review of literature - inflibnetshodhganga.inflibnet.ac.in/bitstream/10603/77504/7/07... · 2018....

Review of Literature

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ulberry (Morus spp. L.), the sole food plant of silkworm (Bombax

mori L.) is a perennial crop and intensively cultivated for its

leaves for feeding the silkworm larvae. Diseases are the major constraint in the

production of leaves in the development of healthy plants. There are many

diseases but root rot is the most dangerous disease due to its epidemic nature and

potential to kill plant completely. The disease is observed in almost all mulberry

growing areas. The rapid death of plats was observed by Fusarium oxysporum,

F. solani. The disease initially appears in few plants in isolated patches with

symptoms of sudden wilting and blackening of leaves starting from the shoot tips

leading to the death (Philip et al., 1997; Chowdary, et al., 2009). The disease is

reported throughout the year in all types of soil under different agro-climatic

conditions resulting in more than 30 % mortality of plants (Sharma et al.,

2003, 2009).

Restrictions on the use of chemical pesticides have been increasing and

will undoubtedly continue to do so in the foreseeable future. Disease management

through biocontrol may be part of an effective response to this challenge. The use

of antagonistic microorganisms is the best control of root rot disease of any crop.

Rhizosphere microorganisms have proved to be effective biocontrol agents against

root diseases of many crop plants (Weller, 1988; Meena et al., 2001), their

antibiotic production now recognized as an important factor in disease suppression

(Fravel, 1988).

M


6

Studies on rhizosphere

From the very beginning in the 19th

century, rhizosphere-research was

characterized by multidisciplinary approaches and paralleled diversification and

development of novel disciplines in natural sciences. The term ‘Rhizosphere’ was

introduced by Lorenz Hiltner (1904) as soil compartment, influenced by root

excretions with impact on activity of beneficial and pathogenic microorganisms. It

is closely linked with the development of soil microbiology and phytopathology.

Many microbial interactions, which are regulated by specific molecules/signals

(Pace, 1997). Many studies have demonstrated that soil-borne microbes interact

with plant roots and soil constituents at the root-soil interface (Bowen and Rovira,

1991, Barea et al., 2002).The region in the vicinity of the roots can be

distinguished into many microhabitats viz. sub-surface, rhizoplane, rhizosphere,

and non rhizosphere. Among them, rhizosphere and rhizoplane habitats play a

major role for interactions among the microbes and plant root to get nutrition,

growth and productivity of the plant. The term ‘rhizosphere-effect’ indicates the

overall influence of plant roots on soil microorganisms. The suitability of soil for

a crop depends not only on its chemical and physical properties but also on its

load of beneficial microflora. Because rhizosphere and rhizoplane zones of all

plants provide rich nutrients with the help of beneficial microbes particularly plant

growth-promoting microbes like Azatobacter spp. Azospirillum spp.,

Pseudomonas flourescens, Bacillus spp., Trichoderma spp., etc. by producing the

growth promoting substances, which increase the availability and uptake of the

nutrients. These microbes are also having the ability to aupress the soil-borne

plant pathogens (Pal and Jalali, 1998). Therefore, the rhizosphere and rhizoplane

are interesting habitats for the study of microbial activity due to their relevance to


7

crop production. Understanding the microflora with special regards to regards to

beneficial and pathogenic to mulberry in the habitats of rhizosphere and

rhizoplane may help to formulate / adopt an effective management for soil borne

disease. Hence in this chapter the related aspects are reviewed.

Mansfield and Brown (1985) observed the most of the Phyto-pathogenic

bacteria depend on colonization of the host plant for survival and over wintering.

These bacteria vary in their effectiveness as saprophytes and usually do not

survive in the soil in the absence of a plant. This has been established for

Agrobacterium tumefaciens, Pseudomonas solancerum. Other survive as

components of the rhizoplane microflora, including P. syringae pv. Syringae

(Blakeman and Fokkema, 1982). These workers noted the lack of information on

host specificity for colonization, in terms of pathogen growth on plant surfaces.

Successful disease establishment involves entry by variety of mechanisms and

four stages of interaction between the microbe and plant viz., avoidance of

resistance processes, establishment of nutritional relationships, colonization of

tissue and development of disease symptoms.

The Gram-negative bacteria are dominated in rhizosphere region (Rouatt

and Katznelson 1961) particularly by fluorescent pseudomonads (Vancura, 1980).

Due to their ability to colonize the plant root. The fluorescent pseudomonads have

frequently been considered as biocontrol agents against various root diseases

(Weller and Cook, 1983; Weller, 1988). The ability of an introduced organism to

colonize a root is less dependent on plant type than on the ability of the organism

to survive in a given soil and complete with the indigenous microflora (Nesmith

and Jenkins, 1985). Various soil factors have been shown to influence root

colonization. They are soil texture, soil water matric potential (Nesmith and


8

Jenkins,1985); soil nutrient levels and soil pH (Yuen and Schroth, 1986), soil

calcium levels (Kao and Ko, 1986) and indigenous soil bacterial populations

(Chao et. al. 1986; Yuen and Schroth, 1986). In view of the major role that the

soil properties exerton the colonization of roots by specific bacterial strains, it

may be necessary to develop different bacterial inoculation for various soil types

or geographical regions.

Distribution of soil microflora in rhizosphere and rhizoplane habitats

Growth and development of microbes in habitats like rhizosphere and

rhizoplane depend on the root exudates produced by host plants. The number of

increase / decrease microflora in rhizosphere region depends on the substrate

supply from the roots (Kloepper et. al., 1985). Various workers have reported that

the total number of microflora was higher in rhizosphere as compared to

rhizoplane and non-rhizosphere regions (Singh and Saxena, 1991; Bolton et. al.,

1993; Pal and Jalali, 1998). Several investigations were made to study the

importance of rhizosphere / rhizoplane microflora in relation to resistant /

susceptible varieties in certain crops. In susceptible cultivars, quantitative change

in rhizosphere microflora of crop plant alters to a great extent plants alters to a

great extent by the infection and development of soil borne pathogens while

rhizosphere of resistant cultivars colonized by more antagonistic microbes and

less with pathogenic microbes. They also observed that high microbial load was in

rhizosphere region compared to non-rhizosphere (Singh and Singh, 1982; Rao et.

al., 1990; Pandey and Upadhyay, 2000). Bhattacharya and Bora (1995) conducted

a study on rhizosphere soil of 5, 35 and 75 years old tea plants in different

seasons. Results revealed that fungi were found to dominate in autumn while

bacteria in rainy-summer and actinomycetes in spring. The highest rhizosphere /


9

non-rhizosphere value for fungi was in the 5 year old plants while bacterial and

actinomycetes were in 35 year old plants.

