1. INTRODUCTION

1. INTRODUCTION

1. INTRODUCTION Biodiversity refers to the variability of life all the living species of animals, plants and microorganisms on earth. According to Ha...

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1. INTRODUCTION

Biodiversity refers to the variability of life all the living species of animals, plants and microorganisms on earth. According to Hawksworth (2002), fungi are a major component of biodiversities, essential for the survival of other organisms and are crucial in global ecological processes.

Fungi being ubiquitous organisms, occur in all types of habitats and are the most adaptable organisms. The soil is one of the most important habitats for microorganisms like bacteria, fungi, yeasts, nematodes, etc.

The filamentous fungi are the major

contributors to soil biomass (Alexander, 1977).

They form the major group of

organotrophic organisms responsible for the decomposition of organic compounds. Their activity participates in the biodeterioration and biodegradation of toxic substances in the soil (Rangaswami and Bagyaraj, 1998). It has been found that more number of genera and species of fungi exist in soil than in any other environment (Nagmani et al., 2005). Contributing to the nutrient cycle and the maintenance of ecosystem fungi play an important role in soil formation, soil fertility, soil structure and soil improvement (Pan et al., 2008). Fungi take a very important position in structure and function of ecosystem. They decompose organic matter from humus, nutrients, assimilate soil carbon and fix organic nutrients. An intense study of the abundance and diversity of soil microorganisms can divulge their role in nutrient recycling in ecosystem.

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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1.1 Biological control of Pythium debaryanum in Chilli pepper(Capsicum annuum L.) Systematic Position Class

- Dicotyledons

Order

- Solanales

Family

- Solanaceae

Genus

- Capsicum

Species

- annuum

Capsicum (Capsicum annuum L.) is an important spices crop extensively cultivated throughout the tropics and Southern countries such as Bangladesh, India, Pakistan and Sri Lanka.

The genus Capsicum belongs to the family Solanaceae (Night shade). The members of the Solanaceae family are mostly herbs or undershurbs while some others are climbers. The family contains about 90 genera and nearly 3000 species (Vidhyarttie and Tripathi, 2002; Sterm, 2000). Capsicum is a crop that is widely cultivated because of its spicy nature and nutritional value. The crop accounts for a large portion of Vitamins A and C in many Nigerian diets. Heiser and Smith (1953) distinguished two Capsicum species cultivated as vegetables while varieties are all forms of either Capsicum annuum or C. frutescens. Capsicum annuum is not known in a wild state and species commonly cultivated are Capsicum annuum known as sweet pepper, bell pepper, cherry pepper and green pepper (Messraen, 1992).

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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Chilli pepper is better adopted to hot weather than sweet pepper, but it does not set fruit well when night temperatures are greater than 24°C. Optimum day temperatures for chilli pepper growth range from 20 to 30°C. When the temperatures falls below 15°C or exceeds 32°C for extended periods, growth and yield are usually reduced. Chilli pepper grows best in a loam or silt loam soil with good water-holding capacity, but can grow on many soil types, as long as the soil is well drained. Soil pH should be between 5.5 and 6.8. Small chillies are much hotter because, proportionally, they contain more seeds and veins than large specimens. Those seeds and membranes can contain upto 80 percent of the chillies capsaicin, the potent compound that gives chillies their fiery nature.

1.1.1. Common varieties Aleppo

Dundicut

Niora

Super chilli

Anaheim

Fresno

New Mexico

Tepin

Ancho

Guajillo

Pasilla

TienTsin

Bell pepper

Hungarian wax

Pepperoncini

Cascabel

Italian sweet pepper

Piquin

Cayenne

Jalapeno

Pimento

Chilaca

Japanese

Poblano

Chiltepin

Mirasol

Puya

Cubanelle

Macho chili

Sanaam

Dearbol

Mulato pepper

Serrano

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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Red chillies contain high amounts of Vitamin C and Carotene (Pro vitamin A). Yellow and especially green chillies (which are essentially unripe fruit) contain considerably a lower amount of both the substances. In addition, peppers are a good source of Vitamin B and Vitamin B6 in particular. They are very high in potassium, magnesium and iron. Their high Vitamin C content can also substantially increase the uptake of non-heme iron from other ingredients in a meal, such as beans and grains. India is the leading country in the world in chilli production with an area of 9,08,400 ha and the production of 9,70,800 tonnes of dry chillies. Recently, chilli is gaining greater importance in the global market because of its value – added products and diverse uses.

This important chillies suffer from many diseases caused by fungi, bacteria, viruses, nematodes and also abiotic stresses. Pythium species are essentially soil-borne pathogenic fungi, that cause seed rot and damping off of many crops including chilli and tomato (Shah-Smith and Burns, 1996).

1.2 Causal organism - Pythium debaryanum Systematic Position Class

:

Phycomycetes

Order

:

Pythiales

Family

:

Pythiaceae

Genus

:

Pythium

Species

:

debaryanum

Pythium species are fungal like organisms (Oomycetes), commonly referred to as water moulds, which naturally exist in soil and water as saprophytes, feeding on organic matter.

Some Pythium species can cause serious diseases on greenhouse

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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vegetable crops resulting in significant crop losses. Pythium infection leads to damping off in seedlings and crown and root rot in older plants.

The genus Pythium is a complex genus containing over 200 described species that occupy a variety of terrestrial and aquatic ecological habitats (Dick, 2001). Perhaps the most economically important members of this genus are plant pathogens (Hendrix and Campbell, 1973), many of which have a broad host range and cause losses by both pre and post emergence damping off (Erwin and Ribeiro, 1996), as well as by reduction in plant growth and yield, due to root rot (Vander Plaats-Niterink, 1981). Pythium damping off is a very common problem in fields and green houses, where the organisms kill newly emerged seedlings.

(Jarvis, 1992).

Pythium debaryanum parasitizes

seedlings of many plants cause a destructive disease known as damping off. The fungus live saprophytically in the moist, humus soil and infect the hypocotyl of seedlings. Thereafter they live as parasites.

1.2.1. Damping off The single term used to describe underground, soil line or crown rots of seedlings due to unknown causes is damping off. The term actually covers several soil borne diseases of plants and seed borne fungi. Pythium root rot (Pythium sp.) is similar to Rhizoctonia in that it causes damping off of seedlings and foot rot of cuttings. However, infection occurs in cool, wet, poorly drained soils, and by over watering. Infection results in wet odourless rots. When severe, the lower portion of the stem can become slimy and black. Usually, the soft to slimy rotted outer portion of the root can be easily separated from the inner core. Species of Pythium can survive for several years in soil and plant refuse.

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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1.2.2. Symptoms of Damping-off Seeds were infected as soon as moisture penetrates the seed coat or a bit later as the radicle begins to extend, all of which rot immediately under the soil surface (preemergence damping-off). This condition, results in a poor, uneven stand of seedlings, often confused with low seed viability cotyledons may break the soil surface and only looking seedlings may suddenly fall over (post-emergence damping-off). Infection results in lesions at or below the soil line. The seedlings will discolour or wilt suddenly, or simply collapse and die. Weak seedlings are especially susceptible to attack by one or more fungi when growing conditions are only slightly unfavourable. Damping-off is easily confused with plant injury caused by insect feeding, excessive fertilization, high levels of soluble salts, excessive heat or cold, excess or insufficient soil moisture, or chemical toxicity in soil.

1.2.3. Disease cycle Pythium spreads by forming sporangia, sack-like structures, each releasing hundreds of swimming zoospores. Zoospores that reach the plant root surface encyst, germinate and colonize the root tissues by producing fine thread-like structures of hyphae forming masses of mycelium. These hyphae release hydrolytic enzymes destroy the root tissue and absorb nutrients as a food source. Pythium forms Oospores and Chlamydospores on decaying plant roots which can survive prolonged adverse conditions in soil, greenhouse growing media and water, leading to subsequent infections.

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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1.2.4. Control of plant disease The edaphic environments of plants are inhabited by a wide range of microorganisms including various fungi, bacteria, mycorrhizae etc., which may be pathogenic or non pathogenic. Certain microbes produce compounds which affect the growth of other microbes and may sometime beneficial to the plants. Such microbes act naturally as biocontrol agents by producing certain compounds which act as growth promoting substances as well as plant protectants.

Thus they form a very good

alternative over chemical fertilizers.

1.2.5. Biological methods These methods aim at direct protection of plant from pathogens or at eradication or reduction of inoculum by using antagonistic microorganisms. Biological methods include reduction of pathogen inoculums by antagonist, which is achieved by the use of suppressive soil that contains many kinds of antagonistic microorganisms like Trichoderma, Aspergillus, etc.,

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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1.2.6. Mechanism of biological control agents A basic knowledge on the mode of action may reveal certain facts including identification of enzymes (or) metabolites that actually impart biocontrol activity to the antagonistic microorganisms under investigation, and can direct its use to study the biochemical aspect involved in the process to a great extent. 1.2.7. Trichoderma as a biocontrol agent Trichoderma is a green coloured, fast growing beneficial fungal species. It has multiple use in crop production as a biocontrol and decomposing agent. It also acts as a growth promoter by producing growth hormones. It controls the soil borne pathogens through either antibiosis (or) mycoparasitism or competition or harmful fungus. It acts against species like Fusarium, Phytophthora, Pythium and Botrytis etc. Biological control based on myco-parasitism and hyper-parasitism between some microbial organisms provide an alternative to chemical control. Several fungi such as Aspergillus flavus, A. ochraceus, Penicillium aurantiogriseum, Coniothyrium minitans, Alternaria alternata, Epicoccum purpurascens, Coniothyrium olivaceum, Gliocladium sp. and Trichoderma sp. (Royse and Ries, 1978; Sinaga 1986; Adebanjo and Bankole, 2004; Rabeendran et al., 2006) have been used as biocontrol agents.

Amongst the fungi, Trichoderma sp. are the most widely used. For example, T. harzianum, T. koeningii and T. viride are known to control damping off caused by Rhizoctonia and Pythium sp. in the laboratory, glass house and in the field (Papavizas, 1985). Rhizoctonia solani causing damping-off disease of seedlings as well as root and stem rot in transplants is a major soil-borne pathogen of chilli. Among the antagonistic fungi, Trichoderma harzianum has shown promise as a biocontrol agent of R. solani in chilli (Bunker and Mathur, 2001). Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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1.2.8. Botanicals as biocontrol agent The inappropriate use of agrochemicals especially fungicides which found to pose more of carcinogenic risk than insecticides and herbicides together may give rise to undesirable side effects. Additionally, resistance by pathogen to fungicides has rendered certain fungicides infective. It may be needed to develop new management systems to reduce the dependence on the synthetic pesticides. Nowadays, plant extracts as natural products are widely used to control pests. Plant extracts and essential oils show antifungal activity against a wide range of fungi.

Microorganisms and medicinal plants are the rich sources of secondary metabolites which are potential sources of useful bioactive products. (Dung and Loi, 1991). The biosynthesis of the metabolites were controlled genetically and affected strongly by environmental influences and therefore, there are fluctuations in the concentration and quantities of secondary metabolites (Deans and Svoboda, 1989; Vining, 1990; Steele and Stowers, 1991). 1.3. Molecular characterization Molecular markers can be definitive in confirming the correctness of any morphological taxonomic system and provide additional tool characterizing fungal genotypes. The DNA technology has opened several new avenues of investigation. The molecular data is also useful for taxonomic purpose and identification of unknown isolates with morphological identification is time consuming and difficult. Especially in some species that are asexual or heterothallic, making identification with sexual structural characterization. In recent years, several PCR based molecular techniques have been used to detect and discriminate among microorganisms. In additional, the analysis of DNA sequences from multiple genetic variations has been used to establish Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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the phylogenetic relationship of the species within Trichoderma (Chaverri et al., 2003; Kullnig et al., 2002). 1.4 Application of Trichoderma sp. From several studies, it has been confirmed that Trichoderma sp. have antagonistic and biological control potential against the diversity of soil borne pathogens. (Grondona et al., 1997; Hanson and Howell, 2004; Bajwa et al., 2004).

The application of Trichoderma species can control a large number of foliar and soil borne fungi i.e., Fusarium sp., R. solani, Pythium sp., S. sclerotium, S. rolfsii in vegetables, fruits and industrial crops (Tran, 1998; Ngo et al., 2006). These results were similar to previous studies in other countries.

Trichoderma sp. was used

successfully to control fungal pathogens. Trichoderma products can be applied to the soil, used as seed treatment, seedling root dip or added to organic fertilizers (or) compost. (Ha, 2010).

The use of Trichoderma product has both short term effects, immediate control of diseases and growth enhancement of crops as well as long-term effects which are demonstrated by the decrease in fungal pathogen inoculum in the field. Presently, Trichoderma based products are considered as relatively novel biological control agents which can help farmers to reduce plant diseases and increase plant growth.

OBJECTIVES OF THE STUDY •

To isolate fungi from chilli field soil samples by serial dilution technique during the period of June 2009 – May 2010.



Isolation of Pythium debaryanum causal agent of soil sample of chilli field

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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Monthly variations of soil mycoflora in Chilli field of Thiruvarur District.



To analyse the physico – chemical parameters of the soil sample (June 2009 to May 2010) in Thiruvarur District talukwise for a period of one year.



Pathogenicity test for the pathogen causing damping off of disease in chilli.



Effects

of

physico-chemical

factors

on

the

saprophytic

survival

of

P. debaryanum •

Control of pathogen using antagonistic fungi by dual culture technique



Antifungal activity of some medicinal plants against Pythium debaryanum.



To separate the antifungal mycelial compounds of potential antgonistic of T. viride by using TLC (Thin Layer Chromatogrphy).



The functional groups of bioactive compounds were carried out by the Fourier Transform Infra Red Spectroscopy and Ultra violet scanning (FTIR & UV).



To characterize extra cellular compounds from the potential antagonistic of T. viride by using GCMS (Gas Chromatography – Mass Spectrometry).

Molecular Characterization •

Isolation of DNA from pathogen.



Isolation of DNA from potential antagonistic of Trichoderma viride.



Amplification using PCR for the potential antagonistic of T. viride.



Gene sequencing for potential antagonistic of T. viride.

Application of Trichoderma sp. Seed treatment Root dipping

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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2.

REVIEW OF LITERATURE

2.1. Fungal Diversity Ploetz et al., (1985) studied, soil sample to a depth of 5 cm in a reduced – tillage, multicropped field to assayed for plant pathogenic and non pathogenic fungi. Soil samples were collected on 22nd day over a period of 27 month. Of the 16 genera of fungi identified, species of Aspergillus, Fusarium, Penicillium, Rhizopus and Trichoderma recorded for up to 75% of the total fungal population detected. Plant pathogens in the genera Rhizoctonia and Pythium recorded for a much lower proportion of the total fungal population detected in the soil. Anastomosis group four and a binuclete anastomis group of Rhizoctonia were the predominant members of Rhizoctonia and Pythium irregulare and P. acanthicum were the most common species of Pythium isolated from soil in Florida.

