growth of several fruit-rotting fungi (Choudhary et al., 2014). Recently, Thirumurugan
(2015) screened Streptomyces isolates for chitinase production and antifungal activity
against phytopathogens. Karthik et al. (2015) purified acidic chitinase of 40kDa size
from Streptomyces sp. which showed activity against phytopathogens. In greenhouse
experiments, chitinolytic actinobacterium Streptomyces vinaceusdrappus S5MW2
suppressed disease caused by R. solani, in plants treated with colloidin chitin (CC) as
compared to untreated control and without CC treated plants (Yandigeri et al., 2015).

Valois et al. (1996) reported glucanolytic activity in actinobacteria which


reduced root rot in raspberry seedlings by inhibiting the pathogen, Phytophthora
fragariae var. rubi. Similarly, El-Tarabily et al. (2009) described the biocontrol
potential of 3 endophytic glucansae producing strains of Streptomyces spiralis,
Micromonospora chalcea and Actinoplanes campanulatus which protected cucumber
seedlings and mature plants from P. aphanidermatum. These three strains when used in
combination resulted in better suppression of damping-off and crown and root rot
diseases of cucumber than the metalaxyl, a chemical fungicide. S. cavourensis strain
SY224 also displayed biocontrol potential towards anthracnose in pepper by producing
chitinase and glucanase enzymes (Lee et al., 2012).

In addition to chitinases and glucanases, Singh and Chhatpar (2011) demonstrated


the involvement of proteases in antifungal activity. Protease enzyme of 20 KDa size,
produced by Streptomyces sp. strain A6, showed antagonistic activity towards Fusarium
udum by inhibiting spore germination. Recently, Palaniyandi et al. (2013a) reported a
novel mechanism of biocontrol exhibited by a Streptomyces sp. ExPro138, which produced
various extracellular proteases that prevent spore adhesion, germination and appressorium
formation in Colletotrichum coccodes and thus resulted in reduced incidence of anthracnose
on tomato fruits. Cell-free culture filtrate of S. violaceusniger MTCC 3959 exhibited broad

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spectrum antifungal activity against both brown rot and white rot fungi (Nagpure et al.,
2014). The zymogram analysis of cell-free culture filtrate revealed presence of five
chitinase isoenzymes, one β-1,3-glucanase and four protease isoenzymes.

In addition to chitin, cellulose is the major component of cell walls of some


phytopathogenic fungi like Pythium spp. and Phoma spp. So, actinobacteria which
produce cellulolytic enzymes also constitutes the potential to be used as biocontrol agents.
Chamberlain and Crawford (1999) demonstrated the efficacy of lignocellulolytic
Streptomyces strains YCED9 and WYE53 as biocontrol agents to control R. solani and
Pythium ultimum, fungal agents of turf grass. Cellulolytic actinobacterium, Streptomyces
rubrolavendulae S4 also showed antagonism towards P. aphanidermatum (Loliam et al.,
2013). Kanchanasin and Chaiyanan (2010) investigated the involvement of cellulolytic
enzymes of Streptomyces isolate S22 in antagonism against P. aphanidermatum (The 8th
International Symposium on Biocontrol and Biotechnology).

Some strains produce both hydrolytic enzymes and antibiotics and may provide
better disease control than the strains producing cell wall-degrading enzymes alone. Once
hydrolytic enzymes damage the cell wall, then pathogen is more likely to be susceptible
to attack by other biological, physical and chemical agents (El-Mehalawy et al., 2004).
Therefore, use of more than one mechanisms for biocontrol of pathogens is always
preffered. S. lydicus WYEC108, a potent biocontrol agent towards Pythium seed and root
rot was reported to produce both antifungal antibiotics and extracellular chitinase
enzymes (Yuan and Crawford, 1995). The antifungal activity of S. hygroscopicus against
C. gloeosporioides and S. rolfsii was also attributed to both hydrolytic enzymes and
thermostable antifungal compounds (Prapagdee et al., 2008). In 2012, Lee et al.
demonstrated the association of chitinase, glucanase and 2-Furancarboxaldehyde in
biocontrol mechanism of S. cavourensis. Jayamurthy et al. (2014) also documented the
involvement of both antifungal antibiotics and hydrolytic enzymes in the biocontrol
potential of Streptomyces NII 1006. S. exfoliatus strain MT9 showed strong and broad-
spectrum antagonism towards several fruit-rotting fungi by producing fungal cell-wall
lytic enzymes and antifungal metabolites (Choudhary et al., 2014).

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Table 2.2: Antagonistic actinobacteria suppressing plant pathogens by the


production of hydrolytic enzymes
Antagonist Disease Causal agent Hydrolytic Reference
enzymes
S. lydicus WYEC Pythium Pythium sp., R. Chitinase Mahadevan and
108 seed rot, solani Crawford, 1995
damping off
Micromonospora Root-rot P. cinnamomi Cellulase El-Tarabily et
carbonacea al., 1996
S. violaceusniger P. infestans Chitinase and β- Trejo-Estrada et
strain YCED-9 glucanase al., 1998a
M. carbonacea Basal drop Sclerotinia minor Chitinase and β- El-Tarabily et
disease 1,3-glucanase al., 2000
of lettuce
Actinoplanes Lupin root Plectosporium Chitinase El-Tarabily,
missouriensis rot tabacinum 2003
S. halstedii AJ-7 F. oxysporium Chitinase Joo, 2005b
S. violaceusniger Wood rot P. chrysosporium, Endo-chitinase Shekhar et al.,
XL-2 P. placenta, C. 2006
versicolor,
and G. trabeum
S. hygroscopicus Anthracnose C. gloeosporioides Chitinase Prapagdee et al.,
2008
Actinoplanes Damping- P. aphanidermatum β – glucanase El-Tarabily et
campanulatus, off and al., 2009
Micromonospora crown
chalcea and root rot
S. spiralis
Streptomyces S. sclerotiorum Chitinase, Froes et al.,
sp.80 protease, 2012
glucanase
S. cavourensis Anthracnose C. gloeosporioides Chitinase, Lee et al., 2012
SY224 in pepper glucanase

2.2.1.3 Production of Volatiles

“The volatile organic compounds (VOCs) are low-molecular-weight


compounds that easily evaporate at normal temperature and pressure, conferring the
characteristic of diffusion through the atmosphere and soil” (Morath et al., 2012; Wu et
al., 2015). The production of VOCs by microorganisms is known for many years
(Zoller and Clark, 1921; Stotzky and Schenck, 1976), but the studies on the diversity
and potential functions of these compounds have been reported only since the last
decade (Cordovez et al., 2015). Till date, approximately 1000 microbial VOCs have
been identified (Piechullaand Degenhardt, 2014). The VOCs released by microbes

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present in soil has been reported to inhibit the growth of phytopathogenic fungi,
promote plant growth, show antinematode activity, and induce host systemic resistance
(Yuan et al., 2012; Raza et al., 2013; Schmidt et al., 2015). In the year of 1973, Moore-
Landecker and Stotzky demonstrated that volatile substances from actinobacteria can
cause several morphological alterations in hyphae and conidiophores of several fungi
such as F. oxysporum, Aspergillus giganteus, Penicillium viridicatum, Zygorhynchus
vuilleminii and Trichoderma viride.