Rhizosphere & microbial antagonism against plant pathogens

In the early 1970s several researchers identified potential of microbial

populations in the minimizing the pathogen infection. Some soil-borne plant

pathogens including Fusarium, Gaeumannomyces, Rhizoctonia, Pythium and

Phytophthora are interested areas in this aspect. The groups of microorganisms

with antagonistic properties towards plant pathogens are diverse. Among the

prokaryotes, a wide range of bacteria such as Agrobacterium, Bacillus spp. (e.g.

Bacillus cereus, B. pumilis, and B. subtilis), Streptomyces and Burkholderia have

been shown to be effective antagonists of soil-borne pathogens. The most widely

studied bacteria by far in relation to biocontrol are Pseudomonas spp., such as P.

aeruginosa and P. fluorescens, which are probably amongst the most effective

root colonizing bacteria. Among the eukaryotes, there are a variety of fungal

species and isolates that display antagonistic properties and have been applied in

biocontrol, but the ubiquitous Trichoderma spp. clearly dominate. Direct effects

on the pathogen include competition for colonization or infection sites. An

effective biocontrol agent often acts through the combination of several different

mechanisms (Whipps, 2001). Rhizobacteria from the genus Pseudomonas provide

an excellent example of a combination of multiple mechanisms for effective

biocontrol including direct antagonism and induction of plant resistance.

Pseudomonas spp. produces several metabolites with antimicrobial activity

towards other bacteria and fungi (Hass and Keel, 2003). Indeed, the first clear-cut

experimental demonstration that a bacteria-produced antibiotic could suppress

plant disease in an ecosystem was made by Thomashow and Weller (1988). Using


10

an elegant genetic approach, they demonstrated the direct correlation between the

production of a phenazine antibiotic by a fluorescent Pseudomonas spp. and its

biocontrol activity against take-all disease of wheat. Competition is another key

factor in the antagonistic properties of Pseudomonas spp. In addition to

competition for substrates (Couteaudier and Alaboubette, 1990), research on the

siderophores produced by Pseudomonas spp. (pyoverdine, pyochelin) has shown

the involvement of siderophore-mediated competition for iron in the control of

Fusarium and Pythium in soils (Duijff et al., 1994; Raaijmakers et al., 1995).

Another well-studied example illustrating a combination of mechanisms for

successful antagonism of plant pathogens is provided by the filamentous fungus

Trichoderma spp. These ubiquitous soil fungi are well-known for their

effectiveness in controlling a broad range of phytopathogenic fungi such as

Rhizoctonia solani, Pythium ultimum, and Botrytis cinerea. The direct

mechanisms involved in this protective effect include competition, antibiosis

(Howell, 1998), and mycoparasitism (Jeffries, 1997). Trichoderma grows towards

the fungal pathogen and releases toxic compounds (e.g. the antibiotics gliotoxin,

gliovirin, and peptabiols) and a battery of lytic enzymes, mainly chitinases,

glucanases, and proteases. These enzymes facilitate penetration into the host by

Trichoderma and the utilization of the host for nutrition (Lorito et al., 1996)

Direct evidence for the role of cell-wall degrading enzyme in biocontrol in vivo

comes from studies utilizing mutant strains over-expressing or lacking a particular

enzyme, of transgenic plants expressing these enzymes (Baek et al., 1999; Lorito

et al., 1998; Mendoza-Mendoza et al., 2003; Pozo et al., 2004). In addition, recent

studies indicated the importance of the induction of plant defense mechanisms in

biocontrol by Trichoderma (Harman et al., 2004). Several reports show the


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potential of combining different biocontrol agents with different disease-

suppressive mechanisms in the field (de Boer et al., 1999, 2003). The

development of appropriate combinations should provide a higher level of plant

protection, a wider range of effectiveness and a reduction of variability in the

results. Thus, the optimal use of the antagonistic properties of the microbiota will

result in a more effective and more reliable biocontrol of plant pathogens, and

constitutes a very promising research area.

Biocontrol potential of Trichoderma spp.

For about 70 years, Trichoderma spp. has been known to be able to

attack other fungi, to produce antibiotics that affect other microbes, and to act as

biocontrol microbes. There are many reports of Trichoderma spp. used as

biocontrol agent against different plant pathogens shortly reviewed in Table 2.1.

Biocontrol of crop disease by bacteria

Many bacteria are potential to restrict the growth of plant pathogens,

among them Pseudomonas spp. is a common. Some P. fluorescens strains present

biocontrol properties, protecting the roots of some plant species against parasitic

fungi such as Fusarium or Pythium. Now-a-days, it is used a biocontrol agent

against wilts and root rots diseases of many crops provides a short list of recently

referred review biocontrol importance of Pseudomonas and Bacillus spp. in

Table 2.2

Role of actinomycetes in biocontrol of plant pathogens

Whipps and Lumsden (1991) applied Streptomyces griseoviridis against

Pythium spp. a causal agent of damping-off of many crops. The in vitro studies

showed that an 80% concentration of the culture filtrate of either Streptomyces

pulcher or S. canescens significantly inhibited spore germination, mycelial growth