Persiani et al., (1998) reported variation in diversity of fungi isolated from soil was studied in a ferrallitic and a hydromorphic soil. Several experimental disturbances were produced and change in species diversity were observed for seven years. The disturbances, as well as changes in soil moisture produced by variation of rainfall, act as extrinsic factors to the community, influencing the level of diversity. The variation of diversity with in the community in time, species with a generalist or specialist behaviour were identified in accordance with their occurrence in dry and wet periods. Knowledge of the fungi isolated from soil is of considerable interest in tropical forests, where traditional shifting cultivation is practiced. Cultivation is currently considered responsible for ecosystem degradation. This review shows traditional cultivation, when

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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practiced as a short time- space disturbance, does not appear to cause serious disturbance to the fungal diversity.

Tsui et al., (1998) studied the effect of human

disturbance on fungal diversity in the tropics is of paramount importance and to conserve renewable resources effectively. In this paper an attempt is made to examine the effect of human disturbance on fungal diversity in the tropics. Fungal diversity appears to be related to plant diversity may also be related to disturbance.

The variety and galaxy of fungi and their natural beauty occupy prime place in the biological world and India has been the cradle for such fungi. One third of fungal diversity of the globe exists in India. Out of 1.5 million of fungi, only 50% are characterized until now. Unfortunately, only around 5-10% of fungi can be cultured artificially. Fungi are not only beautiful but play significant role in the daily life of human beings besides their utilization in industry, agriculture, medicine, food industry, textiles, bioremediation, natural cycling, as biofertilizers and many other ways. Fungal biotechnology has become an integral part of the human welfare (Manoharachary et al., 2005).

Suhail et al., (2006) were investigated the mycoflora from the bed of river at three locations viz., Right Bank, Center and Left Bank from June 2004 to May 2005. Twenty four soil samples were collected from surface, 10, 20 and 30 cm depth. The fungi were isolated by using soil dilution and soil plate method. Of the 73 strains of fungi isolated, 10 species of Penicillium were identified. Greater number of species were isolated on soil plate method than on dilution plate method. Higher number of

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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species were recovered from left bank as compared to right bank while in center isolates were in low frequency.

Meriles et al., (2008) to determine the short-term effects of glyphosate and crop residues on the population dynamics of soil-borne fungi under field conditions. Soil samples were randomly collected from a peanut – corn – soyabean rotation field in order to quantify native populations of Fusarium, Pythium, Trichoderma, Gliocladium and culturable total fungi populations.

Highest population of Trichoderma and

Gliocladium were recorded in soil with corn residue. Pythium populations increased after glyphosate treatment.

Trichoderma, Gliocladium and culturable total fungal

populations were not affected by glyphosate applications. Population responses of various important soil-borne fungi after glyphosate treatment is currently limited since it was dependent on numerous parameters such as soil condition, type of hosts involved and soil microbial interactions. The use of corn residue appeared as an interesting alternative to increase the population of potential antagonistic fungi, and reduce crop diseases.

Kostadinova et al.,

(2009) investigated isolation and identification of

filamentous fungi from soil. Among 12, belonging to the phylum Ascomycota (7 genera), Deuteromycota (2), Zygomycota (2) and Basidiomycota (1).

Mucor,

Cladosporium, Alternaria, Aspergillus and Penicillium were predominant genera. Lecanicillium, Botrytis, Geomyces, Monodictys and Rhizopus were the most frequently isolated genera. Most of the fungal isolates proved to be cold-tolerant.

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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Jamiolkowska (2009) pointed out species of fungi colonizing underground part of hot pepper (Capsicum annuum) plants cultivated field. Mycological analyses showed that roots and stem base were colonized mostly by Colletotrichum coccodes and Fusarium sp.

The effect of fungal communities on Fusarium oxysporum growth was

evaluated by means of the biotic series method. Communities originating from underground part of hot pepper could not reduce F. oxysporum growth.

Fifteen soil samples were collected from three different stations by dilution plating method on PDA medium to assess fungal diversity and the population diversity. The total number of 22 species representing a genera were recorded. Of them many (11 species) were the members of the genus Aspergillus, Penicillium was represented by four species while all others were represented by one species each. Physicochemical characteristics of the soil was analysed and to find out the impact on fungal populations. (Senthilkumar et al., 2009).

Panda et al., (2009) studied a total of 141 species belonging to 64 genera of fungi were isolated from coastal sandy belt of Orissa. The most dominant genera were Aspergillus, Penicillium, Trichoderma and Fusarium sp. Higher fungal and bacterial population was encounted in soil B than in soil A. Surface layer possessed higher fungal population, more soil nutrients and less moisture.

Fungal population was

positively correlated with total organic carbon, moisture content and total soil respiration but negatively correlated with soil temperature.

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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The need for new and useful compounds to provide assistance and relief in all aspects of the human life is evergrowing. There are enormous difficulties in raising enough food on certain areas of the earth to support local human populations. Environmental degradation, loss of biodiversity and spoilage of land and water also add to the problems faced by mankind. In the continual search by both pharmaceutical and agricultural industries for new products, natural selection has been found superior to the combinatorial chemistry for discovering novel substances that have the potential to be developed into new industrial products. The plant is an extraordinarily common source of organic energy. It is thus likely that a huge array of microbes like bacteria and fungi inside the plant tissues and interact with them. Of these a range can be isolated from apparently healthy tissues, many of which have never been documented to be associated with disease; others may cause disease when environmental conditions change. The intent of this review is to prove insights in to the presence of fungi in nature and their diversity, their interaction with the host plant and their role in plant growth promotion (Pandya and Saraf, 2010).

Danial Thomas and Ambikapathy (2010) investigated the fungal diversity of the soil of the forest floor at four sites in South Western Ghats.

Forty two species

belonging to 11 genera were isolated and identified. The total count of genus or species did not always follow the number of cases of isolation. Most of the genera detected belonged to the Ascomycotina with the fewer proportion belonging to Deuteromycotina. The genera of highest incidence and their respective number of species were Aspergillus (33.33%, 14 species) followed by Mucor, Penicillium,Trichoderma (11.9%, 5 species

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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each), Cladosporium (4.76% 2 species), Curvularia, Emericella, Hormodendrum and Sarocladium (2.3%, one species each).

Madhanraj et al., (2010) assessed the fungal diversity, population density and also physico-chemical characteristics of the soil were analysed and to find out their impact on fungal population. A total number of 24 species representing 12 genera were recorded from all the sand dune samples (40) at 8 different stations. Physico chemical analysis revealed the moisture content in the range from 3.6 to 3.1 organic carbon from 4 to 20 mg/g and available nitrogen from 0.014 to 0.046 mg/g in different stations. Correlation analysis made between fungal population density and physico-chemical factors of the soil revealed no factor as responsible for the population density changes in different stations.

Madhanraj et al., (2010) studied Meghamalai forest soils are found to be rich in cellulolytic organisms.

The physico chemical parameters such as temperature,

humidity, soil pH and organic matter present in soil with the growth of microbes. From the soil sample different type of fungus were isolated and identified.

They are

Aspergillus niger, Curvularia lunata, C. geniculata, Penicillium lanosum and Neurospora crassa.

Nilima Wahegaonkar et al., (2011) analysed twenty three soil samples of three ecosystems for number of organisms and the specific composition of hyphomycetous fungi. Totally 45 genera distributed in 85 species were isolated, maximum being in agricultural soils. The relationship between the genera of fungi and different ecosystem

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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type was analyzed. No obvious variation was observed in the different soil types. The dominant genera in all the ecosystem types were also studied.

Aspergillus was

dominant in all the three types of soils followed by Alternaria, Cladosporium, Trichoderma, Gliocladium and Gloeosporium. Species diversity and diversity indices of these soil types were calculated.

Iram et al., (2011) studied the micro-fungal flora of heavy metals contaminated peri-urban agricultural field of Pakistan by soil serial dilution method. A total of 30 micro-fungi isolated from 6 sampling sites of these isolates 24 belong to Phylum Ascomycota, 3 to phylum zygomycota, 2 phylum Basidiomycota and 1 to phylum Deuteromycota. The most wide spread genus was Aspergillus and common species Aspergillus niger. Frequency percentage showed that Kasur is rich in fungal population as compared to other peri urban areas while Wah Cantt showed maximum fungal Colony Forming Unit (CFU).

Prince et al., (2011) studied the seasonal variations in soil fungal populations of traditional sugarcane field. About 49 different species belonging to Phycomycetes and Deuteromycetes were isolated by using PDA medium and identified by using standard manual. The dominant species were Aspergillus niger, A. flavus followed by Botrytis cinera, Trichoderma viride, T. harzianum, T. koeningii, T. glaucum, Penicillum chrysogenum and P. citrinum from the sugarcane field soils of Orathanadu in various seasons, whereas in Pattukottai soils the dominant species were A. niger, Botrytis cinera

followed

by

A.

oryzae,

Fusarium

oxysporum,

Gliocladium

virens,

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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P. chrysogenum and T. viride respectively.

Kalaiselvi and Panneerselvam (2011) studied the seasonal and depthwise variations in soil fungal population in relation to the soil nutrient variability in Paddy field of Thanjavur district. About 30 different species belonging to Ascomycetes and Phycomycetes were isolated using PDA medium and identified by using standard manual. During rainy season, maximum fungal count was recorded in the subsoil layer. The dominant species were Aspergillus niger, Cunninghamella sp. followed by T. viride, T. harzianum, Penicillum janthinellum, P. claviforme, A. terreus and Aspergillus conecium from the paddy field soils of Nadur soil in various stations, whereas in Orathanadu soils the dominant species were A. niger, T. viride, T. harzianum followed by P. janthinellum, P. citrinum and Rhizopus sp. whereas in Punnainallur, the dominant species were A. niger, T. harzianum and Cunninghamella sp. followed by Fusarium oxysporum, P. janthinellum, T. koeningii and T. viride respectively.

2.2 Saprophytic survival The pathogen in general survives in soil in the absence of host either as dormant propagules or as saprophytes in the dead host tissues / crop residues or as parasites in non-crop host or competitive colonizers of the organic substrates. The host tissues infected by the pathogen during its parasitic phase serve as its main source of survival was emphasized by Baker and Cook (1974); Lockwood (1981); Cook and Baker (1983); Duczek et al., (1999). Sclerotium rolfsii causing southern blight of Chinese yam showed its growth most rapidly at 25 – 30°C (Kusaba et al., 2001).

Thus the pathogens are capable of two kinds of saprophytic behaviour (i) they may competetive with obligate saprophyte and with some other root infecting fungi for colonization of the crops of dead plant tissue lying in or on the soil, and (ii) survival of Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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the pathogen is sometimes prolonged in the dead host tissues invaded during the parasitic phase (Garrett, 1975).

Saprophytic colonization refers to the entry of the pathogen in to already dead organic materials in the presence of antagonistic microbes. Sadasivam (1939) and Walker (1941) isolated Fusarium culmorum from fresh wheat straw substrate units buried in different types of soil. Subramanian (1946) showed that the cotton wilt fungus F. vasinfectum also colonized the dead host tissues in soil as that of F. culmorum, Garrett (1956) proposed the term „competitive saprophytic ability‟ (CSA) and defined as the summation of physiological characters that makes for success in the competitive colonization of dead organic substrates. The method CSA was later adopted by Macer (1961); Burges and Griffin (1967); Rai and Upadhayay (1983); Dasgupta (1989). Garrett, was the pioneer in the studies of Competitive Saprophytic Ability but much remains to be done (Lockwood, 1986).

Garrett (1970) suggested that the production of antifungal antibiotics is not essential for saprophytic survival of root infecting fungi. It has been reported that the tolerance to antibiotic substance was not a deciding factor of the competitive saprophytic status of F. oxysporum and Sclerotium rolfsii (Mehrotra and Claudius, 1973). However, Upadhyay and Rai (1983) reported that the colonization of pigeon pea substrates by F. udum as highly suppressed by antagonism from Penicillium citrinum, A. flavus, A. niger, A. terreus, Micromonospora globosa and T. viride. Lockwood (1986) highlighted the importance between the groups of microorganisms in determining the survival of fungi as saprophytes.

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Role of inoculum density and incubation period on saprophytic behaviour of Pythium ultimum and S. rolfsii have been studied by Pandey et al., (1999). However, it has been reported that the survival of the pathogen as saprophytes depends upon the interactions of numerous environmental factors and indigenous microbial population. The elimination of the pathogen from the substrate, the mechanism called „substrate possession‟ or „prior colonization‟ is one of the important approaches suggested for the biological control of the soil borne plant pathogens (Lockwood, 1988).

2.3. Biological control 2.3.1. Antagonistic activity The fungus Gliocladium virens is an important biological control agent against plant pathogenic fungi, such as Pythium ultimum and Rhizoctonia solani that cause damping-off disease. G. virens strain G20 has been commercially formulated in to the disease suppressing product Gliogard (Grace & Co., CT). One possible mechanism of G. virens biocontrol may be the production of the fungistatic metabolite gliotoxin. The presence of this metabolite has been associated previously with disease suppressive activity towards P. ultimum. This study represents strong genetic evidence supporting a major role for antibiosis in the suppression of a plant disease by a biocontrol agent. (Wilhite et al., 1994).

Ali Shtayeh and Saleh (1999) have been investigated using a newly modified dual plate culture method, the three mycoparasites showed varying antagonistic performance against several Pythium host species under a range of in vitro conditions. However, P. periplocum and P. oligandrum were found to be active biocontrol agents against P. ultimum, the damping-off organisms of cucumber.

This pathogen was

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antagonized, on thin films of water agar, by the three mycoparasites, and was moderately susceptible to P. periplocum while slightly susceptible to P. acanthicum and P. oligandrum. In direct application method in which antagonistic mycoparsites were incorporated into peat/sand mixture artificially infested with P. ultimum under growth room conditions.

Pythium oligandrum and P. periplocum significantly improved

seedling emergence and protected seedlings from damping-off. In the seed coating method, biocontrol of two types of seed dressing (homogenate or Oospore coated seeds), was comparable to that achieved by direct application.

The most prevalent fungi were A. alternata, A. niger, A. sydowi, A. versicolor, C. herbarum, C. lunatus, P. herbarum, S. rostrata and U. botrytis on the two types of media. The prevalent fungi were screened for the antagonistic activity against the pathogenic fungus P. herbarum in vivo and in vitro.