Nearly a decade later, Herrington et al. (1987) reported inhibition of


conidiospore germination in C. cladosporioides by methyl vinyl ketone, a volatile
compound produced by Streptomyces griseoruber. In another study, Gurtler and
Pedersen (1994) reported antibacterial activity of a volatile substance produced by
Streptomyces against Bacillus subtilis. Later, Wan et al. (2008) reported reduced
incidence of leaf blight/seedling blight of rice caused by R. solani, fruit rot of
strawberry caused by B. cinerea and leaf blight of oilseed rape caused by S.
sclerotiorum after treatment with volatiles produced from Streptomyces platensis F-1.
After 2 years, Li et al. (2010) found antifungal activity of different volatiles such as
dimethyl sulfide, acetophenone and dimethyltrisulfide (obtained from Streptomyces
globisporus) and demonstrated inhibition of infection of Citrus microcarpa caused by
Penicillium italicum. Volatiles produced by S. globisporus JK-1 suppressed B. cinerea
both in vitro and in vivo and showed potential to control post-harvest grey mold of
tomato fruit (Li et al., 2012). In another study, volatile substances from Streptomyces
philanthi RM-1-138 inhibited the growth of R. solani, Fusarium fujikuroi, Bipolaris
oryze and P. grisea. These volatile substances reduced rice sheath blight disease caused
by R. solani PTRRC-9 by damaging its cell wall (Boukaew et al., 2013). Similarly,
volatile compounds from Streptomyces alboflavus TD-1 inhibited the growth of
different fungi such as Aspergillus ochraceus, Aspergillus flavus, Aspergillus niger,
F. moniliforme, Penicillum citrinum. Among 27 different volatile compounds obtained
from S. alboflavus, dimethylsulfide demonstrated inhibitory activity towards F.
moniliforme in vitro (Wang et al., 2013). Danaei et al. (2014) reported different volatile
substances from S. griseus which suppressed spore germination and mycelium growth
in Penicillium chrysogenum and B. cinerea. Recently, Wu et al. (2015) reported the

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production of 13 volatiles from strain Streptomyces albulus strain NJZJSA2 among


which three relatively abundant volatile compounds viz. 4-methoxystyrene, 2-
pentylfuran, and anisole were proved to have antifungal activity against F. oxysporum
and S. sclerotiorum both in vitro and in soil. In another report, VOCs produced by 12
Streptomyces isolates, isolated from Rhizoctonia suppressive soil, showed antifungal
activity towards R. solani and also significantly enhanced plant shoot and root biomass
in Arabidopsis thaliana seedlings exposed to the volatiles. Five different volatiles viz.
methyl butanoate, methyl 2-methylpentanoate, methyl 3-methylpentanoate, 1,3,5-
trichloro-2-methoxy benzene, and 3-octanone having inhibitory activity against R.
solani were then identified from two Streptomyces strains (W47andW214) (Cordovez et
al., 2015). Volatiles produced by Streptomyces spp. have great potential in agriculture
as biofumigants alternative to chemical fumigants such as methyl bromide, 1, 3-
dichloropropane, or chloropicrin” (Palaniyandi et al., 2013b).

2.2.1.4 Root colonization

Evidences indicate that actinobacteria are important in the rhizosphere (both


quantitatively and qualitatively), where they protect plant roots against attack by fungal
root pathogens and thus stimulate plant growth (Lechevalier 1988; Miller et al., 1990;
Barakte et al., 2002; Doumbou et al., 2002). These bacteria are also found to be
associateted with the roots of various crops. According to Weller (1988) a
microorganism having ability to colonize the roots is an ideal biocontrol agent for
controlling soil borne pathogens of plants and there are reports which documented the
root colonizing ability of actinobacteria (Kortemma et al., 1994; Yuan and Crawford,
1995; Tokala et al., 2002). Among actinobacteria, S. griseoviridis, an antagonistic
microorganism effective in biocontrol of plant diseases such as the damping-off of
Brassica, the Fusarium wilt of carnation, and the root rot disease of cucumbers is a
good example of colonization of plant rhizosphere (Tahvonen and Lahdenpera, 1988).
S. lydicus WYEC108, another antifungal biocontrol agent, is capable of mycoparasitic
colonization of fungal root pathogens and excretion of antifungal metabolites within
plant rhizospheres. It suppressed Pythium seed rot and root rot in P. ultimum infested
soils and was found to colonize the roots of pea plants emerged from streptomycete
inoculated seeds (Yuan and Crawford, 1995). Early colonization by a biocontrol agent
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often is required to fill the critical niches and to effectively compete against pathogenic
fungi. The seed bacterization method has been proved to be more effective as the
biocontrol agent can rapidly and extensively grow and cover the surface of the seeds
and can protect the plants from invading soil-borne pathogens (Martin and Loper, 1999;
Lu et al., 2004; Kanini et al., 2013).

2.2.1.5 Hyperparasitism

Several bacteria and fungi display hyperparasitism, in which they feed on the
pathogenic microorganisms. A strain of S. griseus was reported to parasitize
Colletotrichum lindemuthianum and showed growth on surface of hyphae. Due to
internal parasitism showed by the Streptomyces strain, the formation of several blebs
was also observed in parasitized hyphae. Degenerated hyphae resulted in sponge-like
texture and holes in cell walls of C. lindemuthianum (Tu, 1988). Another Streptomyces
species, S. griseoviridis strain K61 (main constituent of the Mycostop, a commercial
biofungicide) also showed parasitism on several phytopathogenic fungi. It penetrated
the fungal cell wall with little fragmentation of the hyphae in case of Pythium but
hyphae of R. solani and F. oxysporum were affected slightly. The strain K61 showed
more profound destroying effect against conidia of Alternaria due to heavy
colonization.

Recently, mycoparasitism as a mechanism of antagonism against fungal


phytopathogens was observed in Streptomyces phaeopurpureus ExPro138,
Streptomyces rochei X-4, Streptomyces cyaneofuscatus ZY-153, Streptomyces
flavotricini Z-13 and Streptomyces kanamyceticus B-49 (Palaniyandi et al., 2013a;
Xue et al., 2013). These parasitic strains showed a combination of coiling as well as
lysis as mechanism of mycoparasitism. Non-streptomycete actinobacteria have also
been reported to show mycoparasitism. A strain of Nocardiopsis dassonvillei showed
antagonistic, parasitic and mycolytic activities towards the hyphae of Fusarium
oxysporum f.sp. albedinis (Sabaou et al., 1983). Few years later, Upadhyay and Rai
(1987) observed mycoparasitic activity of Micromonospora globosa which penetrate
and grow inside the hyphae and then lyse hyphae in F. udum. Loliam et al. (2013) also
reported the mycoparasitism of cellulolytic S. rubrolavendulae S4 as mechanism of
antagonistic activity towards P. aphanidermatum.

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However, antagonistic microbes showing mycoparasitic activity in vitro not


necessarily exhibit same mechanism in the soil to suppress plant disease” (Palaniyandi
et al., 2013c). Sutherland and Papavizas (1991) reported the inefficacy of actinobacteria
under greenhouse conditions to control crown rot of pepper caused by P. capsici.
However, the actinobacterial strain was able to parasitize the oospores of the pathogen
in in vitro experiments. In literature, there is no report which has shown mycoparasitism
as a sole mechanism exhibited by antagonistic microbes for disease suppression in
plants. Antibiotics and hydrolytic enzymes produced by the antagonistic microbes make
the fungal hyphae susceptible to parasitization by the antagonist (El-Tarabily and
Sivasithamparam, 2006).