12

and sporulation of Fusarium oxysporum f.sp. lycopersici, Verticillium albo-atrum

and Alternaria solani (El-Abyad, 1993). The actinomycete S. lydicus WYEC108

showed strong antagonism against Pythium ultimum and Rhizoctonia solani (Yuan

and Crawford, 1995). Kurtboke (2000) stated the higher antagonistic activity of

Streptomyces spp. and Micromonospora spp. against Rhizoctonia solani and

Phytophthora cinnamomi and P. drechsleri in Australia. Streptomyces are one of

the most attractive sources of biological control (Shahidi, et al., 2004). Soybean

seeds were coated with Actinoplanes missouriensis, A. utahensis,

Amorphosporangium auranticolor, Micromonospora sp., and Hyphochytrium

catenoides survived greater than those from uncoated seeds. It suggests that the

hyperparasites had reduced Phytophthora glycinea inoculum in soil (Filonow and

Lockwood 1985). Pythium ultimum, which causes root rot and damping-off of

many floricultural crops. Strains of Actinoplanes spp. that are hyperparasites of

oospores were evaluated for their biological control of Pythium root rot of plants

grown in a greenhouse (Filonow and Dole, 1999). El–Tarabily (2000) isolated 94

Streptomycetes and 35 non-streptomycete actinomycetes were obtained and

examined in vitro for their ability to suppress the growth of Sclerotinia minor, a

pathogen causing basal drop disease of lettuce. The three most suppressive

isolates were showed antifungal activity as well as their ability to colonize the

roots and rhizosphere of lettuce. The three isolates, Serratia marcescens, S.

viridodiasticus and Micromonospora carbonacea, significantly reduced the

growth of S. minor in vitro, and produced high levels of chitinase and [beta]-1,3-

glucanase. Streptomyces viridodiasticus also produced antifungal metabolite(s)

that significantly reduced the growth of the pathogen in vitro. Bressan (2003)

observed the effectiveness of two Streptomyces spp. against Aspergillus spp.,


13

Curvularia lunata and Drechslera maydis. Yasuhiro Igarashi (2004) investigated

new bioactive compound ‘fistupyrone’, from Streptomyces sp. TP-A0569 an

inhibitor of spore germination of Alternaria brassicicola was isolated and purified

which is completely inhibits the infection of A. brassicicola cause of black leaf

spot of cabbage. Cedarmycin, an antifungal butyrolactone isolated from

Streptomyces sp. TP-A0456 and used against Candida glabrata with the MIC of

0.4 mg/ml. Tahtamouni, et al., (2006) controlled Sclerotinia sclerotiorum

biologically by application of 70 different Streptomyces spp. Ana Cristina et al.,

(2006) studied leaf spot pathogen and its management of Yam (Dioscorea

cayennensis) produced by Curvularia eragrostide and Colletotrichum

gloeosporioides. The effect of six actinomycete namely S. thermotolerans, S.

griseus subsp. griseus, Streptomyces sp. N0035, S. purpurascens and two isolates

identified as Streptomyces sp. were used against pathogen. There was significant

interaction between the actinomycete isolates and the phytopathogenic fungi. The

actinomycete strains evaluated in this study considered as potential biological

agents for controlling phytopathogenic fungi associated with leaf spot diseases.

Soil actinomycetes including 178 isolates were assayed for assessing antagonistic

activity against Pythium aphanidermatatum. Amongst them 43 isolates were

effective but two strains are showed high antifungal activity in agar disc and well

diffusion method (Sharifi, et al., 2007). Ha and Huang (2007) used Streptomyces

spp. with crab shell powder against Fusarium oxysporum f. sp. tracheiphilum and

increased populations of antagonistic rhizosphere microorganisms including fungi,

bacteria and actinomycetes; thereby reduced severity of Fusarium wilt of

asparagus bean and promoted growth and nodule formation of this crop.

Indigenous actinomycetes Streptomyces hygroscopicus (strain SRA 14) isolated


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from rhizosphere soils were assessed for in vitro antagonism against

Colletotrichum gloeosporioides and Sclerotium rolfsii. It has a potential antagonist

against both plant pathogenic fungi. The strain SRA14 highly produced

extracellular chitinase and β-1,3-glucanase during the exponential and late

exponential phases, respectively (Prapagdee, et al., 2008). Loqman (2009) isolated

142 different actinomycete strains from rhizosphere soil of Vitis vinifera for the

evaluation against five fungi namely Pythium ultimum, Fusarium oxyysporum f.

sp. albedinis, Sclerotium rolfsii, Verticillium dahliae and Botrytis cinerea. Results

showed that 24 isolates had an in vitro inhibitory effect toward at least 4 of the

indicator fungi, but only 9 inhibited all these phytopathogens. The findings

indicated the potential of actinomycetes for the biological control of

Botrytis cinerea.

Management by fungicides

Fungicides, despite some limitations serve as an important method of

controlling several crop diseases including chickpea. There were many reports of

application of fungicides against plant pathogens including Fusarium oxysporum.

Mussa and Abdulsalam (1991) used carbendazim, dithane M- 45 and

dithiocarbmate against Alternaria alternate and F. oxysporum causing per-

emergence damping -off of chickpea. Carbendazim was significantly effective.

Benomyl, carbendazim, thiophanate methyl, thiram, vitavax and captan were used

by Kumar and Dubey (2001) against F. solani causing collar rot of pea. They

were observed the synergistic effects with bioagents. Benomyl, captan and

carbendazim totally inhibited the growth of pathogen. Synergistically

Trichoderma harzianum + captan influenced the incidence of pathogen Thind et

al.,(2002) effectively managed the black scurf of potato caused by Rhizoctonia


15

solani. Dutta and Das (2002) applied thiram and dithane M-45 against Sclerotium

rolfsii, collar rot of French bean caused by R. bataticola managed integratedly

with biocontrol against T. viride and with thiram, vitavax and captaf by Dubey

(2002). The web blight of winged bean (R. solani) controlled by vitavax,

carbendazim, blitox-50, contaf, tilt,captan,SAAF, kavach, dithane M- 45, thiram

and thiophanate methyl by Kumar and Dubey (2002). Use of fungicides with plant

extracts decreases the severity of development of fungicide resistance (Singh and