The culture filterates of

A. alternata, A. niger, C. lunatus and E. nidulans caused high inhibition of P. herbarum. Colony interactions in solid cultures indicated that maximum inhibition of P. herbarum was caused by A. alternata, A. niger, C. lunatus, S. rostrata and S. chartarum. (Abdel-Sater, 2001).

Natural and agricultural ecosystems harbor a wide variety of microorganisms that play an integral role in plant health, crop productivity, and preservation of multiple ecosystem functions.

Interactions within and among microbial communities are

numerous and range from synergistic and mutualistic to antagonistic and parasitic. Antagonistic and parasitic interactions have been exploited in the area of biological control of plant pathogenic microorganisms. Microbial antagonists utilize a diverse arsenal of mechanisms to dominate interactions with pathogens, pathogens have

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surprisingly diverse responses to counteract antagonism.

These responses include

detoxification, repression of biosynthetic genes involved in biocontrol, active efflux of antibiotics, and antibiotic resistance. Understanding pathogen self-defence mechanisms for croping with antagonist assault provides a novel approach to improving the durability of biologically based disease control strategies and has implications for the development of transgenes. (Duffy et al., 2003).

Jun and Kim (2004) have undertaken a study to examine the control effect of damping-off on radish caused by Pythium sp. researchers used the isolates of a fungivorous nematode Aphelenchus avenae, and antagonistic fungi, Trichoderm sp. These were used as biocontrol agents, either alone or in combination.

Antibiotic

activity of T. virens and T. harzianum to Pythium sp. was stronger than that of T. koeningii. Control efficacy against damping-off of radish was most enhanced under the treatment using the nematode – Trichoderma combination.

Twenty eight microorganisms showing in vitro antagonistic activity against Pythium ultimum were used for their ability to reduce root rot on mature tomato plants grown in a greenhouse under hydroponic conditions. This study led to the selection of potential biocontrol agents against root rot of Pythium diseases in hydroponic systems that not only protect the crop but also have a beneficial effect on the plant growth and development in the absence of pathogens. (Gravel et al., 2006).

Sahi and Khalid (2007) studied, five species of Trichoderma, Trichoderma viride T. harzianum, T. koeningii, T. aureoviride and T. pseudokoningii were evaluated for their in vitro antagonistic potential against Fusarium oxysporum, the cause of wilt disease in sweet peppers (Capsicum annuum).

Among the Trichoderma species

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T. viride showed the best performance in vitro biological control of Fusarium oxysporum

followed

by

T.

harzianum,

T.

aureoviride,

T.

koeningii

and

T. pseudokoningii respectively.

Intana et al., (2007) reported, three mutant and two wild type strains of Trichoderma harzianum were tested for efficacy to inhibit and overgrow mycelial of Colletotrichum capsici, a causal agent of anthracnose disease of chilli on Potato Dextrose Agar (PDA) at room temperature.

All strains effectively inhibited and

overgrow mycelial of the pathogen. Antifungal metabolites completely inhibited both the mycelial growth and spore germination of the pathogen.

Three species of Trichoderma were evaluated against five isolates of soils borne phytopathogenic fungi in dual culture techniques and through production of volatile and non-volatile inhibitors, pH and temperature effects on Trichoderma mycelial growth were also evaluated. All Trichoderma isolates had a marked statistical inhibitory effect on mycelial growth of the pathogens in dual culture compared with control. (Hajieghrari et al., 2008).

Muthukumar et al.,

(2008) observed that the four fungal antagonist viz.,

Trichoderma sp. Trichoderma viride, T. harzianum, T. hamatum were evaluated in vitro for the management of damping-off in chilli caused by Pythium aphanidermatum. All the antagonists showed their antagonism against the pathogen. Among the fungal antagonists, Trichoderma sp. showed maximum inhibition of the growth of the pathogen followed by Trichoderma viride compared to control. Imtiaj and Lee, (2008) studied three Trichoderma species such as T. harzianum, T. pseudokoningii and Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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T. virens were extensively studied on the inhibition of Alternaria porri pathogen of onion blotch diseases.

Three concentrations of liquid culture filtrate of these

Trichoderma species were tested against mycelial growth (MG) and conidial growth (CG) of A. porri. All the Trichoderma species were also showed good inhibitory effect on A. porri.

Siameto et al., (2010) recorded sixteen selected isolates of T. harzianum were selected for antagonism against five soil borne phytopathogenic fungi (Rhizoctonia solani, Pythium sp., Fusarium graminearum, F. oxysporum, f.sp. phaseoli and F. oxysporum f.sp. Lycopersici) using dual culture assay. All T. harzianum isolates had considerable antagonistic effect on mycelial growth of the pathogen in dual cultures compared to the controls. Maximum inhibitions occurred in Pythium sp. compared to other pathogens. Since all T. harzianum isolates evaluated were effective in controlling colony growth of the soil borne pathogens both in dual cultures and in culture filtrates they could be tried as a broad spectrum biological control agent in the green house and under field conditions.

The strains of Trichoderma species were screened against Pythium aphanidermatum by dual culture method. Efficacy of culture filtrates of the strains was also determined.

Since mycoparasitism plays important role is antagonism of

Trichoderma species, extracellular enzymatic activity of the strains was assayed. Among

the

strains

tested,

T.

viride

was

found

most

effective

against

P. aphanidermatum. (Mishra, 2010). Amin et al., (2010) have isolated five species of Trichoderma were tested for their ability to produce volatile metabolites against seven fungal plant pathogens viz., Fusarium oxysporum, Rhizoctonia solani, Sclerotinia Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

25

sclerotiorum, Colletotrichum capsici, Helminthosporium oryzae and Alternaria brassicicola.

Studies indicates that T. viride was most effective in reducing the

mycelial growth of above fungal pathogens.

Volatile metabolities from T. viride

caused maximum reduction in mycelial growth of C. capsici and A. brassicicola was recorded with T. viride where as, in H. oryzae, T. harzianum accounted for maximum reduction in mycelial growth.

Mahalingam et al., (2011) have been reported the antagonistic potentiality of some soil fungi against Ceratocystis paradoxa a pathogen causing pineapple disease in sugarcane was studied by dual culture method. The pathogen Ceratocystis paradoxa and some individual species of the soil fungi viz., Aspergillus awamori, A. niger, Gliocladium virens, Penicillium citrinum, Trichothecium sp., Trichoderma glaucum, T. harzianum, T. hirsuta, T. koeningii and T. viride were grown on PDA medium individually. The colony interactions between the pathogen and the soil fungi were assessed the following model proposed by Porter (1924) and Dickinson and Broadman (1971).

2.3.2. Antifungal activity Kaur et al., (2005) studied chloroform and methanol extracts of root and shoot of the herb Heracleum candicans wall, showed antifungal activity against six species of fungi viz. Alternaria, Aspergillus, Fusarium, Penicillium, Phytophthora and Pythium was observed in petroleum ether and chloroform root extracts. Petroleum ether extract of shoot showed antifungal effect against the five fungal species, viz., Aspergillus, Alternaria, Fusarium, Phytophthora and Pythium. Methanol root extracts also showed

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antifungal activity against Alternaria species only. Similarly, methanol shoot extract showed inhibitory activity against Aspergillus and Pythium species only. The results suggest significant antimicrobial activity of the extracts against test fungi. This study justifies the claimed use of this herb in the traditional system of medicine to treat various diseases.

Saadabi (2007) collected, leaf samples of Lawsonia inermis to examine their antimicrobial potential.

Water, ethanol and chloroform crude extracts in different

concentrations were obtained and bioassayed in vitro for its bioactivity to inhibit the growth of 6 human pathogenic fungi and 4 types of bacteria. The differences in bioactivity of the 3 types of extracts were analyzed. Despite extreme fluctuations in activity, the extract of water was clearly superior followed by methanol and chloroform. The growth of all pathogen inhibited varying degrees by increasing the concentrations of the extract. Phytochemical analyses showed the presence of anthraquinones as major constituents of the plant leaves and are commonly known to posses antimicrobial activity. These results confirm the antibacterial and antifungal activity of henna leaves and support the traditional use of the plant in therapy of bacterial infections. The possibility of therapeutic use of Sudanese henna as antimicrobial agents is worthy of note.

Sathish et al., (2007) reported aqueous extract of fifty-two plants from different families were tested for antifungal potential against eight important species of Aspergillus such as A. candidus, A.columnaris, A. flavipes, A. flavus, A. fumigatus, A. niger, A. ochraceous and A. tamari. Among fifty-two plants tested, aqueous extract of

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Acacia nilotica, Achras sapota, Dautura stramonium, Emblica officinalis, Eucalyptus globules, Lawsonia inermis, Mimusops elengi, Peltophorum pterocarpum, Polyalthia longifolia, Prosopsis juliflora, Punica granatum and Syzygium cumini have recorded significant antifungal activity against one or the other Aspergillus species tested. A. flavus recorded high susceptibility and hence solvent extracts viz., petroleum ether, benzene, chloroform, methanol and ethanol extracts of all the twelve plants were tested for their antifungal activity against it. Among the solvent extracts tested, methanol gave more effective than ethanol, chloroform, benzene and petroleum ether, except for Polyalthia longifolia, where petroleum ether extract recorded highly significant antifungal activity than other solvent extracts.

Mahmud et al., (2009) reported in vitro antifungal activity of fruits of Vitex negundo Linn., was examined against 5 common fungal strains, Candida albicans, Candida glabrata, Aspergillus flavus, Microsporum canis

and Fusarium solani.

Ethanol extract of fruit seeds showed significant activity against Fusarium solani and moderate response against Microsporum canis with no effect on Candida albicans.

Vitex negundo belongs to the family Verbenaceae. It is a large aromatic shrub distributed throughout the greater part of India upto an altitude of 1500 m in the outer Himalayas. It is widely planted as a hedge plant along the roads and between the roads. Traditionally it is having the flok claims like useful in treatment of rheumatism, insecticidal, antimicrobial, anticancer, tranquillizer, tonic, febrifuge, expectorant and diuretic properties. Different extracts of Vitex negundo leaves were investigated for its antimicrobial and antifungal activity on five bacterial species and three fungal species

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these are Staphylococcus aureus, Proteus vulgaris, Bacillus subtilis, E. coli, Pseudomonas aerugenosa, Aspergillus niger, Aspergillus flavus and Candida albicans respectively.

Among all extracts water ethanol (50:50) extract showed maximum

antimicrobial and water extract showed maximum antifungal activity against all tested species (Aswar et al., 2009).

Valarmathy et al., (2010) studied the antimicrobial activities of extract of leaves against four common bacterial isolates.

Musa paradisiaca (Banana) Azardiractca

indica (Neem), Solanum melongena (Kathirikai), Cynodon dactylon (Grass), Alternanthera

sessilis

(Ponnangkani),

Anisochilus

carnosus

(Karpooravalli),

investigated individually for antimicrobial activity by disc diffusion method. These were investigated against selected species of Eschericia coli, Bacillus subtilis, Vibrio cholerae, Klebsiella pnemoniae to find out the inhibitory activities of the microbes.

Lawsonia inermis L. is a much branched glabrous shrub or small tree, cultivated for its leaves although stem bark, roots, flowers and seeds have also been used in traditional medicine.

The plant is reported to contain carbohydrates, proteins,

flavonoids, tannins and phenolic compounds, alkaloids, terpenoids, quinones, coumarins, xanthones and fatty acids. The plant has been reported to have analgesic, hypoglycemic, hepatoprotective, immuno stimulant anti-inflammatory, antibacterial, antimicrobial, antifungal, antiviral, antiparasitic, antitrypanosomal, antidermatophytic, antioxidant, antifertility, tuberculostatic and anticancer properties. It is now considered as a valuable source of unique natural products for development of medicines against

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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various diseases and also for the development of industrial products. (Chaudhary et al., 2010).

Sharma and Sharma (2011) investigate, Lawsonia inermis Linn. and Eucalyptus citriodora Hook. Leaf extracts were evaluated against 10 pathogenic and 2 human pathogenic fungal species viz. Alternaria solani, Drehalodes (Helminthosporium halodes), Rhizoctonia solani, Fusarium solani, Curvularia lunata, Dreschlera graminae, Fusarium moniliformae, Aspergillus flavus, A. parasiticus var. gobossus, Trichophyton rubrum, Aspergillus fumigatus and Candida albicans. The dried and powdered leaves were successively extracted with petroleum ether, benzene, chloroform, acetone, ethanol and water using soxhlet assembly. The antifungal activity was done by poison food technique.

Acetone extract of

L. inermis leaves and

petroleum ether extract of E. citridora leaves showed highest activity against all tested fungi. The inhibitory activity was significant and better than the synthetic fungicides used as most of the strains showed resistance against flucanazole and amphotericin B.

Inhibition of spore/conidial germination of four fungi viz., Bipolaris sorokiniana, Fusarium oxysporum f. sp. Vasinfectum, Rhizopus artocarpi and Botryodiploida theobromae was tested using the extracts of different parts of Vinca rosea and Azadirachta indica and smoke of rice straw, wheat straw, tobacco leaf and „dhup‟ (incense) and showed good results in their inhibition. Vinca rosea root extract inhibited 100% spore germination of Bipolaris sorokiniana and Rhizopus artocarp when it was immersed from 5.303 minutes at 5:1:25 (w/v) concentration. A. indica (leaf, root and seed) extracts showed good (100%) inhibition results on B. sorokiniana,

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and R. artocarpi. Smoke of rice straw, wheat straw, tobacco leaf and dhup had a great antifungal effect against these fungi. (Alam et al., 2002).

Murugesan et al., (2011) studied, the antifungal activity of eleven different medicinal plants namely Aloe vera, Alpinia calcarata, Acalypha indica, Carum copticum, Leucas aspera, Ocimum sanctum, Piper betle, Phyllanthus niruri, Solanum trilobatum, Memycelon umbellatum and Tridox procumbens were tested against plant pathogenic fungus F. oxysporum by agar well diffusion method. The plant leaves were extracted with various solvents like ethyl acetate, diethyl ether and water (aqueous). Among the different plants tested, all the 3 solvent extracts of the Memycelon umbellatum showed maximum (21 mm) antifungal activity against F. oxysporum. The other plant extracts were showed moderate to minimum antifungal activity.

Prince and Prabakaran (2011) reported, the antifungal activity of eight different medicinal plants namely Aloe vera, Ocimum sanctum, Centella asiatica, Piper betle, Calotropis gigantea, Vitex negundo, Ocimum basilicum and Azadirachta indica were tested against plant pathogenic fungus (red rot disease causing agent) Colletotrichum falcatum by agar well – diffusion method. The plants leaves were extracted with various solvents like chloroform, ethanol and aqueous. Among the different plant tested, all the three solvents of the Vitex negundo showed maximum antifungal activity (25 mm) against the plant pathogen tested.