2.2.1.6 Induction of systemic resistance in host plants

Plants show resistance to a broad spectrum of pathogens because they exhibit


non-specific defense. The responses of non-specific defense are of two kinds: systemic
acquired resistance (SAR) and induced systemic resistance (ISR). The genes
responsible for SAR and ISR can be present elsewhere. The resistance induced by
pathogen and salicylic acid (SA) is called SAR and the one induced by beneficial
rhizosphere microbes (rhizobacteria) is called ISR (Schuhegger et al., 2006). Among
these rhizobacteria, actinobacteria are also reported to induce systemic resistance in
plants (Hassanin et al., 2006; Conn et al., 2008; Martinez-Hidalgo et al., 2015).
“Moreover, ISR involves jasmonate (JA) and ethylene (ET) signaling within the plant
and these hormones stimulate the host plant’s defense responses against a variety of
plant pathogens” (Glick, 2012; Ahemad and Kibret, 2014). In 2006, Hassanin et al.
(2006) described that the amendment of soil with a mixture of the four bacterial species
and four Streptomyces species induce resistance in cotton plants to the R. solani and
was the most effective against the pathogen. Later, Lehr et al. (2008) presented that
inoculation of roots with Streptomyces GB 4–2 protect spruce seedlings from infection
of needle pathogenic fungus B. cinerea and root rot fungus Heterobasidion abietinum
by modifying the cell wall structure (fortified cell walls of root cells) and by inducing
systemic resistance.

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Conn et al. (2008) reported induction of defence pathways (SAR and JA/ET
pathways) in Arabidopsis by antagonistic endophytic actinobacteria isolated from wheat
tissues. Normally, induction of these pathways was low, however, in the presence of
pathogen, actinobacteria treated plants displayed high-level of gene expression related
to defence as compared to non-treated controls. Moreover, they also documented that
resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora was related
to the induction of JA/ET pathway whereas resistance to the fungal pathogen F.
oxysporum was related to induction of SAR pathway. Additionally, these authors also
stated that culture filtrates induce different pathways when obtained from
actinobacterial strain grown under different conditions. For example, Micromonospora
sp. strain EN43 induced SAR pathway and JA/ET pathway when grown in minimal
medium and complex medium, respectively.

Another endophytic Streptomyces sp. strain EN27 induce resistance in defense


impaired mutants of Arabidopsis against E. carotovora ssp. carotovora and F.
oxysporum via an NPR1-independent pathway and NPR1-dependent pathway,
respectively. In another report by Zhao et al. (2012), culture filtrate from Streptomyces
bikiniensis HD-087 induced systemic resistance in cucumber against F. oxysporum f.sp.
cucumerinum causing Fusarium wilt. The treatment with fermentation broth of strain
HD-087 increased the activities of defence related enzymes such as phenylalanine
ammonia-lyase, peroxidase, and β-1,3-glucanase in cucumber plants, and both
chlorophyll and soluble sugar level were also increased (Zhao et al., 2012). Recently,
Martinez-Hidalgo et al. (2015) demonstrated the capability of Micromonospora strains
to control phytopathogenic fungi and to stimulate plant immunity. Root inoculation of
tomato plants with these strains induced systemic resitance and provided protection
against fungal pathogen B. cinerea. Moreover, high abundance of actinobacteria
selectively of streptomycetes was also reported in the endophytic compartments of
Arabidopsis (Lundberg et al., 2012). This indicates that actinobacteria as effective
colonizers of endophytic compartments overcome the defense system of host plants.
This phenomenon of host-endophyte interactions must provide a selective advantage to
both the host plant and its endophytic colonizer.

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2.2.2 Insecticidal potential of actinobacteria

In addition to fungal phytopathogens, pest insects, being plant disease vectors


also reduce crop output by 10-30 % either by reducing the quality or quantity of the
crop production (Ferry et al., 2004). Controlling these insect pests becomes the
challenging work in agriculture field because of development of resistance in insects to
the synthetic pesticides and the toxicity problems associated with these agrochemicals
(Armes et al., 1997; Isman, 2008; Wang et al., 2009a). Therefore, microbial metabolites
as potential pesticides have attracted great interest in agricultural sector due to their
potential activity and low toxicity.

There are many reports which indicate the important role of secondary
metabolites of actinobacteria, especially Streptomyces spp. in the biological control of
insect pests including S. liltoralis (Bream et al., 2001), H. armigera (Xiong et al., 2004;
Osman et al., 2007; Arasu et al., 2013), S. litura (Arasu et al., 2013) Ostrinia furnacalis
(Bayot-Custodio et al., 2014), Aphis gossypii (Zhang et al., 2010) Mythimna separate
(Wang et al., 2011) Tribolium castaneum (Anwar et al., 2014). Following the discovery
of highly potent insecticidal, nematicidal and acaricidal avermectins from Streptomyces
avermitilis (Burg et al., 1979; Putter et al., 1981), several other secondary metabolites,
with insecticidal activity, such as prasinons, doramectin, milbemycin, tetranectin,
nanchangmycin, dianemycin, macrotetrolides and faerifungin and spinosad have been
obtained from genus Streptomyces and established as potential protective agents against
a variety of pest insects (Montesinos, 2003; Omura, 2008). Among these, the
avermectins and milbemycin obtained from S. avermitilis and S. hygroscopicus subsp.
aureolacrimosus, respectively are of significant commercial importance (Dybas, 1983;
Mishim et al., 1983; Nolan and Cross, 1988). They are structurally related and belong to
family of macrolide antibiotics. The avermectins (abamectin and ivermectin) have
demonstrated high potential against agricultural and household insect pests in several
orders. The ivermectin is commercially available in many countries. Milbemycins
produced in submerged cultures exhibit a broad spectrum of activity against agricultural
pests such as aphids, mites, tent caterpillars, intestinal worms, and other parasites that
prey on crops and livestock. They are promising as agricultural pesticides because of

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their potent activity without toxicity to plants and animals at effective dosages
(Takiguchi et al., 1980).

The tetranectin obtained from Streptomyces aureus is a first commercial


pesticidal macrotetrolide antibiotic which is in use as an agricultural miticide in Japan
since 1973 (Ando, 1983). The nikkcinycins from Streptomyces tendae are nucleoside
peptide antibiotics which suppress the growth of various fungi, mites and insects by
inhibiting the enzyme chitin synthase (Dahn et al., 1976: Muller et al., 1981; Delzer et
al., 1984; Fiedler et al., 1982). The nikkomycins are structurally related to the polyoxins
(agricultural antifungal agents) and are potential alternatives to the traditional
insecticides used in agriculture (Demain, 1983). In 2001, Bream et al. (2001) reported
potent larval and pupal mortality in S. littoralis due to secondary metabolites of
Streptomyces and Streptoverticillum. Lewer et al. (2003) purified tartrolone C, a new
member of the tartrolone series of macrodiolides from a Streptomyces sp. on the basis
of its insecticidal activity. Gadelhak et al. (2005) isolated chitinase producing
actinobacteria, belonging to genus Actinoplanes, from Arabia Gulf area which showed
biocontrol potential against insects.