Bhat, 2002). Formulated neem leaf extracts with tridimefon, mancozeb,

carbendazim and hexaconazole, helps in successful management of rust and leaf

spot of groundnut. Synergistic effect of pesticides increases the potential of

fungicide against pathogen Matco+phorate, Bavistin+ phorate, COC+phorate,

Mat+ COC+ Bavistin +Phorate helps in decreasing the root and rhizome rot of

ginger caused by pythium spp., Fusarium spp. and Phytophthora spp. Rajan et al.,

(2002). Mathur et al., (2000) studied integration of soil solarization and pesticides

effect against rhizome rot of ginger. They have applied ridomil in complex of

pathogen.Sclerotium rolfsii is important plant pathogen causing collar rot to

Elephant’s foot yam (Amorphophallus campanulatus). Various treatments of

captan + T. harzianum, captan + Bacillus subtilis were effective against pathogen

(Gogoi et al., 2002). There were many reports of application of fungicides in

management of crop diseases; some recent quotations were discussed in

the Table 2.3.

Physiology of Fusarium spp.

The role of different nutritional factors on the growth of F. oxysporum in

survival to develop cultural disease management practices. The suitable nutritional

sources favoring the mycelial growth. The present studies revealed the growth of


16

F. oxysporum on the various nutritional elements. Many workers studied this

aspect. Sowmya (1993) amongst different carbon sources tested against

F. oxysporum f.sp. cubense, glucose was the best carbon source for isolates I & II.

Glucose, xylose and D-galacturonic acid, carboxymethyl cellulose, xylan and

pectin were used by Steinberg et al., (1999) to study F. oxysporum pathogenic on

tomato. Carbon utilization was best on sucrose followed by maltose, starch,

glucose, fructose and cellulose successively by F. oxysporum causing cotton wilt

(Naim and Sharoubeem, 1963). The fungus may convert certain forms of complex

carbon compounds into simple form, which may be readily metabolized (Bais et

al., 1970). Fructose, mannose and galactose are needed for the growth of F. solani

(Schuerger, et al., 1993). Desai et al., (1994) recorded that, the carbon sources

supported better growth of race one, except maltose and succinic acid which

supported good growth of race three of F. oxysporum f .sp. ciceri. Nitrogen is an

important component required for protein synthesis and other vital functions. The

study revealed that the maximum growth of the pathogen was observed in calcium

nitrate, magnesium nitrate, potassium nitrate and urea. whereas magnesium

nitrate, calcium nitrate were also proved to be effective. The results are in

agreement with Naim and Sharoubeem (1963) regarding use of ammonium nitrate

to F. oxysporum causing cotton wilt. Out of the 10 nitrogen compounds tested

against F. oxysporum f.sp. elaeidis; good growth and sporulation were recorded

on sodium, ammonium and potassium nitrates, peptone and DL-leucine

(Oritsejafor, 1986). While moderate growth of the fungus was recorded on

ammonium sulphate, calcium nitrate, L-asparagine and DL-aspartic acid,

sporulation in these compounds was poor and ammonium sulphate induced

chlamydospore formation (Naim and Sharoubeem, 1963). The results are in


17

agreement with the reports of Bhatnagar et al., (1968) in case of F. oxysporum

f.sp. aurentifoliae which showed good growth on D-leucine and aspargine. Patel

(1990) reported that aspargine supported maximum mycelial growth of F. solani.

High nitrogen levels, which decreased disease severity, increased the protein

content in leaf tissues. Of 17 amino acids only proline content increased with

increasing nitrogen supply (Sarhan et al., 1982). Growth of F. solani, F.

avenaceum, and F. oxysporum on an agar medium minus K. salts was retarded; P

deficiency prevented sporulation. KaNO3 and Ca (NO3)2 were most favourable,

while NH4Cl and (NH4)2SO4 inhibited growth (Korobeinikov, 1960). Prasad

(1972) studied the effect of vitamins on sporulation in F. oxysporum and F.

moniliforme v. subglutinansm. Thiamine, biotin, inositol are selective in

accelerating macro-conidial production in F. moniliforme.The effect of eight

water-soluble vitamins on germination, germ-tube extension, growth, and

sporulation of F. oxysporum f.sp. vasinfectum was studied by El-Abyadm and

Ramadan (1979). Among the vitamins used, the fungus appeared to be highly

sensitive to thiamine and pyridoxine, moderately sensitive to inositol and

pantothenate, and least affected by folic acid. Role of oxides could be attributed to

killing the fungus by heat or mineral ash arising from soil burning. Sun and Huang

(1985) worked with Fusarial wilt in sandy soil and used mineral ash composed of

calcium oxide. This results are similar with our work. F. oxysporum var.

nicotianae grew well with sucrose and ammonium nitrate, potassium, phosphorus,

magnesium, sulfur and calcium. In addition to the micronutrients; iron, zinc,

copper, manganese, and molybdenum (Steinberg, 1950).


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Effect of different media

An understanding of the role of different media and environmental

conditions on the infection and survival of Fusarium oxysporum is necessary to

develop cultural disease management practices. There were many reports on

media study. Ingole (1995) who reported that PDA and Richard’s agar supported

best mycelial growth of F. udum. Jamaria (1972) also reported maximum growth

and sporulation of F. oxysporum f. sp. vanillae on potato dextrose agar, Richard’s

agar and Czapek’s Dox agar. Khare et al., (1975) reported maximum growth of

Fusarium oxysporum f. sp. lentis on PDA followed by lentil extract and Richard’s

agar. Anjaneya Reddy (2002) observed maximum growth of F. udum on

Richard’s agar and potato dextrose agar. Gangadhara, et al., (2010) studied effect

of temperature on growth of F. oxysporum f. sp. vanillae isolates. The fungus

showed best growth on Richard’s agar and potato dextrose agar media. Maximum

growth was at 250C after seven days of inoculation, which was reduced drastically

below 150C and showed zero growth at 40oC. The most suitable pH level for

growth of fungus was 5.0 and 6.0. Recently Imran Khan et al., (2011) studied

effect of media on F. oxysporum f.sp. ciceri and found that PDA is best for the

growth of different isolates.