Herbs have been one of the important and unique sources of medicines even since the dawn of human civilization. In spite of tremendous development in the field

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of allopathy since the 19th century, plants still remain one of the major sources of drug in the modern as well as traditional system of medicine throughout the world. Over 70% of all medication marketed are natural or semi synthetic plant derived. Numerous researches on plants have been carried out for eradicating ailments. Plants and its phytoconstituents are used not only to prevent but also cure various disorders like fungal infections. The incidence of fungal infections is increasing at an alarming rate, presenting a gigantic challenge to healthcare professionals. Many of plants and their phytoconstituents have been studied for their antifungal activity, especially on fungi Candida albicans. (Gupta et al., 2012).

Antifungal activity of solvent extract of Heterostemma tanjorense (wight and Arn.) have been investigated against human pathogenic fungi, such as Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Candida albicans and Fusarium moniliform.

The various solvents extracts were found to be effective against test

organism but the ethyl acetate and ethanol extracts appeared to be most effective antifungal agents as compared to aqueous and chloroform extract. Infectious diseases represent a critical problem to health and they are one of the main causes of morbidity and mortality worldwide. The resistance to antibiotics and with the toxicity during prolonged treatment with several drugs due to this medicinal plants are widely used by the traditional medical practitioners for curing various diseases in their day to day practice. Since ancient times, plants have been an exemplary source of medicine. The presented review summarizes the information concerning the new profile of antifungal drugs obtaining from medicinal plants. (Thevasundari and Rajendran, 2012).

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2.3.3. Compound identification and purification Inventory of biologically active compounds have gained importance in the recent years.

This involves the process such as extraction, separation and

characterization. The compounds resulted in the process are proved to be interesting in their structure and effective in their activity against various pathogens. Moreover the compounds (both inter and intracellular) are considered as a key factor to identify the organisms.

Jang et al., (2001) have undertaken a study to separate and identify antifungal substances produced by Gliocladium virens G1, a biocontrol agent used for the control of plant diseases caused by Rhizoctonia solani. They found that the culture filtrate of G. virens G1 effectively inhibited the growth of R. solani, Colletotrichum gloeosporioides and Phytophthora capsici.

The n-hexane extract of the G. virens

culture, strongly inhibited R. solani and C. gloeosporioides, but not P. capsici, although the n-butanol extract was effective against all the pathogens tested. An antifungal substance from the n-hexane extract was purified by Silica gel column chromatography and HPLC. The substance was examined for purity by HPLC and for nature by UV spectrometry. The compound was differed from known antibiotic compounds such as gliotoxin, viridian and gliovirin. The antifungal substance was very lipophilic based on its solvent-solubility and Rf values on TLC, and more inhibitory to C. gloeosporioides than other fungal pathogens tested.

Yong et al., (2003) isolated a compound (compound 1) and purified the liquid fermentation metabolites of the taxoids, produced by an endophytic fungus (Alternaria

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alternata var. taxi 1011 Y. Xiang et LU An-guo) from the bark of Taxus cuspidate Sieb.et Zuce through the methods of organic solvent extraction, Thin Layer Chromatography (TLC) and column chromatography. Compound I was identified as one kind of taxoids type III, based on the ultraviolet spectroscopy, infra red spectroscopy, mass spectrometry and nuclear magnetic resonance spectroscopy. This study provided a complete method for separation and purification of the endophytic fungi as well as structure identification of its fermentation metabolites.

A comprehensive inventory of the organic compounds and aroma active compounds produced by Antrodua camphorate during growth in submerged culture has been established by extracting culture fluids using three different organic solvent systems and subjecting the extracts to Gas Chromatography Mass Spectrometry (GC-MS) and Gas Chromatography-Olfactometry (GC-O).

Forty-two organic

compounds, of which esters, alcohols, acids and ketones were the most prevalent substances identified in pentane / ether and ether extracts (eleven and nine aroma-active compounds, respectively) by GC-O.

Among them, ethyl acetate, γ-undecalactone,

linalool and 3-hydroxy-2-butanone were assessed to be present at the highest intensity (Liu et al., 2007).

Senthilkumar et al., (2011) isolated 42 species of fungi belonged to 20 genera were recorded. A preliminary screening of all the species isolated from soils were made for antifungal activity against Fusarium oxysporum. Among the species tested the Trichoderma harzianum inhibited the pathogenic fungus to the maximum both in dual culture and food poisoning technique. Gas Chromatography Mass Spectrum analysis of

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acetonitrile extract of the filtrate of T. harzianum revealed the presence of six compounds represents six major peaks. The peaks correspond with diethyl phthalate, tetradecanoic acid 9,12 – octadecadienoic acid (z,z) oleic acid, 1,2 – benzene dicarboxylic acid, diisooctyl ester and squalene.

2.4. Molecular Studies Species in the genus Trichoderma are important commercial source of several enzymes, biofungicides and growth promoters. The most common biological control agents of the genus are strains of T. harzianum, T. viride and T. virens. In this study, sixteen selected isolates of T. harzianum from different land use and were tested for antagonistic action against five soil borne phytopathogenic fungi (Rhizoctonia solani, Pythium sp., Fusarium graninearum, F. oxysporum, F.sp., Phaseoli and Fusarium oxysporum, f. sp. Lycopersici) using dual culture assay and through production of nonvolatile inhibitors. Seven isolates were further characterized using RAPD – PCR procedure to determine genetic variability. All Trichoderma isolates had considerable antagonistic effect on mycelial growth of the pathogens in dual cultures compared to the control. Maximum inhibitions occurred in Pythium sp. the culture filtrates obtained from Czapek‟s liquid medium reduced the dry weight of the mycelial significantly while those from the Potato dextrose broth showed minimum inhibition growth. Pythium sp. was most sensitive compared to other pathogens. Since all T. harzianum isolates evaluated were effective in controlling colony growth of the soil borne pathogens both in dual cultures and in culture filtrates they could be tried as a broad spectrum biological control agent in the green house under field conditions. (Siameto et al., 2011).

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Chakraborty et al., (2010) isolated Trichoderma viride and T. harzianum from rhizosphere soil of plantation crops, forest soil and agricultural fields, were studied using RAPD and ITS-PCR. The genetic relatedness among eleven isolates of T. viride and eight isolates of T. harzianum were analyzed with six random primers. RAPD profiles showed genetic diversity among the isolates with the formation of eight clusters. Analysis of dendrogram revealed that similarity co-efficient ranged from 0.67 to 0.95. ITS – PCR of rDNA region with ITS1 and ITS4 primers products in all isolates.

Rubio et al., (2005) demonstrated the use of sequence – characterized amplified region markers to detect genomic DNA from Trichoderma harzianum 2413 from soil. Two primers and 27 Trichoderma sp. and amplified a 990 – bp fragment from T. atroviride 11 and a1.5 – Kb fragment from T. harzianum 2413, using an annealing temperature of 68°C.

These fragments showed no significant homology to any

sequence deposited in the databases. Total DNA was extracted from sterile and nonsterile soil samples, inoculated with spore or mycelium combinations of Trichoderma sp. strains indicated that the BR1 and BR2 primers could specifically detect T. harzianum 2413 in a pool of mixed DNA. T. harzianum 2413 could be used in real – time PCR experiments, new primers conjugated with a TaqMan fluorogenic probe were designed. Real time PCR assays were applied using DNA from sterile and non sterile soil samples inoculated with a known quantity of spores of Trichoderma sp. strains.

Zhang et al., (2007) reported, the combination of phenotypic characteristics and gene analysis of ITS1 and 2. Tef 1 and rpb2 gene sequences. Distinctive morphological

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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characters of Trichoderma taxi are its white small subglobose conidia and pachybasium like conidiophores aggregated into compact pustules. Phylogenetically, Trichoderma taxi forms an independent branch in vicinity to the Lutea (Hypocrea lutea, H.

melanomagna)

and

Pachybasioides

(Hypocrea

pachybasioides,

Hypocrea

minutispora, H. pilulifera, H. parapilulifera, H. lacuwombatensis and H. stellata) clades.

Siddiquee et al., (2007) studied the sequence analysis of internal transcribed spacer 1 region of the rDNA can be used to detect species level of Trichoderma harzianum.

Internal transcribed spacer 1 region (ITS1) of the ribosomal DNA was

amplified by polymerase chain reaction (PCR). To test the selected universal primers (ITS1 and ITS2) and conditions of the PCR, thirty – six of Malaysian Trichoderma isolates were used. The results of PCR product were positively performed purification. The PCR purification products were proved possible to amplify the ITS 1 region of all Trichoderma strains. The amplified DNA was sequenced and aligned against using extype strains sequencings from Tricto BLAST GenBank and established Trichoderma taxonomy. Thirty six isolates were positively identified as Trichoderma harzianum (32 strains) Trichoderma virens ( 3 strains) and Trichoderma longibrachiatum (1 strain) formed clearly defining phylogenetic analysis.

Initially a fungus of Macrophomina phaseolina was isolated from diseased roots of Citrus reticulate which was morphologically identified as Macrophomina phaseolina. Genomic DNA of M. phaseolina isolated from mandarin rhizosphere was purified and PCR amplification of 18S rDNA was done using genus specific ITS-1 and

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ITS-4 primers. Amplified product (550 bp) was sequenced and aligned against ex-type strain sequences of M. phaseolina from NCB1 GenBank using BLAST and phylogenetic analysis was obtained using MEGA4 software. The evolutionary history was inferred using the UPGMA method. Amplification of ITS 1 region of the rDNA can be considered as a rapid technique for identifying the organisms successfully in all cases. (Chakraborty et al., 2011).

Thangaraj and Meenupriya (2011) studied, four Aspergillus species internal transcribed spacer region 1 (ITS 1) secondary structure was predicted and compared. By observing the structures, some topologies were conserved in all the species and some were varied. These characters could be used for the identification fungi at genetic level. Secondary structure topologies such as first stalk and junctions were varied between species.

The phylogenetic trees based on both sequence and secondary structure

showed similar clad in the four Aspergillus species.

2.5. Application of Trichoderma sp. Nelson et al., (1988) reported that the biological control activity of Trichoderma koeningii and T. harzianum against Pythium seed rot and pre emergence damping-off of pea was increased by adding various compounds to seed treatments. Biological control activity of T. koeningii was increased up to 48% while activity of T. harzianum was increased up to 44% by incorporating specific compounds in seed treatments. Compounds promoting T. koeningii were generally in effective in promoting biological control activity of T. harzianum. Organic acids were most promotive to the activity of T. koeningii where as polysaccharides and polyhydroxy alcohols were most promotive

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to T. harzianum. There was no relationship between the ability of the compounds to support in vitro growth and proliferation of Trichoderma strains in the spermosphere and increased biological control activity by the antagonist.

Whipps and Lumsden, (2008) studied, rapid germination of sporangia of Pythium species in response to seed or root exudates followed by immediate infection, and the ability to cause long-term root rot make biological control of these pathogens very difficult. Pythium suppressive soils exist and these may be good sources of suitable biological control agents. Both bacterial and fungal antagonists are known to affect Pythium species by producing antibiotics, competing for space or nutrients or by direct parasitism. Antagonists have been incorporated in to soil or applied to seeds and in some instances, control of damping-off, equivalent to standard fungicide applications, has been achieved. However, reproducible cost effective biological control in the field is

rare.

Nevertheless

Pseudomonas

fluorescens,

Streptomyces

grieseoviridis,

Gliocladium virens, Pythium oligandrum and Trichoderma harzianum, have been used commercially for the control of diseases caused by Pythium.

Aziz et al., (1997) reported, application of Trichoderma lignorum as a seed coating or wheat bran preparation at a rate of 20g/kg soil, greatly reduced the number of bean seeds infested by Rhizoctonia solani, and the percentage of healthy seeds reached 92%.

Germination of conidia of Rhizoctonia solani in bean rhizosphere soil was

inhibited after soil or seed application with Trichoderma lignorum. Bean seedlings exudate increased the mycelial growth rate of Rhizoctonia solani and Trichoderma lignorum in vitro. Under green house conditions, the addition of germinating bean seed

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exudates in soil infested with Rhizoctonia solani and planted with bean, reduced the disease control capability of the antagonist. Plants grown from seeds treated with Trichoderma lignorum have the roots with lower levels of Rhizoctonia solani in their rhizosphere than the roots of untreated seeds. Trichoderma had little effect on the survival of Rhizoctonia solani in non rhizosphere soil.

However, application of

Trichoderma lignorum as a wheat-bran preparation, conidial suspension or seed coating reduced the pathogen counts in the rhizosphere soil of beans. Pythium ultimum, which causes root rot and damping-off of many floricultural crops grown in Oklahoma green house produces Oospores for survival and to initiate disease. Strains of Actinoplanes sp. that are hyperparasites of Oospores were evaluated for their biological control of Pythium root rot of plants grown in green house. (Filonow and Dole, 1999).

Yang et al., (2004) reported, formulation of Trichoderma species were tested for their ability to control pre-emergence and post-emergence damping-off caused by Pythium ultimum in green house grown Echinacea angustifolia seedlings. Over 400 Trichoderma strains were obtained from the rhizosphere of vegetable crops in which plants had survived Pythium damping-off. In co-culture trials in the laboratory, 24 isolates of Trichoderma sp. displayed the ability to steadily colonize and aggressively attack mycelial of P. ultimum and to produce numerous conidia on Pythium colonies. An “aggression index” was developed to quantify the

in vitro antagonism of the

isolates. Inoculum of each Trichoderma strains was added to Pythium infested potting mix to evaluate the efficacy of antagonistic isolates in reducing the incidence of damping-off of Echinacea. The effectiveness of the isolates was correlated with their aggression index. A wettable powder formulation of T. harzianum isolate, significantly

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reduce pre and post emergence damping-off caused by P. ultimum to a level comparable to that in non infested potting mix, or in infested potting mix influenced the persistence of Trichoderma and the rate at which it increased in the mix. Propagule density in the potting mix was strongly associated with suppression of Pythium damping off.

Ozbay and Newman (2004) reported, biocontrol with antagonistic microbes such as the fungus Trichoderma is one area of research. Trichoderma sp. are among the most common saprophytic fungi. Trichoderma sp. are well documented as effective biological control agents of plant diseases caused by both soilborne fungi leaf and fruit infecting plant pathogenic fungi. Trichoderma sp. are often very fast growing and rapidly colonize substrates, thus excluding pathogens such as Fusarium sp. several of these fungi are also parasitic to other fungi including plant pathogens. Trichoderma harzianum Rifai is an efficient biocontrol agent that is commercially produced to prevent development of several soil pathogenic fungi T. harzianum alone or in combination with other Trichoderma species can be used in biological control of several plant diseases. T. harzianum increases growth of various plants.