Xiong et al. (2005) isolated S. avermitilis strain 173 from marine source which
showed potent insecticidal activity against serious pests such as Spodoptera exigua, H.
armigera, aphids and Plutella xyllostlla L. Later, Liu et al. (2008) also reported the
insecticidal potential of secondary metabolites of strain of Streptomyces sp. against S.
exigua, Plutella xylostella, Aphis glycines etc. Spinosad derived from fermentation
broth of soil actinobacterium, Sacchropolyspora spinosa, caused significant reduction
in the population of H. armigera and other pests (Mandour, 2009; Wang et al., 2009)
and has been accepted in organic farming. With continuous research for bioinsecticidal
agents, Wang et al. (2011) isolated four doramectin congeners from S.
avermitilis NEAU1069 which showed acaricidal and insecticidal activity against
Tetranychus urticae Koch and M. separate, respectively. El- Khawaga and Megahed
(2012) demonstrated insecticidal activity of soil actinobacteria against S. littoralis
where S. bikiniensis A11 exhibited 100% larval mortality. The culture filtrate of
Streptomyces lavendulae showed highly toxic effect against 2nd-instar larvae of S.

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littoralis with 84 % mortality. A year later, Arasu et al. (2013) isolated novel polyketide
from Streptomyces sp. AP-123 which displayed antifeedant, larvicidal and growth
inhibitory bioactivities against S. litura and H. armigera. In another report,
Vijayabharthi et al. (2014a) also reported the insecticidal activity of 15 actinobacteria
isolates against H. armigera, S. litura and Chilo partellus.

Since the discovery of avermectins, biochemists tried to synthesize new


avermectin like compounds or its derivatives with chemical modification. Recently, two
new avermectin derivatives, 25-methyl and 25-ethyl ivermectin are reported to have
enhanced insecticidal activity against second-instar larva of M. separate as compared to
milbemycin A3/A4 (Zhang et al., 2015). Similarly, spinetoram, a new semi-synthetic
chemical belonging to spinosyn class of insecticides is discovered by modifying the
fermenting substances of S. spinosa. It has broad insecticidal spectrum and have been
registered in many countries. Very recently, a new insecticidal compound having
activity against H. armigera was purified from the extracellular extract of Streptomyces
griseoplanus SAI-25 and was identified as cyclo(Trp-Phe) belonging to class of
diketopiperazines. It showed 67% larvicidal and 59% pupicidal activities against H.
armigera (Sathya et al., 2016).

2.2.3 Nematicidal potential of actinobacteria

Among various plant pathogens, nematodes are also one of the important
microscopic obligate biotrophic plant parasites with worldwide distribution and cause
severe damage to a wide variety of economically important agricultural crops. As there
is an increasing trend towards the use of microorganisms as biocontrol agents for
controlling various plant pests, the search for new potential microbial nematicides has
also gained momentum. Actinobacteria, especially Streptomyces spp. have also been
reported for production of various nematicidal compounds (Burg et al., 1979; Mishra et
al., 1987a; Park et al., 2002; Sun et al., 2006; Ruanpanun et al., 2011b; Yang et al.,
2013; Zeng et al., 2013). For example avermectins, insecticidal compounds
(macrocyclic lactone derivatives) obtained from fermentation broth of S. avermitilis,
and their derivatives have been used worldwide to control parasitic nematodes and pests
(Burg et al., 1979). Later, Mishra et al. (1987a) also screened a large number of

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metabolites of aerobic actinobacteria and found that 12 isolates showed toxicity against
free-living nematode Panagrellus redivivus. Dicklow et al. (1993) investigated
nematicidal properties of Streptomyces species isolated from nematode suppressed soil
and observed that the metabolites produced by these bacteria inhibited reproduction of
Caenorhabditis elegans which was used as a screening model in the laboratory test. In
greenhouse experiment, the Streptomyces strain was also found to reduce tomato root
galling caused by M. incognita. This antinematode streptomycete was then identified as
Streptomyces dicklowii.

Another actinobacterium, Streptoverticillium albireticuli also showed


nematicidal and pathogenic activity against C. elegans (Park et al., 2002). Shiomi et al.
(2005) isolated an antibiotic, antimycin A9, from culture broth of Streptomyces sp.
K01-0031 which exhibited potent nematicidal activity against C. elegans. Later on, Sun
et al. (2006) isolated 30 actinobacteria from root-knot samples and found that 47% of
them were virulent to eggs and juveniles of Meloidogyne hapla. Jayakumar (2009)
studied the bio-efficacy of S. avermitilis culture filtrates and found that isolate Manp
induced the highest mortality of 68 and 79 % against M. incognita and Rotylenchulus
reniformis, respectively. Ruanpanun et al. (2011a) reported the isolation of three
nematicidal compounds viz. Methoxy-2-methyl-carbazole-1,4-quinone, carbazomycins
D and carbazomycins F from the crude extract of Streptomyces 005. In the same year
Ruanpanun et al. (2011b) purified two compounds, fervenulin and isocoumarin from
Streptomyces sp. CMU-MH021 which inhibited egg hatch and increased juvenile
mortality in M. incognita. Fervenulin caused 100 % juvenile mortality at concentration
of 250 µg/ml whereas isocoumarin showed only weak activity. Yang et al. (2013)
demonstrated nematicidal activity in two nemadectin congeners obtained from the
fermentation broth of a mutant strain (Y-3) of Streptomyces microflavus neau3 against
C. elegans. Zeng et al. (2013) discovered nematicidal compound (fungichromin B) from
fermentation broth of Streptomyces albogriseolus with LD50 values of 7.64 and 7.83
µg/ml for 2-stage juveniles of M. incognita and M. javanica, respectively. Tsyhankova
et al. (2012) prepared a formulation, averkom, using bioactive substances from S.
avermitilis which also displayed antinematode activity against M. incognita. 25-methyl
and 25-ethyl ivermectin, derivatives of avermectin, showed enhanced activity against C.

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elegans as compared to commercial ivermectin (Zhang et al., 2015). Recently,


Ruanpaum and Chamswarng (2016) demonstrated nematicidal activity in 12
Streptomyces isolates (isolated from commercial earthworm castings in Thailand)
against M. incognita and their efficiency to protect the chili plants against root-knot
disease in greenhouse experiments.

2.3 ACTINOBACTERIA AS PLANT GROWTH PROMOTERS

Kloepper and Schroth (1978) were the first who defined plant growth promoting
bacteria (PGPB) as those root colonizing bacteria where following their inoculation
onto seeds, enhanced plant growth was observed. These microorganisms, isolated from
diverse environments, positively influence many plant growth parameters and yield
(Kloepper et al., 1980; Bashan and Holguin, 1998; Vacheron et al., 2013) and thus
possess the potential to be used as substitute of chemical fertilizers which have several
ill effects. Different groups of bacteria (Bacillus, Pseudomonas etc.) have been
developed commercially as plant growth enhancers. Similarly, there are reports which
also documented streptomycetes as potent plant growth promoters (Yuan and Crawford,
1995; Mishra et al., 1987b; Kunoh et al., 2002; Nassar et al., 2003; El-tarabily, 2008;
Sousa et al., 2008; Gopalakrishnan et al., 2013; Palanyandi et al., 2013c; Passari et al.,
2015). However, despite their well-documented history as biocontrol agents, these
species have been poorly investigated specifically for their potential in plant growth
promotion. In 1974, Merriman et al. reported that treatment of carrot seeds with the S.
griseus (Krainsky) Waksman and Henrici isolate in two separate field trials resulted in
higher marketable yields over controls by 17% and 15%. Nearly two decades later, El-
Abyad et al. (1993) observed significant improvement in tomato growth after the
coating of tomato seeds with each of three antagonistic Streptomyces strains.
Gopalakrishnan et al. (2013 and 2015ab) reported plant growth promoting potential of
Streptomyces spp. which significantly enhanced the agronomic traits in streptomycete
inoculated sorghum, rice and chickpea over uninoculated control plants. Similarly, Jog
et al. (2014) also demonstrated that Streptomyces cultures significantly improved
growth in inoculated wheat as compared to an uninoculated control after 4 weeks.
Recently Passari et al. (2015) studied in vitro as well as in vivo plant growth promoting

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Review of Literature

potential of endophytic actinobacteria obtained from medicinal plants. They reported


two potential strains Streptomyces sp. (BPSAC34) and Leifsonia xyli (BPSAC24) which
enhanced a range of growth parameters in chilli (Capsicum annuum L.) under
greenhouse conditions.