Effect of different pH

pH of soil pays important role in pathogenesis. The findings of Moore

(1924) who reported that two strains of Fusarium coeruleum could tolerate a pH

range of 3.0 to 11.0. The studies conducted by Jamaria (1972) on F. oxysporum f.

sp. nivium indicated that, as the pH decreases or increases from the optimum, the

rate of amount of growth gradually decreases. Gangadhara, et al (2010) studied

effect of pH levels on growth of F. oxysporum f. sp. vanillae isolates. The fungus


19

showed best growth pH at 5.0, Least growth of all the isolates was recorded at 9.0

pH. Imran Khan et al., (2011) showed optimum pH for growth of F. oxysporum

f.sp. ciceri ranged from 6.5 to 7.0.

Effect of different temperature:

Studies conducted by Chi and Hansen (1964) indicated that Fusarium

solani isolates grew well at higher temperature of 280C. the fungus grew at the

temperature range of 10–350C. However, growth of the fungus was drastically

reduced below 150C and started to decline above 30

0C and become zero at 40

0C,

as these temperatures did not favour for growth of the fungus. It was observed that

at 250C and 30

0C, the fungus attained the maximum growth 76.8 and 85.4 mm

while at 250C, it was 59.3 mm after seven days of inoculation. Soil temperature

relationship indicated that suitable temperature for development of chickpea wilt

is 25-300C. Gupta et al., (1986) reported similar findings regarding temperature

requirements to this fungus. These studies are in confirmation with Anjaneya

Reddy (2002) who reported that growth of 40 isolates of F. udum differed in their

temperature requirement which varied from 200C to 35

0C. The effects of

temperature of F. oxysporum f. sp. ciceris was studied by Landa et al.,

(2001).They found the disease development was greater at 25°C compared with

20 and 30°C. Scott et al., (2010) studied effect of temperature on Fusarium wilt

of lettuce (Lactuca sativa), caused by F. oxysporum f. sp. lactucae, were observed

to increase from 10°C up to an apparent maximum near 25°C. Results are in

confirmation with Imran Khan et al., (2011) showed the F. oxysporum f.sp. ciceri

grew highest at 300C.


20

Molecular characterization by RAPD

Fungi are a large group of with diversified population. The genus

Fusarium is one of the most important in plant pathogen often causing wilt in

different crop plants (Booth, 1971). Fusarium oxysporum Schlecht has a greater

variability of species within the genus Fusarium populations possessing

pathogenic and non pathogenic form species (f. spp.) for one or other groups of

hosts (Booth, 1971). Molecular techniques are important aids in taxonomy and

classification of isolates of Fusarium and have supported the species

identification. These techniques have also allowed the analysis of the variability of

DNA by molecular markers. They have long been used to characterize populations

and also assess levels of genetic diversity and inter-and intra-specific phylogenetic

relationships, including identifying particular races and pathotypes. More recently,

the advent of PCR-based techniques presented a new option to the use of

molecular markers. The technique was developed in the mid-80 (Mullis and

Faloona, 1987) and achieved widespread and extensive use in many areas of

biology almost immediately (White et al., 1989). The technique involves the in

vitro enzymatic synthesis of millions of copies of a specific segment of DNA in

the presence of DNA polymerase and specific primers. These primers delimit the

sequence of double-stranded DNA to be amplified, whose results are millions of

identical copies (Mullis and Faloona, 1987, White et al., 1989). The RAPD

technique, developed by Williams et al., (1990) and Welsh and Mc Clell (1990),

involves the simultaneous amplification of several regions in the genome using

primers of arbitrary sequence, and has been used for genetic, taxonomic and

ecological studies of several fungi (Vilarinho et al., 1995; Fungaro et al., 1996;

Muthumeenakshi et al., 1998; Abbasi et al., 1999; Paavanem-Huhtala et al.,


21

2000).In addition to analyzing the genetic variability, these markers can determine

phylogenetic relationships, but also assist in identification of a particular race or

pathotype. There is also the possibility of being closely associated with avirulence

genes, and collaborate on cloning of genes (Majer et al., 1996). In addition, have

the advantage of being generated regardless of the phenotype and reveal highly

informative polymorphisms in DNA sequences of organelles and nucleus. The

genetic variability in isolated form species of F. oxysporum has also been assessed

using the RAPD technique (Assigbetse et al., 1994, Bentley et al., 1995; Manuli et

al., 1993, Nelson et al., 1997; Woo et al., 1996; Woudt et al., 1995, Wright

et al., 1996).

Role of actinomycetes in biocontrol of plant pathogens

Whipps and Lumsden (1991) applied Streptomyces griseoviridis against

Pythium spp. a causal agent of damping-off of many crops. The in vitro studies

showed that an 80% concentration of the culture filtrate of either Streptomyces

pulcher or S. canescens significantly inhibited spore germination, mycelial growth

and sporulation of Fusarium oxysporum f.sp. lycopersici (El-Abyad, 1993).

Kurtboke (2000) stated the higher antagonistic activity of Streptomyces spp. and

Micromonospora spp. against Rhizoctonia solani and Phytophthora cinnamomi

and P. drechsleri in Australia. Soybean seeds were coated with Actinoplanes

missouriensis, A. utahensis, Amorphosporangium auranticolor, Micromonospora

sp., and Hyphochytrium catenoides survived greater than those from uncoated

seeds (Filonow and Lockwood 1985). Pythium ultimum, which causes root rot and

damping-off of many floricultural crops. El–Tarabily et al., (2000) isolated 94

Streptomycetes and 35 non-streptomycete actinomycetes were obtained and

examined in vitro for their ability to suppress the growth of Sclerotinia minor.