Microorganisms such as fungi, bacteria, viruses and nematodes are integral parts of agroecosystems. Some of them are harmful plant pathogens, whereas others are neutral or beneficial in their effects on plant growth. Control of disease – causing organisms is an essential component in every crop production system. Since World War II, numerous synthetic pesticides have been developed and used for control of crop pests. Many of the chemical pesticides killed not only the target species of pests but also other non-harmful or beneficial organisms. One chemical kills all approach for

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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management of plant diseases is detrimental to the microbial biodiversity in agroecosystems, and is therefore no longer acceptable in modern agriculture which emphasizes the importance of using sustainable technologies for food production. Numerous reports suggest that control strategies such as biocontrol used as viable alternatives to chemical control. (Huang and Chou, 2005).

Rini

and

Sulochana

(2006)

isolated

Trichoderma

and

Fluorescent

pseudomonads (Pseudomonas fluorescens P28 and P51) were evaluated under green house and field conditions for efficacy in suppressing Rhizoctonia root-rot incidence and promoting plant growth in chilli. The combination of T. harzianum, P. fluorescens, were most effective in reducing disease incidence – 66.7% more efficient than the control.

El-Shami (2008) reported effect of the antifungal antibiotic gliotoxin on root – rot diseases caused by Fusarium solani and its influence on population of fungal flora in soil were investigated.

Bean seeds were treated with different concentrations of

gliotoxin before sowing. The results obtained from the green house application of bioagent indicated that soaking seeds in different concentration of gliotoxin significantly reduced the percentage of damping off and root rot as compared with control.

Deepak et al., (2008) have been carried out to assess their possible use as bioagents for several antagonistic fungi on growth of two cumin fungal pathogens under in vitro and field conditions. Under in vitro conditions maximum inhibition of radial

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growth of Fusarium oxysporum f.sp. cumini was observed with the treatment of Trichoderma harzianum strain I, whereas maximum inhibition of mycelial growth of Alternaria burnsii was observed in the presence of T. harzianum strain II.

The

antagonists who showed maximum inhibition of the pathogen in laboratory conditions were applied in field conditions as soil treatment / seed treatment or as foliar spray.

Ekefan et al., (2009) have been determined the potential of Trichoderma harzianum isolates as biocontrol agents of Colletotrichum capsici, causing anthracnose of pepper. This work conclude that the Trichoderma harzianum isolates to suppressed the growth of Colletotrichum capsici and reduced the incidence of the pathogen on seeds and soil and finally the biocontrol organism (T. harzianum) for field and post harvest application to control anthracnose of pepper.

Ha, (2010) surveys conducted on food crops, industrial crops, vegetable crops and fruit crops in the north and south of Vietnam indicate that Trichoderma are common and can be isolated easily from soil, root and plant organic matters.

T. viride,

T. harzianum, T. hamatum were predominant species than other species. Laboratory and field trials also proved that Trichoderma species had ability to suppress the growth of fungal plant pathogens and enhance plant growth and development. Experiments conducted on several crops such as: Peanut, tomato, cucumber and durian indicates that selected Trichoderma strains could reduce significant diseases caused by fungal pathogens including Phytophthora palmivora, Rhizoctonia solani, Fusarium sp. Sclerotium rolfsii and Pythium sp. The efficacy of Trichoderma species on soil borne fungal disease is higher than fungicides and maintain longer. The value obtained

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through development, exploitation and use of Trichoderma products are not only plant disease control but also gave the local people opportunities to reduce health risks, costs and environmental damage due to over fungicide usage. Moreover, crop treated with Trichoderma grown better and had higher yields to compare with the one without application. Trichoderma product can be used in many ways including: seed treatment, applied direct to the soil before planting and added to organic fertilizers.

Among the antagonistic micro-organisms isolated from rhizospheric soil of healthy broccoli plants two fungal isolate Trichoderma harzianum and T. viride were used as biocontrol agents for controlling broccoli root rot disease caused by Pythium ultimum and Rhizoctonia solani pathogens. Moreover, their effect on broccoli plant growth and yield were also studied. In the same record, application of biocontrol agents as a combination of soil mixing plus root dipping method was generally more effective than applied individually for suppressing Pythium and Rhizoctonia rots incidence. In addition, using of soil mixing plus root dipping method gave the highest values of all measured parameters followed by soil mixing and root dipping methods. Concerning the interaction effect used between antagonistic micro-organisms and method of treatments, there was a highly significant effect. These results suggested that using of T. harzianum as a bio-control agent through soil mixing plus root dipping treatment could be provided not only additional protection against crop loss due to Pythium and Rhizoctonia diseases but also significantly increased vegetative growth head parameters ie. head yield (Plant (g), head yield (ton/fed), head diameter (cm), stem diameter (cm) and number of florets per plant (El-Mohamedy et al., 2011).

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3. MATERIALS AND METHODS

3.1 Description of the study site Thiruvarur District was formed on 1st January 1997 by carving out of certain portions of erstwhile Nagappatinam and Thanjavur Districts. Accordingly nine blocks from Nagappatinam district and 1 block from Thanjavur district were taken out and Thiruvarur district was formed with 10 blocks. For administrative purpose the district was divided in to two divisions i.e., Mannargudi and Thiruvarur. The district was bound by palk straight in the south, Thanjavur in the west and Nagappatinam district in the east and parts of Nagappattinam and Thanjavur district in the north.

At

present

Thiruvarur was located approximately between 10° 20° N and 11°07‟S of the north latitude and between 79°15‟E and 79° 45‟ W of east longitude. The total geographical area of the district was 2097.09 Sq.km. It has 2,37,715 hectares of cultivated area which constituted 69 percent of the total geographical area of the district.

This district was essentially a deltaic plain comprising of old and new delta. The old delta has a net work of canals and channels of the river Cauvery (Nannilam parts of Valangaiman, Kodavasal and Thiruvarur taluks) and Vennar (Thiruthuraipoondi, Needamangalam and parts of Thiruvarur, Kodavasal, Valangaiman taluks) of new deltaic area was irrigated by Grand Anaicut canal. Thiruvarur District was made up of tertiary and alluvial deposits.

The Cuddalore sand stones of tertiary age are well

developed as seen near Mannargudi. These sand stones are covered by a thin layer of wind blown sandy clays, unconsolidated sands, clay bound sands and mottled clay with lignite seams.

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The Map shows sampling sites of Tamilnadu Tamilnadu

Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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The Map shows sampling sites of Thiruvarur district

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Table shows sampling sites of Thiruvarur district each Taluk wise Tiruvarur Division

Firkas

Villages

1.

Thiruvarur (Thirunaippur)

2

48

2.

Nannilam (Mulamangalam)

4

73

3.

Kodavasal (Amaravathi)

5

106

4.

Valangaiman(Narthangudi)

3

71

Mannargudi Division 5.

Mannargudi (Neduvakottai)

6

128

6.

Needamangalam (Needamangalam)

3

70

7.

Thiruthuraipoondi (Karambayam)

4

77

27

573

Total

The alluvial deposits of the river Cauvery and its tributaries lie over the tertiary sand stone.

They consist of medium to firm sands, clays and sandy clays.

The

thickness of these formations range from 30 m to 400m. The minimum temperature was 22.6° and the maximum temperature was 39.7°C. The field was under cultivation of chilli during the months of June to May. In Thiruvarur district the prime crop production was Paddy, Green gram, Blackgram, Ground nut, Gingelly, Brinjal, Bhendi and Chillies.

3.2. Population dynamics of soil fungi in the Chilli field 3.2.1. Collection of soil samples In each Taluk, the soil samples were collected at a depth within 10 cm using a metal spatula. The spatula was sterilized every time with 70 per cent alcohol. At each station 5 to 7 samples were collected randomly and were pooled together. The samples were kept in new polythene bags, sealed and transported to the laboratory immediately

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for the mycological examination. For the analysis of soil nutrients, one kg of soil was separately collected in polythene bags from each station.

3.2.2. Isolation of fungi from soil (Warcup, 1950) Soil sample (10g) was taken in a 250 ml conical flask containing 100 ml sterile distilled water. The flask was shaken on an electric shaker to get a homogenous suspension and serial dilutions of the soil sample such as 10-1. 10-2 and 10-3 were prepared. One ml of 10-3 dilution was plated in petridishes containing PDA medium. The pH of the medium was adjusted to 5.6. Streptomycin sulphate (100 mg-1) was added to the medium to prevent the bacterial growth. The plates were incubated at 25±2°C for five days and the fungi appearing on the medium were recorded.

Population of fungi g-1 Mean number of propagules in dilution plate dry wt. of the soil. = factor Wt. of the dry soil

X

dilution

No. of soil samples from which fungi were recorded Percentage frequency 100

=

X No. of soil samples

3.2.3. Observations The colonies growing on PDA plates with different morphology were counted separately. A portion of the growing edge of the colony was picked up with the help of a pair of needles and mounted on a clean slide with lactophenol cotton blue stain. The slide was gently heated in a spirit lamp so as to facilitate the staining and remove air

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bubbles, if any. The excess stain was removed with the help of tissue paper and then the cover slip was sealed with transparent nail polish. The slide was observed under a compound microscope.

Microphotography of the individual fungal species was also taken using Nikon optiphot microscope (Nikon, Japan).

3.2.4. Identification Colony colour and morphology were noted besides hyphal structure, spore size, shapes and spore bearing structures of the fungi were identified by using standard manuals, such as Manual of soil fungi (Gillman, 1957), Dematiaceous Hyphomycetes (Ellis, 1971), More Dematiaceous Hyphomycetes (Ellis, 1976), Hyphomycetes (Subramanian, 1971).

3.2.5. Test Organism The fungus Pythium debaryanum Hesse one of the soil borne, broad spectrum pathogen that causing damping-off disease in chilli were isolated from the chilli field soil and used as a test organism.

3.3. Physico-chemical properties of the soil Moisture content of the soil was estimated by finding the weight difference of known quantity of soil before and after drying in a hot air oven at 60°C for 6 h. Soil samples after removing the debris were suspended in distilled water (1:2 w/v) and allowed to settle down the sand particles. The pH of the suspension was read using pH

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meter (systronics, India), to find out the soil pH. Electrical conductivity of soil was determined in the filtrate of the water extract using conductivity bridge as described by Jackson (1973), Cation exchange capacity (CEC) of the soil was determined by using 1 N ammonium acetate solution as described by Jackson, (1973).

The total organic carbon was estimated by rapid titration methods of Walkley and Black (1934) as described by Piper (1944). The total organic matter was calculated by multiplying organic carbon with constant factor 1.7241 as it is presumed that the organic matter of soil contains 58% carbon (Robinson, 1952). Available nitrogen was estimated by alkaline permanganate method as described by Subbiah Asija (1956) and available Phosphorus by Brayl method as described by Bray and Kutz (1945), Available Potassium was extracted from soil with neutral 1N ammonia acetate (1:5) and the Potassium content in the extract was determined by using flame photometer. (Standfold and English 1949), Calcium (Neutral 1N NH4 OAC extractable 1:5) was extracted and determined by versenate method (Jackson, 1973). Available micronutrients such as Zinc, Copper and Manganese were determined in the diethylene triamine pentaacetic extract of soil using Perklin-Elmer model 2280. Atomic absorption spectrophotometer (Lindsay and Norwell, 1978). Other nutrients such as magnesium, sodium and available iron were also analysed following method of Muthuvel and Udayasoorian (1999).

3.4. Statistical Analysis Pearson‟s correlation analysis was used to assess the relationship between physico-chemical parameters, total fungal colonies (Population density) and total fungal

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species (species diversity). The data were computed and analysed using statistical package for social sciences (SPSS) software.

3.5. Pathogenecity test Pythium debaryanum isolated from infected chilli field soil. The pathogenicity was confirmed adopting Koch‟s postulates.

3.5.1. Barley seed inoculum preparation Barley seed 150g together with 100ml distilled water was deposited in an conical flasks and autoclaved. After autoclaving each flask was inoculated with five 5 mm agar disc cut from a fresh PDA culture of Pythium debaryanum. The inoculum was incubated at 27 ± 2°C for 7 days.

3.5.2. Planting of Chilli Chilli seeds were surface sterilized with 70% ethanol for 5 min, 1% sodium hypochloride for 1 min and rinsed three times with sterile distilled water. The seeds were then pregerminated in sterile vermiculite for four days at 30°C in a growth cabinet. Prior to seedling transplanting, the barely seed inoculum of Pythium debaryanum was mixed in to steam pasteurized soil in 12 cm by 10.5 cm diameter plastic pots at the rate of 30 g/kg. For the control treatments, sterile barley seed was mixed into the soil at the same rate. Eight chilli seedlings were transplanted into each pot and the pots were maintained in the green house at 30 ± 2°C.

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3.6. Effect of soil physicochemical factors on the saprophytic survival of P. debaryanum 3.6.1. Preparation of pre – colonized substrate units Saprophytic survival of the test organism was studied using pre-colonized substrate units of the chilli straw with the pathogen. The pre-colonized substrate units were prepared following the method described by Garrett (1956). The chilli straw was collected from the chilli field after harvesting and brought to the laboratory. The straw was cut in to standard size (3 cm with a node) units. The substrate units were washed thoroughly in running water and oven dried at 80°C. The dried bits were then placed in 500 ml conical flask and sterilized in an autoclaving at 121°C for 15 min. Suitable moisture was adjusted in sterilized substrate units using sterile distilled water. Number of conical flasks containing straw bits were prepared in this way. They were inoculated with 5 agar blocks (5mm diameter) cut from the actively growing margin of P. debaryanum. The inoculated substrate units were incubated at 25±2°C for 4 weeks, to get the bits colonized uniformly with the pathogen. These pre-colonized substrate units were used for further investigation.

3.6.2. Effect of soil moisture The hand picked and air dried field soil containing organic carbon (0.59%), organic nitrogen (0.041%) and pH (7.2) was distributed in plastic containers. The moisture content was adjusted to 10, 25, 50 and 75% MHC on oven dry weight basis. Hundred chilli straw bits pre-colonized with test organism was buried in each container. The containers were incubated at laboratory temperature for about three months. The bits were recovered after 4, 8 and 12 weeks, washed in sterile distilled water, surface

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sterilized with 0.1% mercuric chloride solution and the percentage of survival was assessed on PDA medium. S50 values were also determined.

3.6.3. Effect of soil pH The pH of the soil distributed in plastic containers was adjusted to 4, 5, 6, 7, 8 and 9 with lime and gypsum. The moisture content of the soil was maintained at 45% MHC. The percentage survival of the pathogen in pre-colonized substrates buried in soil was assessed as described earlier. S50 values were also calculated.