To date, there has been only limited commercial use of plant growth promoting
microorganisms as their large scale application has been restricted due to insufficient
understanding of the mechanisms responsible for enhanced plant growth, rhizosphere
incompetence, and the inability of microbial strains to flourish in different types of soil
and environmental conditions. With recent progress towards understanding the
mechanisms that these organisms utilize to facilitate the plant growth, the use of plant
growth promoting microorganisms are expected to continue to increase worldwide.
Interactions between plant and plant growth promoting bacteria involve a combination
of direct and indirect mechanisms (El-Tarabily, 2008; Sousa et al., 2008; El-Tarabily et
al., 2009; Franco-Correaa et al., 2010; Glick, 2012; Gopalakrishnan et al., 2012;
Goudjal et al., 2014). Direct attributes for promotion of plant growth by these bacteria
include several mechanisms such as production of plant growth regulators such as
auxins, gibberelins and cytokinins (Aldesuquy et al., 1998; Sousa et al., 2008; El-
Tarabily, 2008; Khamna et al., 2010; Alam et al., 2012; Sadeghi et al., 2012;
Harikrishnan et al., 2014a), production of siderophores (Khamna et al., 2009; Nakouti
et al., 2012; Wang et al., 2014), fixation of atmospheric nitrogen (Prakash and
Cummings, 1988; Gadkari et al., 1992; Madhaiyan et al., 2009), solubilization of
phosphate (Hamdali et al., 2008ac; Balakrishna et al., 2012; Hamdali et al., 2012), and
alleviation of stress by producing the enzyme ACC deaminase which lowers ethylene
levels in plant rhizosphere (El-Tarabily, 2008; Siddikee et al., 2010; Palaniyandi et al.,
2014). The indirect mechanisms of plant growth promotion include production of
antibiotics and cell wall lytic enzymes, competition for space and induction of systemic
resistance (Doumbou et al., 2002). PGPR strains may use one or a combination of these
growth promoting attributes in the rhizosphere and influence plant growth (Glick et al.,
2007; El-Tarabily, 2008; Palaniyandi et al., 2014).

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2.3.1 Indole acetic acid production

Production of plant growth hormones is an important trait exhibited by plant


growth promoting microorganisms. Diverse genera of actinobacteria including
Streptomyces and Micromonospora inhabiting rhizosphere and internal tissues of plants
are reported to enhance plant growth by synthesizing auxins, gibberelins and cytokinins
(Kaunat, 1969, Brown, 1972, Merckx et al., 1987; Joshi and Loria, 2007; El-Tarabily,
2008; Ting et al., 2008; Ghodhbane-Gtari et al., 2010; Verma et al., 2011; Lin and Xu,
2013). The rich supply of substrates available in plant root exudates generates the
potential conditions for the Streptomyces to produce and release plant hormones
(Wheeler et al., 1984; Kravchenko et al., 1991). Among auxins, IAA is the most studied
natural plant growth hormone which influences plant growth and development by
stimulating germination of seeds, cell division, elongation of roots and stems, and
seedling growth (Bonner et al., 1952; Patten and Glick, 1996; El-Tarabily et al., 2008).
In addition, it also leads to initiation of roots with more number of root hairs and lateral
roots, thus improving the capability of the plant to absorb soil nutrients.

With tryptophan as main starting precursor for IAA production in


microorganisms, five different pathways have been described (Patten and Glick, 1996;
Spaepen et al., 2007). Among these pathways tryptophan dependent production of IAA
occurs through indole-3-acetamide in Streptomyces spp. (Manulis et al., 1994; Khamna
et al., 2010; Lin and Xu, 2013). Several Streptomyces species, such as Streptomyces
violaceus, Streptomyces olivaceoviridis, Streptomyces scabies, S. griseus, S. rimosus, S.
exfoliatus, Streptomyces viridis, Streptomyces coelicolor and S. lividans, producing IAA
showed the ability to improve plant growth by increasing seed germination, root
elongation and plant dry weight (Aldesuquy et al., 1998; Tokala et al., 2002; El-
Tarabily et al., 2008; Khamna et al., 2010; Goudjal et al., 2014). In 1998, Aldesuquy et
al. reported stimulated growth and yield of wheat when treated with culture filtrates of
S. olivaceoviridis containing IAA. Later, El-Tarabily, (2008) reported the ability of
Streptomyces spp., possessing IAA activity, to promote plant growth by enhancing seed
germination, root length, and root weights in tomato plants. Khamna et al. (2010)
observed similar results when maize and cow pea seeds were treated with IAA
containing culture filtrate of Streptomyces CMU-H009, a rhizospheric isolate.

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Streptomyces strains (isolated from internal tissues of Azadirachta indica) which


produce high quantity of IAA have been reported to promote the growth of tomato
plants (Verma et al., 2011). Two years later, Lin and Xu (2013) documented the plant
growth promoting potential of IAA producing endophytic Streptomyces En-1, which
resulted in a significant increase in biomass of Arabidopsis plantlets as compared to non
IAA producing streptomycete strains. Recently, Goudjal et al. (2013, 2014) also
observed that treatment of tomato seeds with the culture supernatant of Streptomyces sp.
PT2 containing IAA resulted in enhanced seed germination and root elongation.

2.3.2 Siderophore production

Iron is a vital element for plant growth. “Despite its relative abundance in
nature, the amount of bioavailable iron is very limited (about less than 10-18 M) because
atmospheric oxygen rapidly oxidizes iron into sparingly soluble ferric oxyhydroxides”
(Wang et al., 2014). Plants and microorganisms can readily uptake iron in Fe2+ which is
the more soluble form (Francis et al., 2010). Microorganisms having capability to
reduce Fe3+ to Fe2+ improves plant growth by increasing the bioavailability of iron in
the plant rhizosphere. In 2007, Valencia-Cantero et al. reported an actinobacterial
strain, Arthrobacter maltophilia which reduced Fe3+ to Fe2+ and thereby stimulated
growth of common bean by improving the bioavailability of iron in soil bean.