22

Bressan (2003) observed the effectiveness of two Streptomyces spp. against

Aspergillus spp., Curvularia lunata and Drechslera maydis. Yasuhiro Igarashi

(2004) investigated new bioactive compound ‘fistupyrone’, from Streptomyces sp.

TP-A0569 an inhibitor of spore germination of Alternaria brassicicola was

isolated and purified which is completely inhibits the infection of A. brassicicola

cause of black leaf spot of cabbage. Cedarmycin, an antifungal butyrolactone

isolated from Streptomyces sp. TP-A0456 and used against Candida glabrata with

the MIC of 0.4 mg/ml. Tahtamouni, et al., (2006) controlled Sclerotinia

sclerotiorum biologically by application of 70 different Streptomyces spp. Ana

Cristina (2006) studied leaf spot pathogen and its management of Yam (Dioscorea

cayennensis) produced by Curvularia eragrostide and Colletotrichum

gloeosporioides. The effect of six actinomycete namely S. thermotolerans, S.

griseus subsp. griseus, Streptomyces sp. N0035, S. purpurascens and two isolates

identified as Streptomyces sp. were used against pathogen. There was significant

interaction between the actinomycete isolates and the phytopathogenic fungi. The

actinomycete strains evaluated in this study considered as potential biological

agents for controlling phytopathogenic fungi associated with leaf spot diseases.

Soil actinomycetes including 178 isolates were assayed for assessing antagonistic

activity against Pythium aphanidermatatum. Amongst them 43 isolates were

effective but two strains are showed high antifungal activity in agar disc and well

diffusion method (Sharifi, et al., 2007). Ha and Huang (2007) used Streptomyces

spp. with crab shell powder against Fusarium oxysporum f. sp. tracheiphilum and

increased populations of antagonistic rhizosphere microorganisms including fungi,

bacteria and actinomycetes; thereby reduced severity of Fusarium wilt of

asparagus bean and promoted growth and nodule formation of this crop.


23

Indigenous actinomycetes Streptomyces hygroscopicus (strain SRA 14) isolated

from rhizosphere soils were assessed for in vitro antagonism against

Colletotrichum gloeosporioides and Sclerotium rolfsii. It has a potential antagonist

against both plant pathogenic fungi. The strain SRA14 highly produced

extracellular chitinase and β-1,3-glucanase during the exponential and late

exponential phases, respectively (Prapagdee, et al., 2008). Loqman (2009) isolated

142 different actinomycete strains from rhizosphere soil of Vitis vinifera for the

evaluation against five fungi namely Pythium ultimum, Fusarium oxyysporum f.

sp. albedinis, Sclerotium rolfsii, Verticillium dahliae and Botrytis cinerea. Results

showed that 24 isolates had an in vitro inhibitory effect toward at least 4 of the

indicator fungi, but only 9 inhibited all these phytopathogens. The findings

indicated the potential of actinomycetes for the biological control of

Botrytis cinerea.

Application of vermicompost and oil cakes

There are many reports of application of vermicompost in agriculture

produce. Bhatnagar and Paitta (1996) promoted vermicompost for enhancement

of decomposition process in mulberry field. Angadi and Radder (1996) reported

that vermicompost increases beneficial soil microflora and reduces 50% chemical

fertilizers. Edwards and Steele (1997) applied earthworms in field to increase

grass yield. Boyle et al., (1997) observed 89% higher produce in grass yield

fertilized by earthworms. However Ranwar et al., (1997) applied vermicompost in

high production of grains. Venkatratnam (1997) reported buffering action of

earthworm helpful in high yield. Das et.al. (1997) applied vermicompost in

mulberry field for quality foliage.


24

Integrated disease management with application of different oil cakes like

neem and castor increase the population of biocontrol agents like Trichoderma

spp. Sharma (1999). Krishnamoorthy and Bhaskaran (1994) reported the enhanced

population of biocontrol agents due to neem oil cake into the soil Uphadhaya et.

al. (2004) reported that neem oil cake supported good growth and sporulation of

T. viride.

Use of plant extracts in plant disease management

Now-a- day’s integrated disease management of crop plants by using plant

extracts is upcoming area of research. Application of plant extracts has a potential

to avoid fungicide resistance in plant pathogens and reducing the cost of

production. There are many reports of use of plant extracts against plant

pathogens, which were shortly reviewed in Table 2.4


25

Table 2.1 Biocontrol of crop disease by Trichoderma spp.

Sr.

No.

Crop disease with pathogen Trichoderma spp. Author and year

1 Wilt of Pisum sativum

(F. oxysporum)

Trichoderma spp. Sharma (2011)

2 Diseases on chickpea

(Aspergillus flavus, A.

fumigates)

T. harzianum Agarwal et al.,

(2011)

3 Root rot of Tomato

( Rhizoctonia solani)

T. harzianum , T.

viride

Jaime et al.,(2009)

4 Anthracnose of pepper

(Colletotrichum capsici)

T. harzianum Ekefan et al.,(2009)

5 Tomato wilt

(F. oxysporum)

T. virens

Christopher

et al.,(2010)

6 Anthracnose of pear, apple,

cherry and tomato

(Colletotricum acutatum and C.

gloeosporioides )

T. harzianum

Gliocladium roseum

Živkovic et al.,(2010)

7 Mulberry root rot disease

(Fusarium spp.)

T. harzianum ,

T.viride

Dhahira and Qadri

(2010)

8 Root rot of sage

(F. solani and R. solani)

Trichoderma spp. Mallesh et al.,(2008)

9 Dry root rot of pigeonpea

( M. phaseolina)

T.virens (PDBC

TVS-2)

Lokesha and Benagi

(2007)

10 Peanut root rot

(F. solani )

Trichoderma spp. Gil et al.,(2008)

11 Cotton root rot

(F. oxysporum)

T. hamatum Kamal et al.,(2009)


26

Table 2.2 Use of bacteria as biocontrol agent against plant pathogens

Sr.