S50 value, ie.

The period up to which 50% survival of the test organism buried in the soil and sampled determined by taking the incubation period on the „X‟ axis and the percentage of survival on the „Y‟ axis.

3.6.4. Effect of soil temperature The survival of pathogen in the pre-colonized chilli straw units buried in soil and incubated at 15°, 30° and 42±2°C was studied as described previously. The moisture content of the soil was maintained at 45% MHC. S50 values were also determined.

3.7. Control of the pathogen using antagonistic fungi by dual culture technique Colony interactions between the test organism and the soil fungi were studied in vitro in dual culture experiments. The organism Pythium debaryanum and the soil fungi such as Aspergillus flavus, A. fumigatus, A. sydowi, A. niger, A. sulphureus, A. fumigatus, Penicillium sp. Trichoderma harzianum, T. koeningii and T. viride were grown separately on PDA medium.

Then agar blocks cut from the actively growing

margin of the individual species of soil fungi and test organism were inoculated just

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opposite to each other approximately 3cm apart. Three replicates and respective control for each set were maintained.

The growth rate of both test fungus and antagonistic fungi were recorded at 24 h intervals. Assessment was made when the fungi had achieved an equilibrium after which there was no further alteration in the growth. Since both of the organisms were mutually inhibited, the assessment was made for both organisms.

The percentage inhibition of growth was calculation as follows. r – r1 Percentage inhibition of growth =

X 100 r

r

=

growth of the fungus was measured from the centre of the colony towards the

centre of the plate in the absence of antagonistic fungus. r1 =

growth of the fungus was measured from the centre of the colony towards the

antagonistic fungus. The colony interactions between the test pathogen and the soil fungi were assessed following the model proposed by Porter (1924) and Dickinson and Broadman (1971). Five types of interaction grades as proposed by Skidmore and Dickinson (1976) have been used. They are as follows: 1. Mutual intermingling growth without any microscopic sights of interaction – Grade 1. 2. Mutual intermingling growth where the growth of the fungus is ceased, and is being over grown by the opposed fungus – Grade 2.

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3. Intermingling growth where the fungus under observation is growing into the opposed fungus either above or below – Grade 3. 4. Slight inhibition of both the interacting fungi with a narrow demarcation line (12 mm) – Grade 4. 5. Mutual inhibition of growth at a distance of >2mm – Grade 5.

3.8. Antifungal activity of some medicinal plants against P. debaryanum The plants were collected from the non-irrigated cultivated lands in and around Thanjavur (Dt.), Tamilnadu.

Medicinal plants species such as Aloe vera Linn,

Alternanthera sessilis R. Br., Lawsonia inermis Linn, Mimosa pudica L., Murraya koeningii Spreng, Pithecolobium dulce Benth, Phyllanthus niruri L., Tephrosia purpurea Pers., Vinca rosea L. and Vitex negundo Linn. were taken for the antifungal study.

3.8.1. Sterilization of plant materials The disease free and fresh plants were selected. About 2g of fresh and healthy leaves were taken for each solvent extraction. They were washed with distilled water for three times. Then surface sterilized with 0.1% mercuric chloride for 20 seconds. Again the leaves were washed thoroughly with distilled water (three times).

3.8.2. Preparation of plant extracts Two grams of sterilized plant leaves were kept in the 10 ml organic solvents such as n-butanol, methanol and aqueous. Then they were ground well with the help of Mortar and Pestle. The plant materials were subjected to centrifugation, for 10-15 min

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(at 10000 rpm) again it was filtered through whatman No. 1 filter paper.

The

supernatant was collected and made to known volume, by adding sterile n-butanol, methanol and aqueous stored for further antifungal screening purpose.

3.8.3. Microbial cultures and growth conditions The plant extracts were assayed for antifungal activity against the fungal strain Pythium debaryanum isolated from chilli field soil. This fungus was grown on PDA plate at 28°C and maintained with Periodic sub-culturing at 4°C.

Potato Dextrose Agar (PDA) Medium (pH 6.7) Potato

-

250g

Dextrose

-

15g

Agar

-

18g

Distilled water -

1000ml

The potato tubers were peeled off and weighed for about 250 g tubers were chopped into small pieces into the sterile conical flask. After boiling the supernatant were collected and dextrose (15g) with agar (18g) to dissolve the ingredients. The pH of the medium was adjusted to 6.7. Finally the medium was sterilized in pressure cooker for 20 min.

3.8.4. Screening for antifungal assay Antifungal activity was screened by agar well diffusion method (Perez et al., 1990). The n-butanol, methanol and aqueous extracts of ten different medicinal plants

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were tested against plant pathogen Pythium debaryanum.

The PDA medium was

poured in to the sterile petriplates and allowed to solidify. The test fungal culture was evenly spread over the media by sterile cotton swabs. Then wells (6 mm) were made in the medium using sterile cork borer. 200µl of each extracts were transferred into the separate wells. The plates were incubated at 27°C for 48 – 72 hrs. After the incubation the plates were observed for formation of clear incubation zone around the well indicated the presence of antifungal activity. The zone of inhibition was recorded.

3.8.5 Antibiotic sensitivity test on fungi (Positive control) The antibiotic sensitive test using standard antibiotics (Amphotericin B, Griseofulvin and Fluconazole) were analysed by agar well diffusion method.

3.8.6 Antifungal effect of solvents (Negative control) The antifungal activity of methanol, n-butanol and aqueous were tested against the selected fungal strain.

3.9. Separation of bioactive compounds from T. viride by using TLC (Thin Layer Chromatography) The general principle was similar to that of column chromatography i.e., adsorption chromatography. In this adsorption process, the solution completes with the solvent for the surface sites of the adsorbent.

Depending on the distribution

coefficients, the compounds used to distribute on the surface of the adsorbent. The adsorbent normally used contains a binding agent such as calcium sulphate which facilitate the holding of the adsorbent to the glass plates.

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3.9.1. Thin layer plate preparation The stationary phase (Silica gel) was prepared as a slurry with water (or) buffer at 1:2 ratio and it was applied to a glass plate or it was inserted into a plastic (or) Aluminium sheet by using glass rod (or) pipette (or) using TLC applicator (0.25mm) thickness for analytical separation and 2.5m thickness for preparative separations are prepared.

Calcium sulphate (CaSO4) ½ H2O (Gypsum) (10.15%) was incorporated to the adsorbent it was a binder, as it facilitates the adhesion of the adsorbent to the plate. After application of adsorbent, the plates were air dried for 10-15 min. The process is also known as activation of the adsorbent. The plates could be used immediately or stored in desiccators.

3.9.2. Sample preparation 3.9.2.1. Flavonoids About 2g fungal culture was filtered with 10 ml of ethanol. Then the extract was heated for few min and 100µl of extract was applied on the silica gel plates.

3.9.2.2. Phenols Two gram fungal culture filtrate with 10 ml of methanol on rotary shaker (180 thaws/min) for 24 hours. Then these extract was filtered by using Whatmann No.1 filter paper. The condensed filtrate was used for TLC (Harborne, 1998).

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3.9.2.3. Saponins Two grams of fungal culture filtrate with 10ml of 70% ethanol was refluxed for 10 min. Then these extract was filtered by using Whatmann No.1 filter paper. The filtrate was condensed, enriched with saturated n-butanol and thoroughly mixed (Wagner and Bladt, 1996).

3.9.2.4. Sterols Two gram of fungal culture filtrate with 10 ml of methanol was kept in water bath at 80°C for 15 min. The condensed filtrate was used for TLC (Wagner and Bladt, 1996).

3.9.2.5. Tannins About 2g of fungal culture filtrate with 10 ml of ethanol was refluxed by using Whatmann No. 1 filter paper. The filtrate was condensed and used for TLC (Harborne, 1998).

3.9.3. Sample application Draw a line lightly with a pencil about 1.5 – 2.0 cm from the bottom. If the thin layer is too soft to draw a pencil line place a scale at the bottom and mark a spot at a distance of 1cm. The samples were spotted using capillary tubes at 1.5cm distance between them. For preparative TLC, the sample was applied as a band across the layer, rather than as a spot.

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3.9.4. Solvent preparation 3.9.4.1. Flavonoids Flavonoids were separated by using butanol, acetic acid and water (4:1:5) solvent mixture.

3.9.4.2. Phenols The phenols were separated by using chloroform and methanol (27:3) solvent mixture (Harborne, 1998).

3.9.4.3. Saponins The saponins were separated by using chloroform, glacial acetic acid, methanol and (64:34:12:8) solvent mixture (Wagner and Bladt, 1996).

3.9.4.4. Sterols The sterols were separated by using acetone, glacial acetic acid, methanol and water (64:34:12:8) solvent mixture (Wagner and Bladt, 1996).

3.9.4.5.Tannins The tannins were separated by using n-butanol, glacial acetic acid and water (4:1:5) solvent mixture (top layer) (Harborne, 1998).

3.9.5. Plate development The chromatographic tank was filled with developing solvent to a depth of 1.5cm and equilibrated for about 5 hours. The thin layer plate was placed gently in the

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tank and allowed to stand for about 60 min. Care was taken so that the spots did not touch the solvent directly and the capillary action caused the solvent to ascent as in paper chromatography and the separation of compounds took place. As the solvent front reached about 1.2 cm from the top of the plate, the plate was removed, solvent front was marked with a pencil immediately and allowed to air dry placing the plate upside down.

3.9.6. Component detection Several methods were available to detect the separated compounds. Different types of specifying reagents were used to detect different components.

3.9.6.1. Flavonoids The presence of flavonoid in the developed chromatogram was detected by spraying the folin-ciocalteu‟s reagent. The plates were then heated at 80ºC for 10 min. A positive reaction was indicated by the formation of yellow colour spot.

3.9.6.2. Phenols The presence of phenol in the developed chromatograms was detected by spraying the folin-ciocalteu‟s reagent. The plates were then heated at 80°C for 10 min. A positive reaction was indicated by the formation of blue colour spot.

3.9.6.3. Saponins The presence of saponin in the developed chromatograms was detected by iodine vapours. A positive reaction indicated by the formation of yellow colour spot.

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3.9.6.4. Sterols The presence of sterol in the developed chromatogram was detected by spraying the folin-ciocalteu‟s reagent. The plates were then heated at 80°C for 10 min. A positive reaction was indicated by the formation of blue spot.

3.9.6.5. Tannins The presence of tannin in the developed chromatogram was detected by spraying 10% ferric chloride in ethanol solution. A positive reaction was indicated by the formation of blue colour spot.

3.9.7. Determination of RF value The RF values of the various compounds were calculated by using the following formula. After incubation period, the results were observed and the diameter of the inhibition zone was measured around the isolates.

Distance travelled by solute (measured to centre of the spot) RF = Distance travelled by solvent

3.10.

Detection of various functional groups by FTIR analysis of phenol from

T. viride (Yong et al., 2003) The functional groups of bioactive compounds were carried out by Fourier transform infra red spectroscopy (FTIR) and ultraviolet scanning (UV).

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3.10.1. Ultraviolet scanning The fractionated sample was dissolved in acetonitrite and then detected its UV absorption values with Lambda 35 Ultraviolet scanner.

3.10.2. Infrared scanning A small quantity of solid sample product was collected, ground adequately and was pressed to tablet with KBr method. Infrared spectrum of sample product was recorded in the range of 4000 – 400cm-1 using Perking Elmer R x 1 infrared scanner.

3.11 Gas chromatography – Mass Spectrometry analysis (Liu et al., 2007) 3.11.1 Sample preparation The selected fungal cultures were inoculated in a liquid Potato dextrose agar medium and incubated for 7 days at 24±2°C. After incubation, the mycelial were separated by whatman No.1 filter paper and the filtrates were filtered through Millipore filter. An equal volume of ethanol was added to the culture filtrates in separate flasks, vortex mixed, centrifuged and the supernatants were extracted and used for Gas Chromatography – Mass Spectrometry analysis.

3.11.2. Identification of bioactive compounds Volatile components were identified by GC-MS using a column Elite-1 (100% Dimethyl poly siloxane), 30 x 0.25 mm x 1 µm df equipped with GC clarus 500 Perkin Elmer. The turbo mass-gold-perkin-Elmer detector was used. The carrier gas flow rate was 1ml per min, split 10:1, and injected volumes were 2µl. The column temperature was maintained initially at 110°C for 2 min (hold) followed by increases upto 200°C at

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the rate of 10°C / min (no hold), upto 280°C at the rate of 5°/min-9 min (hold). The injector temperature was 250°C and this temperature was held constant for 36 min. The electron impact energy was 70 eV Jule, line temperature was set at 200°C and the source temperature was set a 200°C. Electron impact (EI) mass scan (m/z) were recorded in the 45 – 450 aMU range.

Using computer searches on the NIST Ver.2.1 MS data library and comparing the spectrum obtained through GC-MS the compounds present in the crude sample were identified.

3.12.

Molecular characterization of fungi

3.12.1. Isolation of chromosomal DNA. Fungal mycelium 0.01 g was ground with mini grinder using 75µ l of STE extraction buffer (320 mM

Sucrose, 10mM Tris-Cl, 20mM EDTA,

75mM NaCl and 2.5ml of 20% SDS) along with 5mg of Polyvinyl pyrolidone and 0.1g of silica powder, incubated at 65°C for 10 minutes. Centrifuged the sample at 13,000 rpm for 10 minutes. To the supernatant, equal volume of chloroform: isoamyl alcohol was added and repeated the centrifugation. To the aqueous layer, added 2/3 volume of isopropanol and centrifuged at 13,000 rpm for 10 min. The pellet was washed with 70% ethanol by centrifuging and the pellet was dried, dissolved in 50µ l TE buffer.

3.12.2. Analysis of DNA purity and quality The DNA stock samples were quantified using UV spectrophotometer at 260 and 280 nm using the convention that one absorbance unit at 260 nm Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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wavelength equals 50 µg DNA per ml. The Ultra violet (UV) absorbance was checked at 260 and 280 nm for determination of DNA concentration and purity. Purity of DNA was judged on the basis of optical density ratio at 260:280 nm. The DNA having ratio between 1.8 to 2.0 was considered to be of good purity. Concentration of DNA was estimated using the formula. Concentration of DNA (mg/ml) = OD 260 x 50 x Dilution factor

Quality of DNA was again checked by agarose gel electrophoresis. The 0.8 % agarose was prepared (0.8 g agarose power / 100ml 1 X TBE), and was melted. 30 ml agarose was poured into the casting tray. The gel was allowed to solidify and the comb and tape was removed. 1 X TBE (Tris-Borate-EDTA; electrophoresis buffer) was added to the chamber until the buffer just covers the top of the gel. The samples were loaded with Bromophenol blue loading dye, taking care not to puncture the well bottoms. The power pack was turned on and run

at

100V. The gel was viewed on a UV transilluminator after

electrophoresis. The DNA was used further for PCR.