Additionally, microorganisms also produce siderophores which are low


molecular weight, 200–2,000 Da, chelating agents with very high affinity for Fe+3, and
play an important role in extracellular solubilization of iron from minerals to make it
available to microorganisms (Schalk et al., 2011). Siderophores are secreted by
microorganisms under conditions of limited iron availability to solubilize environmental
iron (Haas, 2003). Therefore, siderophores produced by microorganisms in the plant
rhizosphere can improve the uptake of iron by plants. Jurkevitch et al. (1986) reported
the plants ability to use catechol-type siderophores, hydroxamate-type siderophores, and
mixed-type siderophores produced by root-colonizing bacteria. Streptomyces spp. are
known to produce hydroxamate and catechol types of siderophores (Tokala et al., 2002;
Gangwar and Kataria, 2013). Tokala et al. (2002) revealed plant growth-promoting
ability of an antagonistic strain, S. lydicus strain WYEC108, in the absence of pathogen

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Review of Literature

stress and attributed that it may be due to production of hydroxamate-type siderophores


and/or other plant growth-promoting metabolites in the rhizosphere. Streptomyces
acidiscabies E13 was reported to produce hydroxamate siderophore and promote the
growth of Vigna unguiculata under nickel stress (Dimkpa et al., 2008). Sadeghi et al.
(2012) also observed enhanced iron uptake and growth promotion in wheat by a
Streptomyces strain which produced siderophores. Verma et al. (2011) related the
possible participation of siderophores in promoted seed germination, root and shoot
growth in tomato seedlings where seeds were inoculated with each of the three
endophytic Streptomyces spp. obtained from A. indica. There are many other reports
which documented the production of hydroxamate-type and catechols-type siderophores
by endophytic, root-colonizing and soil actinobacteria (Khamna et al., 2009; de Oliveira
et al., 2010; Palaniyandi et al., 2013b; Passari et al., 2015)

In addition to their role in plant growth promotion, several workers also reported
involvement of siderophores in indirect biocontrol of soil borne pathogens where they
inhibit their growth by competing for available iron in plant rhizosphere soils (Muller et
al., 1984; Tokala et al., 2002).

2.3.3 Phosphate solubilization

Phosphorus (P) is one of the important nutrients for plant growth and
development. It also plays role in energy transfer, photosynthesis, respiration, signal
transduction and macromolecular biosynthesis (Fernández et al., 2007). However, many
agricultural soils throughout the world are P deficient (Arcand and Schneider, 2006). It
is present in the form of insoluble metallic complexes (with Fe, Al, Mn etc.) in acidic
soil while in alkaline soil it reacts with Ca and form insoluble Ca3(PO4)2. As a result,
only a small portion of P is available for plant growth (1 mg/kg or less) (Hamdali et al.
2008a).

Microorganisms are able to solubilize insoluble phosphates in metallic


complexes or in hydroxyapatite and release free phosphates (Rodríguez and Fraga,
1999; Bojinova et al., 2008; Oliveira et al., 2009). Such microorganisms are able to
promote plant growth by providing the available forms of P to plants. Actinobacteria

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Review of Literature

such as Streptomyces, Micromonospora and Micrococcus (Hamdali et al., 2008a),


Kitasatospora (Oliveira et al., 2009) and Thermobifida (Franco-Correaa et al., 2010)
are among the most fascinating phosphate solubilizing microorganisms (PSM). Hamdali
et al. (2008a) reported the growth promotion of wheat plants in vitro as well as in situ
by rock phosphate-solubilizing actinobacteria. They reported production of
siderophores by the rock phosphate-solubilizing actinobacteria as a mechanism to
solubilize the phosphate. They described that siderophore chelation of phorphorus
adsorbants such as Fe, Al and Ca will result in increased phosphate solubilization.
Sadeghi et al. (2012) demonstrated that inoculation of soil with the phosphate
solubilizing Streptomyces sp. resulted in increased uptake of N, Fe, P and Mn in wheat
shoots and thus promoted its growth. Micromonospora aurantiaca and S. griseus
related strains originally isolated and screened for solubilization of rock phosphate were
shown to inhibit Pythium damping off and promoted the wheat growth in a soil
deficient in P (Hamdali et al., 2008c).

These results direct that plant growth-promoting attributes are beneficial in


addition to biocontrol traits to accomplish biocontrol in nutrient lacking soils. These
studies show that it is possible to use phosphate solubilizing Streptomyces and other
actinobacterial strains as natural and efficient amendments in the rhizosphere soils to
improve plant growth. Therefore, search for efficient phosphate solubilizers is going on.
Oliveira et al. (2009) found that 16.2 % endophytic actinobacterial isolates were
positive for phosphate solubilization. In another study, Franco-Correa et al. (2010)
evaluated actinobacterial strains for various plant growth promoting traits where three
strains viz. Streptomyces (MCR9), Streptomyces (MCR26) and Thermobifida (MCR24)
exhibited the highest phosphatase activity. Later in 2012, Hamdali et al. reported 29
isolates of actinobacteria which were able to weather rock phosphate in synthetic
minimal medium. Palaniyandi et al. (2013c) also observed phosphate solubilizing
ability in 48% of actinobacteria which were isolated from Yam rhizosphere. Recently,
Passari et al. (2015) and Farhat et al. (2015) demonstrated phosphate solubilizing
activity of 14 endophytic and 30 soil actinobacteria, respectively.

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Review of Literature

2.3.4 Nitrogen fixation

In addition to Fe and P, nitrogen (N) is another crucial plant nutrient required for
their growth and development. The N in the form of dintrogen (N2) gas is abundant in
the earth’s atmosphere (Muthukumarasamy et al., 2002) however, most of the soils are
deficient in utilizable N. The inadequate bioavailability of nitrogen and its requirement
for crop growth have spawned a huge N-based fertilizer industry worldwide (Westhoff,
2009; Santi et al., 2013). Plants use >50% of the applied fertilizers and this inefficient
use of nitrogen resulted in soil and underground water pollution with nitrate
accumulation which lead to health hazards and compromising agricultural
sustainability. Some organisms are able to use atmospheric nitrogen using a process
known as biological nitrogen fixation (BNF), which is the conversion of elemental N to
ammonia (NH3), a plant usable form (Lam et al., 1996; Franche et al., 2009; Wagner,
2012). Therefore, BNF constitutes an excellent sound source to substitute N fertilizer
(Pareek et al., 2002). According to Sellstedt and Richau (2013) among actinobacteria
some species of Arthrobacter, Agromyces, Corynebacterium, Mycobacterium,
Micromonospora, Propionibacteria and Streptomyces have nitrogen-fixing capability.
But till date, Frankia is the only most widely studied actinobacterium in terms of
nitrogen fixation. Frankia species live in symbiosis with a large number of dichotomous
plants (known as actinorhizal plants) and form nitrogen-fixing root nodules on their
roots (Palaniyandi et al., 2013b). Another nitrogen fixing actinobacterium Streptomyces
thermoautotrophicus (isolated from burning charcoal pile) is able to utilize N2 as a sole
source of nitrogen when grown under aerobic conditions at a temperature of 65 °C
(Gadkari et al., 1992). But it has not been reported for its involvement in formation of
root nodules in any plant or its direct positive influence on plants. In addition to these
bacteria, Valdés et al. (2005) reported nitrogen fixing property of endophytic bacteria
(that belonged to families Thermomonosporaceae and Micromonosporaceae) obtained
from roots of Casuarina equisetifolia. Franco-Correa et al. (2010) isolated thirty
actinobacterial isolates from rhizosphere of Trifolium repens L where 10 isolates
showed their ability to grow on nitrogen free medium.

2.3.5 ACC deaminase

Stresses, both biotic as well as abiotic often limit the crop productivity. Under
these stress conditions, the level of ethylene production increases in plants, which lead

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Review of Literature

to negative impact on plant growth (Glick, 2005; Glick et al., 2007a). Plant growth
promoting microorganisms are well-known for their growth-promoting effects on
several plants using multiple mechanisms. Until recently, the production of
phytohormones by plant growth promoting bacteria was the most studied mechanism
and a lot of attention has been focused on role of IAA. However, in last few years it has
been found that number of plant growth promoting bacteria stimulate plant growth
through activity of enzyme ACC deaminase. This enzyme cleaves 1-aminocyclopropane
1-carboxylic acid, the ethylene precursor in plants, into ammonia and α- ketobutyrate,
thus lowering stress-ethylene level in the rhizosphere, and increase plant growth
(Jacobson et al., 1984; Glick, 2005; Glick et al., 2007b; Viterbo et al., 2010).