No. Crop disease with pathogen Bacterium Author and year

1 Root rot of soybean

(R. solani)

Bacillus

megaterium

Zheng and Sinclair

(2000)

2 Pigeon Pea wilt

(F. oxysporum)

B. brevis Bapat Sangita and

Shah (2000)

3 Root rot of angelica trees

(P. cactorum)

Enterobacter

cloacae, Serratia

ficaria

Okamoto Hiroshi et

al.,(2000)

6 Safflower root rot

(R. bataticola)

Pseudomonas

fluorescens

Prashanthi et al.,

(2000)

7 Root rot of Asparagus

(P. megasperma)

P. aureofaciens Godfrey et al.,(2000)

9 Collar rot of teak

(R. solani)

P. fluorescens Ramesh (2000)

11 Root rot of seasamum

(M. phaseolina)

P. fluorescens Karunanithi et al.,

(2000)

12 Root rot of plams

(P. ultimus, F. solani, F.

oxysporum )

P. ameofaciens Paulitz et al., (2000)

13 Sclerotium rot of sunflower

(Sclerotium rolfsii)

Pseudomonas spp. Rangeshwaran &

Prasad, (2000)

14 Collar rot of cucumber

( R. solani)

P. putida Ongena et al., (2000)

15 Blue mold and gray mold of

apples (Penicillium expansum

,Botrytis cinerea)

P. syringae, MA-4,

MB-4, MD-3b, and

NSA-6.

Zhou et al., (2001)

16 Bacterial wilt / Brown rot of

potato (R. solanacearum)

Bacillus spp. Dhanbir Singh and

Rana (2001)

17 Damping off of tomato

(P. aphanidermatum )

P. fluorescens Ramamoorthy and

Samiyaappan, (2001)

18 Fruit rot of chilli


P. fluorescens Prasad and

Kulshershtha,(2002)

19 Seedling blight of sunflower

(A. helinathi)

P. fluorescens Prasad and

Kulshreshtha (2002)

20 Bacterial blight of rice

(X. oryzae pv. oryzae)

P. fluorescens Manmeet Manav and

Thind (2002)

21 Dry root rot of chickpea

(R. bataticola)

P. fluorescens Sindham et al., (2002)

22 Bacterial blight of rice

(X. oryzae pv.oryzae)

B. subtilis Manmeet Manav and

Thind (2002)

23 Coconut wilt

(C. gloeosporioides and

Exserohilum rostratum)

Pseudomonas

fluorescens

Srinivasan (2003)

Contd…


27

Sr.

No. Crop disease with pathogen Bacterium Author and year

24 Basal rot of balsam

(S. rolfsii)

Bacillus spp. Girija and

Umamaheshwaran

(2003)

25 Damping off of true potato

(P. aphanidermatum)

B. subtilis Lakra (2003)

26 Root rot of groundnut

(M. phaseolina )

P. fluorescens Charita devi et al.,

(2003)

27 Pigeonpea and Chickpea wilt

in (F. udum and F.

oxysporium f.sp.ciceri)

P. aeruginosa Anjaiah et al., (2003)

28 Banded leaf and sheath blight

of maize (R. solani f.sp.

sasakii )

P. fluorescens Sivakumar and Sharma

(2003)

29 Chickpea wilt (F. oxysporum

f.sp. ciceri)

P. fluorescens Inam et al., (2003)

30 Floury leaf spot and fuscous

blight of rajmash bean

(X.axonopodis pv.phaseoli var

fuscans, Ramulariaphaseoli)

P. fluorescens

RPB14

Mondal (2004)

31 Sheath blight of rice

(R. solani)

P. fluorescens Singh and Sinha (2005)

32 Root rot of brinjal

(M. phaseolina)

P. fluorescens Bhattacharyya and

Ghosh (2006)

33 Chickpea wilt

(F. oxysporum f.sp.ciceri.)

Pseudomonas spp. Kaur et al.,(2007)

34 Pigeonpea wilt of

(F. oxysporum f.sp. cajani)

P. fluorescens Mahajan et al., (2007)

35 Damping off ,root rot and dry

rot of acid lime (Phytophthora

,Pythium spp. and F. soliani)

P. fluorescens Gade et al., (2008)

36 Root rot of chickpea

(M. phaseolina)

P. fluorescens Khan and

Gangopadhyay (2008)

37 Root rot of pea

(F.solani f.sp. pisi.)

P. fluorescens Negi et al.,(2008)

38 Bacterial wilt of chilli

(R.solanacearum)

P. aeruginosa Bora and Deka (2008)

39 Wilt of tomato

(F. oxysporum f. sp. radicis-

lycopersici )

Pseudomonas

chlororaphis

Puopolo et al. (2011)


28

Table 2.3 Management of crop diseases by fungicides

Sr.

No. Crop disease with pathogen Fungicide used Author and year

1 Leaf spot of turmeric


Indofil M-45, Blitox,

Hinosan, Kavach, Bavistin,

Topsin-M, Calixin, Kitazin

Narasimhudu and

Balasubramanian

(2002)

2 Storage fungi of pearl millet Captan, Apron , Thiram Jakhar et al., (2003)

3 Guava wilt (Fusarium spp.) Carbendazim Misra et al., (2003)

4 Stem rot of chickpea

(Sclerotinia sclerotiorum)

Benomyl , Carbendazim,

Chlorothalonil, Thiophanate

methyl, Propineb,

Manozeb,Ziram, Ofurace

Kumawat and Jain

(2003)

5 Web blight of Mung

(Rhizoctonia solani)

Captaf , Topsin –M, T. viride

+Vitavax, Vitavax+Captaf,

Vitavax+ Topsin

Dubey (2003)

6 Rot of brinjal

(S. sclerotiorum)

Antracol, Bayton, Benlate,

Captan, Daconil, Dithane M-

45, Ridomil gold, Tecto-60,

Topsin-M

Iqbal et al., (2003a)

7 Chickpea root rot

(Sclerotium rolfsii, R. solani,

F. solani, FOC )

Vitavax+ Gliocladium virens Tewari and

Mukhopadhyay

(2003)