3.12.3 PCR amplification of 18S rRNA ITS fragment was amplified by PCR from fungal genomic DNA using ITS-PCR universal primers: Details of primers used for PCR ITS 1: 5 ‟-TCCGTAGGTGAACCTGCGG-3‟ ITS4: 5‟- TCCTCCGCTTATTGATATGC-3‟ PCR was carried out in a final reaction volume of 25 µ l in 200 µ l capacity thin

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wall PCR tube. PCR tubes containing the mixture were tapped gently and spin briefly at 10,000 rpm. The PCR tubes with all the components were transferred to thermal cycler. The PCR protocol designed for 30 cycles for the primers used is given below. Preparation of Reaction mix for PCR Components

Step

Vol. per reaction

Deionized water

16.5

Taq buffer without MgC12 (10X)

2.5µl

MgCl2 ( 15 Mm)

1.0µl

Forward Primer (10pm/µl)

2.0µl

Reverse Primer (10 pm/µ l)

0.5µl

Taq DNA Polymerase (5U/µl)

0.5µl

Template DNA(20 ng/µl)

0.5µl

Final Volume

25.0µl

Process

Temperature

Time

1.

Initial denaturation

95°C

5 minutes

2.

Denaturation

94°C

30 seconds

3.

Annealing

55°C

30 seconds

4.

Extension

72°C

45 seconds

Go to step 2 for 29 times 5.

Final Elongation

72°C

10 minutes

End

3.12.3.1. Analysis of DNA Amplification by AGE - Standard DNA Markers Commercially available 100bp ladder was used as standard molecular weight DNA.

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3.12.3.2. PCR-Product electrophoresis Loaded 5 µl of PCR product with 4 µl bromophenol blue (Loading Dye) in 1.5% agarose gel. Ran the gel at constant voltage of 100 V and current of 45°A for a period of 1 hr 20 min till the bromophenol blue

has travelled 6 cms

from the wells. Viewed the gels on UV transilluminator and photograph of the gel was taken.

3.12.3.3. Purification and DNA sequencing of samples Amplified PCR product was purified using column purification as per manufacturer‟s guidelines, and further used for sequencing reaction.

3.12.3.4. Sequencing of purified ITS gene Segment The concentration of the purified DNA was determined and was subjected to automated DNA sequencing on ABI3730xl Genetic Analyzer (Applied Biosystems, USA).

3.12.3.5 ITS sequence analysis Each nucleic acid sequence was edited manually to correct falsely identified bases and trimmed to remove unreadable sequence at the 3 ‟and 5‟ ends (considering peak and Quality Values for each base) using the sequence analysis tools. The

edited

similarity

using BLAST

searches

sequences

(ITS

gene)

(Basic Local

were

then

used

for

Alignment Search Tool)

programme in the NCBI GenBank (www.ncbi.nlm.nih.gov) DNA database for identifying the fungal strains.

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3.12.4. Phylogenetic analysis of ITS gene in T. viride The D2 region of 18S rRNA gene sequence was used to to carry out BLAST with the NR database of NCBI genebank database (URL http://www,ncbi.n/m.nih.g). Based on maximum identity scores first ten sequence were selected and Global pair wise sequence similarity between the sequence were performed using Needleman and Wunsuh algorithm available with the emboss sequence analysis suite.

Multiple

sequence analysis were performed using alignment program CLUSTAL W the phylogenetic tree was constructed using MEGA 4.

The evolutionary history was inferred using the neighbour joining method of Saitou and Nei (1987). The bootstrap consensus tree inferred from 500 replicated was taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicated tree in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches; the evolutionary distance were computed using the Kimura 2- parameter method (Kimura, 1980) and are in the units of the number of base substation gaps substations per site Codon position included were 1st + 2nd + 3rd + Noncoding. All position gaps and missing data were eliminated from the dataset (complete deletion option). There were a total of 663 positions in the final dataset. Phylogenetic analyses were conducted in MEGA 4 (Tamura et al., 2007). Tree visualization was done with the tree view program.

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3.12.5. 18S rRNA Secondary structure prediction of T. viride The secondary structure of Trichoderma viride were predicted using the Bioinformatics

tools

available

in

online

www.genebee.msu.su/service/ma2-

reduced.html.

3.12.6. Restriction site analysis of 18S rRNA of T. viride The restriction sites in 18S rRNA of Trichoderma viride were analyzed using NEB cutter programme version 2.0 in online www.neb.com/NEBcutter2/index.phb.

3.13. Application of Trichoderma sp. 3.13.1. Seed treatment Three petriplates are taken, each plates contain 50 chilli seeds and surface sterilized with distilled water. Then surface sterilized chilli seeds mixed with 10 ml of T. viride (T1), T. harzianum (T2) and distilled water (C) poured in to each petriplates. Then the plates were incubated at 24-48 hrs. at 35°C. After incubation seeds were allowed to air dry over night under aseptic conditions and sown in the growth cabinet. The growth cabinet were watered with tap water when required. Then germinated seedlings were maintained in three growth cabinets. (Three replicates maintained) labelled as C, T1, T2 respectively control, T. viride, T. harzianum. Finally percentage of germination, shoot length and root length were recorded.

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3.13.2. Root dipping (Islam and Bora, 1998) Root dipping method was adopted for the application of biocontrol agent to the chilli plant. The diluted liquid culture was used to dip the roots of chilli seedlings and allowed for 10 min. Then the seedlings were transplanted in to the pots.

3.13.2.1. Time of observation The data were observed between 30th , 60th, 90th and 120th days after planting of chilli crops. The following parameters of the pot culture in chilli crops were observed in the present investigation.

3.13.2.2. Shoot length The shoot length of all plants was measured from the soil level up to the 1st leaf of the plant and the result were recorded in centimeter.

3.13.2.3. Root length The root length of all plants was measured and the results were recorded in centimeter.

3.13.2.4. Weight of fruits The total number of fruits were collected in each plant, weighed and results are tabulated.

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4.

RESULTS

This chapter includes the results of the experiments conducted on physicochemical characteristics, correlation co-efficient between physico-chemical characters and the total number of species, monthly variation in the population of soil fungi in chilli field of Thiruvarur district taluk wise.

4.1. Monthly variation in the population of soil fungi in chilli field. Totally seven taluks were investigated to determine mycoflora diversity in the chilli field of Thiruvarur district. Fungal population density, species diversity and percentage frequency were also determined and recorded for all the taluks individually.

In all the seven taluks, totally 46 species beloning to 18 genera (5 Phycomycetes, 2 Ascomycetes and 39 Deuteromycetes) were identified in the present study (Plate II - IX), they are as follows:

Phycomycetes 1. Absidia glauca Hagem 2. Rhizopus sp. 3. Rhizopus nigricans Ehrenberg 4. Syncephalastrum sp. 5. Pythium debaryanum Hesse

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Ascomycetes 6. Chaetomium globosum Kunze 7. Chaetomium sp. Deuteromycetes 8.

Aspergillus awamori Kawachi

9.

A. conicus Blochwitz

10. Alternaria alternata Keissl 11. Aspergillus flavipes Bainier and Sartory 12. A. flavus Link 13. A. fumigatus Fresenius 14. A. funiculosum Thom 15. A. granulosis Raper and Thom 16. A. humicola Chaudhuri 17. A. luchuensis Inui 18. A. niger Van Tieghem 19. A. nidulans Winter 20. A. ochraceous Wilhelm 21. A. repens (Corda) de Bary 22. A. sydowi Thom and Church 23. A. sulphureus Thom and Church 24. A. terreus Thom 25. A. versicolor Tiraboschi 26. A. wentii Wehmer 27. Acrophiolophora fusispora (S.B. Saksena) Samson

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28. Botrytis cinera Persoon 29. Cladosporium herbarum Link 30. Cladosporium sp. 32. Curvularia lunata (Walker) Boedijn 32. C. geniculata Boedijn 33. Fusarium oxysporum Schlechtendahl. 34. F. semitectum Barkelay and Ravenel 35. Helminthosporium oryzae Breda de Haan 36. Humicola sp. 37. Masoniella sp. 38. Penicillium citrinum Thom 39. P. chrysogenum Thom 40. P. janthinellum Biourge 41. P. javanicum Van Beyma 42. P. turbatum Westling 43. Trichoderma harzianum Rifai 44. T. viride Per-ex.S.F. Gray 45. T. koeningii Oudemans 46. Torula alli Saccardo

4.1.1.Thiruvarur A total of 18 species of fungi was isolated from the soil of chilli field. The total number of colonies isolated varied from 20.1 to 55.2 (X103g-1 dry wt. of the soil). The maximum number of colonies were found in the month of December. There was a

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decline in the number of colonies during the month of July and August. Qualitatively, the species spectrum of fungi was more during the month of November and December when compared with the number of species isolated during other months. Among the different species of fungi, the species Aspergillus, Penicillium was frequently isolated. The percentage frequency is as follows; Pythium debaryanum (100%), Cladosporium herbarum (91.6%), Aspergillus niger, A. wentii and Torula alli (83.3%), Alternaria alternata, A. conicus, A. terreus and Fusarium oxysporum (75.0%) (Table 1).

4.1.2. Nannilam A total of 16 species of fungi was isolated from the soil of chilli field of Nannilam. The total number of colonies isolated varied from 17.8 to 46.6 (X103g-1 dry wt. of the soil). The maximum number of colonies was found in the month of December. There was a decline in the number of colonies during the month of March and August. Qualitatively, the species spectrum of fungi was the maximum in the month of November and December. The percentage frequency is as follows; Pythium sp. (100%), Aspergillus niger (91.6%), A. fumigatus, A. granulosis and Penicillium chrysogenum (83.3%), A. awamori, A. versicolor, Penicillium janthinellum (75%) respectively (Table 2).

4.1.3. Kodavasal A total of 16 species of fungi was isolated from the chilli field soil of Kodavasal. The total number of colonies isolated varied from 17.4 to 40.2 (X103g-1 dry wt. of the soil). The maximum number of colonies was recorded in the month of November whereas minimum number of colonies was found in the month of August and October.

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Among the different species of fungi, the species Aspergillus was frequently isolated. The percentage frequency is as follows; Pythium debaryanum (91.6%), Aspergillus nidulans, A. versicolor, Curvularia geniculata and Penicillium citrinum (83.3%), A. flavus and Curvularia lunata (75%) were isolated respectively (Table 3).

4.1.4. Valangaiman A total of 21 species of fungi was isolated from the chilli field soil of Valangaiman. The total number of colonies isolated varied from 27 to 50.3 (X103g-1 dry wt. of the soil). The maximum number of colonies was recorded in the month of January and the minimum in the month of February and September.

Among the

different species fungi, the species Aspergillus, Pythium was frequently isolated. The percentage frequency of Pythium debaryanum (100%), Aspergillus nidulans, A. niger and Penicillium funiculosum (83.3%), Absidia glauca, A. flavus, A. luchuensis, A. sydowi, A. terreus, Helminthosporium oryzae and Penicillum turbatum. (75%) respectively (Table 4).

4.1.5. Mannargudi A total of 15 species of fungi was isolated from the chilli field soil of Mannargudi. The total number of colonies isolated varied from 20.5 to 39.9 (X103g-1 dry wt. of the soil). The maximum number of colonies was found in the month of December.

There was a decline in the number of colonies during the month of

February and March. Qualitatively, the species spectrum of fungi was more during the month of December and November, when compared with the number of species isolated during other month. Among the different species of fungi, the species of Aspergillus

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and Penicillium

were frequently isolated.

The percentage frequency of Pythium

debaryanum and A. sulphureus (83.3%), A. luchuensis, A. fumigatus, P. janthinellum (75.0%) were isolated respectively (Table 5).

4.1.6. Needamangalam A total of 15 species of fungi was isolated from the chilli field soil of Needamangalam. The total number of colonies isolated varied from 17 to 38.3 (X103g-1 dry wt. of the soil). The maximum number of colonies were recorded in the month of November, where as minimum in the month of July and March. Among the different species Aspergillus sp. was the dominant of all species. The percentage frequency of Pythium debaryanum (83.3%), Acrophiolophora fusispora and Penicillium funiculosum (75%) respectively (Table 6).

4.1.7. Thiruthuraipoondi A total of 19 species of fungi was isolated from the chilli field soil of Thiruthuraipoondi. The total number of colonies isolated varied from 17.2 to 47.4 (X103g-1 dry wt. of the soil). The maximum number of colonies were found in the month of January, There was decline in the number of colonies during the month of May when compared with the number of species isolated during other months. Among the different species of fungi, the species of Aspergillus, Penicillium and Trichoderma were frequently isolated. The percentage frequency of Pythium debaryanum (91.6%), Penicillium chrysogenum, P. janthinellum and Trichoderma harzianum (83.3%), Chaetomium globosum, Rhizopus nigricans, T. viride and T. koeningii (75%) respectively (Table 7).

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4.2. Physico-chemical characteristics of soil The results of Physico-chemical properties of chilli field soil samples of Thiruvarur district talukwise are given below.

4.2.1. Thiruvarur The physico-chemical characteristics of Thiruvarur soil samples revealed that the pH level was recorded between the ranges of 7.3 to 8.2. Electrical conductivity showed about 0.21 to 1.26 dsm-1, Organic carbon ranges from 0.18 to 0.58%, Organic matter ranges of 0.31 to 0.99%, available nitrogen from 86.2 to 142.6 kg/ac, available phosphorus showed about 3.13 to 5.25 kg/ac, available potassium ranges from 111 to 189 kg/ac, The soil fractions such as fine sand ranges showed about 42.25 to 45.69%, coarse sand ranges from 18.96 to 25.69%, silt ranges from 15.69 to 21.65%, and clay ranges from 8.52 to 19.07% where as cation exchange capacity ranges about 12.3 to 24.6 mole proton+/kg respectively. The micronutrients such as zinc 0.52 to 1.26 ppm, copper ranges from 0.72 to 1.36 ppm, iron ranges from 2.41 to 9.64 ppm and manganese ranges from 1.38 to 3.68 ppm. The calcium content recorded between the ranges of 5.8 to 14.6 mg/kg, magnesium ranges from 6.2 to 12.6 mg/kg, sodium ranges from 0.41 to 2.75 mg/kg and potassium ranges about 0.18 to 0.36 mg/kg respectively. (Table 8).