However, to date limited literature is available to demonstrate the potential of


ACC deaminase to promote plant growth by lowering the plant ACC levels in
actinobacteria. In their pot experiments, Jaemsaeng et al.
(http://anchan.lib.ku.ac.th/kukr/bitstream/003/25643/3/TNAR02033c.pdf) observed that
the mungbean plants inoculated with the wildtype ACC producer, Streptomyces sp.
GMKU 336 survived through flooding and salinity (100 mM NaCl) stresses and also
showed significantly improved root and shoot growth and leaf chlorophyll content as
compared to the growth of plants either un-inoculated or inoculated with the ACC
deaminase-deficient mutant strain. In 2007, Sziderics et al. reported significantly
reduced expression of osmotic stress-inducible genes such as CaACCO and CaLTPI in
ACC deaminase possessing endophytic Arthrobacter sp. strain EZB4 isolated from
pepper plants. Halotolerant nonstreptomycetes such as Arthrobacter nicotianae,
Corynebacterium variabile and Micrococcus yunnanensis exhibiting ACC deaminase
activity were reported to promote growth of Canola plants under salt stress condition
(Siddikee et al., 2010). In the same year, Francis et al. (2010) reported low endogenous
levels of ACC and low stress-ethylene accumulation in plants associated with
Rhodococcus spp. exhibiting ACC deaminase activity.

However, there are only two reports of ACC deaminase activity in Streptomyces
spp. El-Tarabily (2008) demonstrated the involvement of ACC deaminase in plant
growth promotion by Streptomyces filipinensis no.15 strain. The inoculation of tomato
plants with this strain significantly lowered ACC levels in shoots and roots and thereby
promoted the growth of the tomato plants. Recently, in 2014, Palaniyandi et al.

38
Review of Literature

observed ACC deaminase activity in six actinobacterial isolates, all belonging to the
genus Streptomyces. This shows that the distribution of ACC deaminase activity may
not be a common growth promoting trait among actinobacteria. They observed
significant increase in biomass and chlorophyll content and a reduction in leaf proline
content in tomato plants inoculated with Streptomyces PGPA39 (a ACC deaminase
producer strain) under conditions of salt stress as compared to control and uninoculated
salt-stressed plants.

2.3.6 Root colonization

Root colonization is related to bacteria which can colonize the whole root
system and survive during several weeks in the presence of the natural microflora
(Weller, 1988). Later, Baker (1991) stated that root colonization is the ability of a
microorganism to colonize the rhizosphere of developing roots when applied by seed
treatment. The root colonization plays an important role in antagonistic as well as plant
growth promoting activities of bacteria (Chin-A-Woeng et al., 2000; El-Tarabily, 2008;
Bouizgarne, 2013). There are reports which also documented the role of root
colonization by Streptomyces species in biocontrol and plant growth promotion.

In 1994, Kortemma et al. studied the root colonizing ability of S. griseoviridis (a


potent biocontrol agent) on turnip, rape and carrot. Yuan and Crawford (1995)
characterized the antagonistic potential of S. lydicus stain WYEC108. Application of S.
lydicus stain WYEC108 to the P. ultimum infested soils planted with cotton and pea
seeds enhanced the agronomic traits of the plants, by colonizing their emerging roots, as
compared to control plants. Seed inoculation with S. lydicus strain WYEC108
significantly increased the nodulation by Rhizobium species in pea plants from an
agricultural field in north Idaho, increasing iron and molybdenum assimilation as well
as root growth. S. lydicus also colonizes and then sporulates within the surface cell
layers of the nodules of a pea plant (Tokala et al., 2002; Seipke et al., 2012) and this
discovery of actinobacteria colonizing the surface of a root nodule demonstrated that
this phenomenon could be frequent in nature (Tokala et al., 2002). Later El-Tarabily
(2008) reported that plant growth promotion in tomato plants was more pronounced
where plants were treated with two rhizosphere-competent streptomycetes, S.
filipinensis and Streptomyces atrovirens isolates as compared to plants treated with non-
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Review of Literature

rhizosphere-competent isolate. Gopalkrishnan et al. (2015ab) demonstrated the effect of


six Streptomyces strains on growth promotion in chick pea. A remarkable degree of root
colonization by all the six Streptomyces strains was observed in scanning electron
microscopic analysis of chickpea roots and this ability of root colonization resulted in
enhanced growth of chickpea plants along with enhanced microbial biomass carbon,
dehydrogenase activity, total nitrogen, available phosphorous and organic carbon over
the un-inoculated control in the rhizosphere of mature crop.

2.4 COMMERCIAL PREPARATIONS FROM ACTINOBACTERIA AS


BIOCONTROL AND PLANT GROWTH PROMOTING AGENTS

Several commercial preparations derived from actinobacteria are available in the


agricultural market for use to protect crops from various different pathogens and to
promote plant growth (Table 2.3).

2.4.1 Mycostop

Mycostop is the first commercial biofungicide containing dried spores and


mycelium of S. griseoviridis strain K61 as active ingredients of the preparation. The
strain K61 was isolated from Sphagnum peat in Finland (White et al., 1990). Mycostop
marketed as wettable powder is used for the control of seed, root and stem rots, and wilt
caused by Fusarium, Alternaria and Phomopsis of container grown ornamentals,
vegetables and tree and forest seedlings. It has also shown suppression of Botrytis Gray
Mold and root rots of Pythium, Phytophthora and Rhizoctonia in the greenhouse.
Mycostop can also be used as a seed treatment for seed or soil-borne damping off and
early root rot of vegetables, herbs and ornamentals planted in the field or greenhouse
(http://www.planetnatural.com/wp-content/uploads/mycostop-label.pdf). It antagonizes
phytopathogenic fungi in at least two ways. First, by colonizing plant roots before the
disease organisms get there, it deprives them of space and nourishment. It also produces
several kinds of chemicals (antibiotics) that may attack the harmful fungi.

2.4.2 Rhizovit

Rhizovit® (formulated in Germany) is another commercial phytoprotectant


biofungicide containing spores of S. rimosus HR071. It can be used to control several
seed-borne and soil-borne fungi including Pythium spp., Phytophthora spp., Botrytis
sp., R. solani and A. brassicicola (Marten et al., 2001).
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Review of Literature

2.4.3 Actinovate®

Actinovate® is well-known commercial biocontrol product of S. lydicus strain


WYEC108 (Crawford et al., 2005; Elliott et al., 2009). It is formulated as water-
dispersible granules, which can be used as soil drench or seed treatment for the control
of soilborne pathogens (Pythium, Rhizoctonia, Verticillium, Phytophthora, Fusarium
and other root decay fungi) and as spray for foliar pathogens (Alternaria, Botrytis and
others). S. lydicus WYEC108 as active constituent control fungal pathogens through
many mechanisms like production of antifungal antibiotics and extracellular chitinase,
and colonization of plant roots and long term viablity in the soil (Yuan and Crawford,
1995; http://www.monsantobioag.com/global/us/Products/Documents/actinovatesp_
fungicide_bklet_usa_10_13062.pdf). Actinovate is marketed by Natural industries, Inc,
Housten, USA and this company also market several other products with the same
strain.