8 Chickpea wilt

(Fusarium oxysporum f. sp.

ciceri)

Carbendazim, Thiophanate

methyl, Propiconzole,

Tebuconazole

Singh et al., (2004)

10 Web blight of Urdbean

(R. solani)

Tilt , Bavistin, Dithane M-45 Shailbala and Tripathi

(2004)

11 Root rot of strawberry

(S. rolfsii)

Bavistin, Dithane M-45,

STOP, Captan, SAAF,

Kavach, Companion

Bhardwaj and

Harender (2004)

12 Late blight of potato

(Phytophthora infestans)

Fenamidone , Secure,

Ridomil, Curzate, Antracol

Thind et al., (2004)

13 Root rot of sugerbeet

(S. rolfsii)

Chitosan , Cycocel,

Cycloheximide

Das and Raj (2004)

14 Guava wilt

(F. oxysporum f. sp. psidii )

Carbendazim, Carboxin John et al., (2006)

Contd…


29

Sr.

No. Crop disease with pathogen Fungicide used Author and year

15 Pea wilt complex

(Fusarium spp., Sclerotinia

spp.)

Carbendazim Kapoor et al., (2006)

16 Foot rot of rice

(F. moniliformae)

Bavistin, Benomyl, Emisan,

Tilt, Topsin –M

Bagga et al., (2007)

17 Gladiolus wilt

(F. oxysporum f. sp. gladioli)

Quintal ,SAAF,

Carbendazim, Thiophanate

methyl

Chandel and Tomar

(2007)

18 Aerial blight of soybean

(R. solani)

Carbendazim, Ipridion,

Hexaconazole, Thiram

Ria et al., (2007)

19 Pigeonpea wilt

(F. udum)

Benomyl, Thiram Gade et al., (2007)

20 Chickpea wilt

(F. oxysporum f. sp. ciceris)

Benomyl, Carbendazim Mukhtar (2007)

21 Chickpea wilt

(F. oxysporum f. sp. ciceris)

Thiram+ Carbendazim,

Thiram+ Captan,

Carbendazim

Jagtap and Sontakke

(2007)

22 Cumin wilt

(Fusarium spp.)

Carbendazim, Thiophanate-

methyl.

Gangopadhyay et

al.,(2009)


30

Table 2.4 Use of plant extracts against plant pathogens

Contd…

Sr.

No.

Plant used Against pathogen Author and year

1 A. sativum, Annona squamosa,

Vitex negundo

F. oxysporum f.

sp. ciceris

Sahayaraj et al.,

(2006)

2 Costus pisonis, Achilla

millefolium and Plectrantus

barbatus

Glomerella

cingulata,

Silva et al.,(2006)

3 Dioscorea spp. A. niger, A. flavus,

F. oxysporum,

Rhizopus

stolonifer

Okigbo and

Ogbonnaya (2006)

4 Lycopersicum esculentum. A. solani Vadivel and

Ebenezar (2006)

5 Azadirachta indica, A. sativum F. oxysporum f.

sp. lycopersici

Agbenin and

Marley (2006)

6 O. gratissimum and Aframomum

melegueta

A. niger, A. flavus,

F. oxsporium, R.

stolonifer,

Botryodiplodia

theobromae and

Penicillium

chrysogenum.

Okigbo and

Ogbonnaya,

(2006)

7 Cymbopogon citrates, Eucalyptus

camaldulensis, A. indica

C.graminicola, P.

sorghina and F.

moniliformae.

Somda et al.,

(2007)

8 A. indica; Ziziphus spina-christi

& Zygophylum coccineum

F. solanmi,

Haikal (2007)

9 Gonzalagunia rosea Candida albicans,

F. solani.

Nino et al., (2007)

10 Camellia sinensis A. brassicae Banerjee and

Laura (2007)

11 Cajanus cajan L. Sclerotium rolfsii Suryawanshi, et

al., (2007).

12 Acacia nilotica, Alstonia

scholaris, A. indica, E.

citriodora, Ficus

bengalenisis, Mangifera

indica, Melia azadirach and

Syzygium cumini

Alternata, F.

solani,

Cladosporium sp.

R. arrhizus, and

A. niger

Shafique et al.,

(2007)

13 A. indica, Datura metal, O.

sanctum, P.hysterophorus

F. oxysporum f.

sp. ciceri.

Mukhtar (2007)

14 Cassia alata and Dennetia

tripetala

S. rolfsii Eunice and Osuji

(2008)


31

Sr.

No.

Plant used Against pathogen Author and year

15 A. indica, Elaeis guineensis, Cola

natida, M. indica

R. stolonifer Nahunnara. (2008)

16 Adhatoda vasica, Jatropha

curcas, Sapindus emarginatus

and V. negundo

F. oxysporum f .sp.

melongenae

Siva et al., (2006)

17 A.indica, D. metal,

O.gratissimum,

C. domestica, Parthenium sp.

Lantana camara,

Solanum nigrum

A. alternata,

F. oxysporum, F.

udum

,Acrocylindrium

oryzae ,

Helminthosporium

sativum,

Phomopsis vexans

,Botrytis cinerea

and Rhizopus spp.

Lakpale et al.,

(2008)

18 A. indica, Tagetes erecta,

A. squamosa.

C. gloeosporioides Jat et al., (2008)

19 Desmostachya bipinnata F.oxysporum f.sp.

lycopersici

Srivastava and

Yadav (2008)

20 Citrullus colocynhis, Calotropis

procera, Nerium oleander,

Pergularia tomentosa

F.oxysporum f.sp.

albedinis.

Boulenouar et al.,

(2009)

21 Ocimum gratissimum, Brassica

oleracea and Ipomoea batatas

Ralstonia

solanacearum

Wagura et

al.,(2011)

22 Garlic, Clove, Dokudami,

Kumasasa, dandelion, Kusagi,

Yomogi, Ginkgo, marigold,

Lavender, Thyme, Hot pepper,

Ginger, Lemon basil.

P. expansum Ikeura

et al.,(2011)

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