2.4.4 Thatch Control

S. violaceusniger YCED9 has been developed as biocontrol agent under a


commercial name “Thatch Control” for removal of thatch from lawn. Spores of S.
violaceusniger YCED9 formulated in powdered milk and zeolite have been shown to
protect the turfgrass from Sclerotinia homeocarpa and R. solani by producing
antifungal compounds and cell wall degrading enzymes (Trejo-Estrada et al., 1998ab).

2.4.5 ArzentTM

ArzentTM, a commercial biocontrol product is a mixture of four distinct


compatible strains of S. hygroscopicus (Jensen) Waksman and Henrici (Innovative
Biosystems, Inc., Moscow, ID). S. hygroscopicus produces antifungal compounds and
chitinase enzymes to inhibit the growth of phytopathogenic fungi. Hamby and Crawford
(2000) also tested its ability as plant growth promoting agent in the greenhouse. Radish
and carrot wet weights were found to be 13 and 19% greater with Arzent™ treatment as
compared to the untreated controls, respectively.

2.4.6 Validamycin

Validamycin (also called Validamycin A or Jingangmycin) is a non-systemic


biofungicide obtained from S. hygroscopicus var. limoneus. It is most effective against

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Review of Literature

soil borne pathogens and is used for the control of R. solani in rice (sheath blight of
rice), potatoes, vegetables, strawberries, tobacco, ginger and other crops. It is also used
to control damping off diseases in vegetable seedlings, cotton, sugar beets, rice and
other plants. It is applied as a foliar spray, soil drench, seed dressing, or by soil
incorporation. Since 1970s, validamycin had been widely used in China against the Rice
Sheath Blight and in Japan and Netherlands to control black scurf of potato caused by
R. solani (Kulik, 1996). The popular trade names of validamycin are Validacin and
Solacol. Validamycin is mainly used in China, Japan, Netherlands, Colombia and India.

2.4.7 Avicta and Agri-Mek

Abamectin under the trade names Avicta and Agri-Mec is commercially used as
nematicide and insecticide, respectively. Abamectin is a mixture of avermectins B1a (>
80%) and avermectin B1b (< 20%) derived from the soil bacterium, S. avermitilis.
Avicta is a contact nematicide used as seed treatment and immediately kills the
nematodes (eg. M. incognita, M. arenaria, M. javenica, Tylenchulus semipenetrans and
R. reniformis) upon contact and does not allow the nematode to feed or reproduce. Agri-
Mek is used as foliar spray to control insect and mite pests of a range of agronomic,
fruit, vegetable and ornamental crops.

Avermectins have been registered in several countries under the trademark


AVID®, VERMITEC®, AGRI-MEK® and AFFIRM® for use against a wide variety of
pests. AVICTA® alone or in combination with other pesticides is commercially used as
seed treatment in corn, cotton, beans, etc. to control a wide variety of plant parasitic
nematodes.

2.4.8 Spinosad

Spinosad, a commercial organic insecticide is a fermentation product of a soil


actinobacterium, S. spinosa. Spinosad is a mixture of two spinosoids, spinosyn A and
spinosyn D that are tetracyclic/macrolide compounds produced by S. spinosa. Spinosad
is highly active, by both contact and ingestion, to numerous insect species which
include Lepidopterans, Dipterans, some Coleopterans, termites, ants and thrips (Bret et
al., 1997). It is a neurotoxin targeting the nicotinic acetylcholine receptor and
apparently the GABA receptors (Salgado, 1997, 1998). Exposure results in cessation of
feeding followed later by paralysis and death.

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Table 2.3: Commercially available biocontrol agents, plant growth promoting agents and agroactive compounds derived from
actinobacteria (Palaniyandi et al., 2013b)
Product name Active Ingredient Application Country Reference
Actinovate® AG S. lydicus WYEC108 Biocontrol of fungal USA Elliott et al., 2009
pathogens
Actinovate® SP S. lydicus WYEC108 Biocontrol of fungal USA http://naturalindustries.com/commercial/Docs/2012/a
pathogens vspbrochure.pdf. Accessed 09 August 2013
Action Iron ® S. lydicus WYEC108 Biocontrol and plant USA http://naturalindustries.com/commercial/Docs/2012/a
growth promotion vspbrochure.pdf. Accessed 09 August 2013
Arzent™ Mixture of four separate, Biocontrol and plant Moscow Hamby and Crawford, 2002
compatible strains of S. growth promotion
hygroscopicus
Micro108® Seed S. lydicus WYEC108 Bicontrol and plant USA http://naturalindustries.com/commercial/Docs/2012/1
Inoculant growth promotion 08seedfactsheet.pdf. Accessed 09 August 2013
Micro108® soluble S. lydicus WYEC108 Biocontrol of fungal USA http://naturalindustries.com/commercial/Docs/2012/1
pathogens 08solublefactsheet.pdf. Accessed 09 August 2013
Thatch Control S. violaceusniger Biocontrol of fungal USA http://www.naturalindustries.com/retail/index.
strain YCED 9 pathogens php?option=com_content&view=article&id=
16&Itemid=7. Accessed 09 August 2013
'Rhizovit® S. rimosus Biocontrol of fungal German Marten et al., 2001
pathogens y
PH-D® Fungicide S. violaceusniger Biocontrol of fungal USA http://www.arysta-na.com/us-agriculture/products/
strain YCED 9 pathogens ph-d/overview.html. Accessed 09 August 2013
Mycostop® S. griseoviridis strain Biocontrol of fungal Finland Suleman et al., 2002
K61 pathogens

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Product name Active Ingredient Application Country Reference


YAN TEN Streptomyces saraceticus Biocontrol of fungal Taiwan http://www.yanten.com.tw/products-3_30633-
KH400 pathogens english.html. Accessed 09 August 2013
AFFIRMWDG Polyoxin D (produced by Fungicide for USA http://www.clearychemical.com/view_product.
Streptomyces cacoi var. turfgrass fungi php?id=4195&cat=24. Accessed 09 August 2013
asoensis)
Validacin/Solacol Validamycin A (S. Fungicide China,
hygroscopicus) Japan,
India
Biomycin Kasugamycin (produced Fungicide and India http://www.biostadt.com/crop-protection/
by S. kasugaensis) Bactericide biomycin.aspx. Accessed 09 August 2013
Omycin Kasugamycin (produced Fungicide and India http://www.biostadt.com/crop-protection/
by S. kasugaensis) Bactericide omycin.aspx. Accessed 09 August 2013
Kasumin™ Kasugamycin (produced Bactericide Canada http://www.arysta-na.com/ca-agriculture/products/
by S. kasugaensis) kasumin/overview.html. Accessed 09 August 2013
Keystrepto™ Streptomycin (produced Bactericide New Rezzonico et al., 2009
by S. griseus) Zealand
Avicta, Agri-Mec Abamectin (produced by Nematicide, USA Khalil, 2013
and Tervigo S. avermitilis) acaricide and
insecticide
Spinosad S. spinosa Insecticide USA http://www.aces.edu/extcomm/timelyinfo/entomolog
y/2012/May/organicInsecticides.pdf
Conserve® and S. spinosa Insecticide https://microbewiki.kenyon.edu/index.php/Saccharop
Entrust® olyspora_spinosa

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