4.2.2. Nannilam The ranges of variation in the pH of the soil was narrow. The soil pH was recorded between the ranges of 7.3 to 8.1. Electrical conductivity of the soil was about 0.21 to 1.15 dsm-1. The organic carbon content of the soil was in the ranges from 0.22 to 0.56%, organic matter ranges from 0.37 to 0.96%, available nitrogen ranges about

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96.5 to 128.9% and available potassium ranges from 102 to 165 kg/ac. Soil fractions such as fine sand ranges about 41.20 to 48.54%, coarse sand 17.69 to 24.57%, silt from 16.68 to 22.35% and clay showed about 5.53 to 19.60% where as cation exchange capacity ranges from 12.6 to 23.4 mole proton+/kg. The micronutrients of soil revealed the zinc ranges about 0.32 to 1.56 ppm, copper 0.53 to 1.52 ppm, iron 2.36 to 8.79 ppm and manganese from 1.37 to 3.65 ppm. The calcium content was ranges from 6.9 to 13.8 mg/kg, magnesium showed about 6.3 to 12.8 mg/kg, sodium content ranges from 0.38 to 2.79 mg/kg and potassium was ranges about 0.18 to 0.32 mg/ka respectively. (Table 9).

4.2.3. Kodavasal The physico-chemical characteristics of Kodavasal soil samples revealed that the pH level was in the ranges from 7.18 to 8.69, EC from 0.32 to 1.36 dsm-1, organic carbon from 0.28 to 0.58%, organic matter from 0.48 to 0.99%, available phosphorus from 3.10 to 5.36kg/ac, available potassium from 112 to 182 kg/ac. The micro nutrients of soil revealed the zinc from 0.45 to 1.45 ppm, copper ranges from 0.47 to 1.58 ppm, iron from 2.45 to 8.78 ppm, manganese from 1.29 to 3.54 ppm. Soil fractions such as fine sand from 41.26 to 47.50%, coarse sand from 18.69 to 26.54%, silt from 15.16 to 20.36% and clay ranges from 11 to 19.85%. The cation exchange capacity from 6.9 to 24.5 mole proton+/kg, where as calcium ranges about 5.9 to 16.6 mg/kg, magnesium ranges from 5.7 to 11.6 mg/kg, sodium ranges about 0.15 to 2.89 mg/kg and potassium from 0.15 to 0.34 mg/kg were observed (Table 10).

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4.2.4. Valangaiman The physico-chemical characteristics of Valangaiman soil samples revealed that the pH level was in the ranges from 7.26 to 8.26, EC from 0.15 to 0.74 dsm-1, organic carbon from 0.18 to 0.52%, organic matter from 0.31 to 0.89%, available nitrogen ranges from 82.2 to 135.9 kg/ac, available phosphorus from 3.01 to 4.69 kg/ac and available potassium from 106 to 159 kg/ac. The micronutrients of soil revealed that the zinc content was in the ranges from 0.29 to 1.38 ppm, copper from 0.45 to 1.36 ppm, iron in the ranges from 0.45 to 7.75 ppm and manganese from 1.29 to 3.58 ppm. The soil fractions such as fine sand ranges from 42.16 to 48.36%, coarse sand from 18.95 to 24.79%, silt from 16.39 to 23.23% and clay from 11.26 to 19.90%.

The cation

exchange capacity ranges from 15.3 to 26.4% mole proton+/kg. The calcium ranges from 6.5 to 14.5 mg/kg, magnesium from 5.5 to 12.9 mg/kg, sodium from 0.29 to 2.56 mg/kg and potassium 0.21 to 0.48 mg/kg. (Table 11).

4.2.5. Mannargudi The Mannargudi soil samples revealed that the pH level was in the ranges from 7.2 to 8.4, EC from 0.32 to 1.25 dsm-1, organic carbon from 0.24 to 0.54% organic matter from 0.41 to 0.93%, available phosphorous from 3.15 to 4.75 kg/ac, available potassium from 118 to 160 kg/ac. The micronutrients of soil revealed the zinc from 0.37 to 1.25 ppm, copper from 0.69 to 1.36 ppm, iron from 2.18 to 9.62 ppm, manganese from 1.32 to 3.93 ppm. The soil fractions such as fine sand from 41.16 to 48.62%, coarse sand from 17.68 to 24.97%, silt from 16.42 to 24.65% and clay 17.12 to 19.14%. The cation exchange capacity ranges from 14.1 to 27.6 mole proton+/kg. Then

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the calcium ranges from 7.0 to 15.6 mg/kg, magnesium from 6.2 to 13.2 mg/kg, sodium from 0.37 to 2.98 mg/kg and potassium 0.13 to 0.36 mg/kg respectively (Table 12).

4.2.6. Needamangalam The physico-chemical characteristics of Needamangalam soil samples revealed that the pH level was in the ranges from 7.2 to 8.4, EC from 0.16 to 1.23 dsm-1, organic carbon from 0.18 to 0.53%, organic matter from 0.31 to 0.91%, available nitrogen ranges from 83.3 to 142.0 kg/ac, available phosphorus from 3.16 to 4.78 kg/ac, available potassium in the ranges from 105 to 162 kg/ac. The micronutrients of soil revealed the zinc content was in the ranges from 0.34 to 1.26 ppm, copper from 0.58 to 1.50 ppm, iron in the ranges from 2.28 to 5.63 ppm, manganese from 1.24 to 3.78 ppm. The soil fractions such as fine sand from 40.29 to 45.21%, coarse sand from 19.25 to 25.45%, silt from 18.21 to 22.58% and clay from 12.23 to 18.83%.

The cation

exchange capacity from 14.9 to 27.4 mole proton+ /kg. The calcium content was in the ranges from 6.3 to 14.3 mg/kg, magnesium from 5.7 to 13.5 mg/kg, sodium from 0.39 to 2.75 mg/kg and potassium from 0.21 to 0.28 mg/kg respectively (Table 13).

4.2.7. Thiruthuraipoondi The physico-chemical characteristics of Thiruthuraipoondi soil samples revealed that the pH level was in the ranges from 7.2 to 8.1, EC from 0.26 to 1.20 dsm-1, organic carbon from 0.27 to 0.56%, organic matter from 0.46 to 0.96%, available nitrogen from 94.6% to 152.3 kg/ac, available phosphorus in the ranges from 3.25 to 4.75 kg/ac and available potassium 119 to 189 kg/ac. The micronutrients of soil such as zinc from 0.32 to 1.35 ppm, copper from 0.38 to 1.30 ppm, iron from 2.35 to 4.63 ppm and manganese

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1.26 to 3.56 ppm. The soil fractions such as fine sand ranges from 41.20 to 45.63%, coarse sand from 18.65 to 26.5%, silt from 16.39 to 20.16% and clay from 9.46 to 21.05%, where as the cation exchange capacity from 15.4 to 27.3 mole proton+/kg. Then the calcium content was in the ranges from 7.6 to 14.8 mg/kg, magnesium from 5.8 to 12.8 mg/kg, sodium from 0.36 to 2.85 mg/kg and potassium from 0.11 to 0.23 mg/kg respectively (Table 14).

4.3.

Correlation coefficient between physico-chemical parameters and the total

number of species The results of statistical analysis between the population of mycoflora and physico-chemical parameters of Thiruvarur district talukwise are given below.

4.3.1. Thiruvarur In Thiruvarur taluk, the correlation co-efficient analysis between the physicochemical properties and the total number of fungal species are statistically not significant at 0.05% level. (Table 15)

4.3.2. Nannilam The correlation coefficient between the physico-chemical character and the total number of species were also made. The significant positive correlation was observed between the total number of fungal species (TNS) and the available potassium (APO) (0.621≥0.05). Total number of species (TNS) and available copper (AC) (0.630≥0.05), total number of species (TNS) and cation exchange capacity (CEC) (0.621≥0.05), total number of species and magnesium (MG) (0.603≥0.05) were observed. (Table 16)

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4.3.3. Kodavasal The correlation co-efficient analysis between the physico-chemical character and total number of species were made. The significant positive correlation was observed between the total number of species (TNS) and the available phosphorous (AP) (0.683≥0.05), available potassium (APO) (0.647≥0.05) (Table 17).

4.3.4. Valangaiman The correlation co-efficient analysis between the physico-chemical parameter and total number of fungal species in chilli field soil was also made. The positive correlation was observed between total number of fungal species (TNS) and available nitrogen (AN) (0.678 ≥ 0.05) (Table 18).

4.3.5. Mannargudi The correlation co-efficient analysis between the physico-chemical character and total number of species was also made. It was statistically not significant at 0.05% level (Table 19).

4.3.6. Needamangalam The correlation co-efficient analysis between the physico-chemical parameter and the total number of fungal species in chilli field soil was also made. The positive correlation was observed between the total number of fungal species (TNS) and clay (0.593 ≥ 0.05) (Table 20).

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4.3.7. Thiruthuraipoondi The correlation co-efficient analysis between the physico-chemical characters and the total number of fungal species in the chilli field soil was also made. The significant positive correlation was observed between the total number of species (TNS) and available potassium (APO) (0.630≥0.05), cation exchange capacity (CEC) (0.619≥0.05) and magnesium (MG) 0.672 ≥ 0.05) (Table 21).

4.4. Identification of causative organism Based on the cultural and morphological characters the pathogen was identified as Pythium debaryanum using standard manual of soil fungi (Plate 1).

Characteristics of the pathogen Hyphae are large, branching, irregular, free and septate.

In old cultures,

sporangia are spherical or oval. Conidia are usually numerous, intra and extramatrical, 15-25µ in diameter, round and oval. Oogonia usually numerous intra-and extramatrical, 20-55µ in diameter, spherical and terminal. Antheridia are upto three in number, from the same or another hypha as the oogonium, often formed close below the latter and not seldom hypogynous. Oospores, 14-18µ in diameter, spherical and smooth.

4.5. Pathogenecity Test The pathogenecity test conducted revealed that the barley seed inoculum used at a rate of 30g/kg was sufficient for the pathogen to grow and cause infection in a few days (Plate X –XI).

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4.6.

Effect of soil physico-chemical factors on the saprophytic survival of

P. debaryanum 4.6.1. Effect of soil moisture on the saprophytic survival The percentage survival of the pathogen in the dead host tissue substrate was maximum (62%) in the soil with moisture content 75% MHC and increased with increase in moisture content. It was 45 CFU in the soil with 10% MHC in the precolonized substrate recovered after 4 weeks. The percentage of survival increased with the increase in moisture content and the period of incubation. S50 values were less than 4 weeks in the soil with moisture content 10% MHC and 10 – 12 weeks in the soil with moisture content 75% MHC (Plate XII; Table 22).

4.6.2. Effect of soil pH on the saprophytic survival The saprophytic survival of test pathogen was more favoured by alkaline range than the acidic range of pH. The percentage survival of the pathogen was 50, 65, 65, 68, 65 and 48 in the pre-colonized substrates recovered from the soil adjusted to pH 4, 5, 6, 7, 8 and 9 respectively after 4 weeks of incubation. The saprophytic survival of the pathogen decreased with an increase in the period of incubation (Plate XII; Table 22).

4.6.3. Effect of temperature on the saprophytic survival The saprophytic survival of the test pathogen was maximum in the soil incubated at 42±2°C after 4 weeks of incubation. The percentage survival of the pathogen was 41, 58 and 65 in the pre-colonized substrates recovered from the soil incubated at 15±2, 30±2 and 42±2°C respectively.

S50 values decreased with the

increase in temperature (Plate XII; Table 22).

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4.7. Control of pathogen using antagonistic fungi by dual culture technique The growth of Pythium debaryanum towards the centre of the plate in the absence of antagonistic fungi (control) was 45mm and the measurement was taken after 72 hours.

The type of interactions of the pathogen with soil fungi was as follows. Aspergillus fumigatus

- Grade 1

Aspergillus sydowi

- Grade 2

A. niger, A. sulphureus, T. harzianum

- Grade 3

Penicillium sp. T. viride, T. koeningii

- Grade 4

A. flavus

- Grade 5

The maximum zone of inhibition were observed in T. viride (64.4%), followed by

T. harzianum (62.2%), T. koeningii (60.0%), A. sulphureus (60.0%), A. niger

(57.7%), Penicillium sp. (57.7%), A. sydowi (55.5%), A. flavus (55.5%), A. fumigatus (53.3%), respectively (Plate XIII – XIV; Table 23; Fig. 1).

4.8. Antifungal activity of some medicinal plants against P. debaryanum Antifungal activity of ten medicinal plants extract was assayed by agar well diffusion method. The result revealed that the extract of ten medicinal plants showed significant reduction in the growth of Pythium debaryanum.

Among all the ten plants extract the n-butanol and methanol extract of Vitex negundo exhibited maximum antifungal activity (15 and 30 mm) followed by Lawsonia inermis (15 and 25 mm), Phyllanthus niruri (15 and 20 mm), Murraya koeningii (15 and 10 mm) compared with other plants extract. The n-butanol and methanol extract of Tephrosia purpurea (10 and 15mm), Aloe vera (10 and 15mm) showed moderate activity against P. debaryanum. The methanol extract of Mimosa pudica (20mm), Pithecolobium dulce (15mm), Alternanthera sessilis (10mm), Vinca rosea (10mm) Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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exhibited least activity against Pythium debaryanum. The results of antifungal effect of aqueous extract of all tested ten plants showed no activity against P. debaryanum (Plate XV – XVIII; Table 24; Fig. 2).

4.8.1. Antibiotic sensitivity test on Pythium debaryanum The antibiotic sensitivity test using standard antibiotics viz., (Amphotericin B, Griseofulvin and Fluconazole) were tested against Pythium debaryanum.

All the

antibiotics used were exhibited antifungal activity. The results confirmed that the solvent extracts of all medicinal plants exhibited a higher antifungal activity against Pythium debaryanum when compared to the standard antibiotics. Antifungal effect of methanol, n-butanol and aqueous revealed no activity against pathogenic fungi.The results of antibiotic sensitive test were presented in Table 25 and Plate XIX

4.9. Separation of bioactive compounds from T.viride by using TLC (Thin Layer Chromatography) TLC of the extract of T.viride showed the presence of the compounds such as saponin, flavonoid, sterol, tannin and phenol and the characteristic of bioactive compounds were identified. The Rf value of bioactive compounds was recorded and given in Table 26 and Plate XX.

4.9.1. Antifungal activity of TLC fraction of mycelial extract Among the five fractions the phenol showed the highest antifungal activity with the inhibition of 12mm dia. No inhibitory effect was observed in flavonoid, sterol, tannin and saponin against Pythium debaryanum (Plate XXI).

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4.10. Detection of various functional groups by FTIR and UV analysis of Phenol from T. viride The functional groups of isolated bioactive compounds were detected by using FTIR spectroscopy analysis. The IR spectra of purified phenol compounds showed as strong bands at 3426.16 cm-1, 2074.12cm-1, 1637.83cm-1, 675.65cm-1. The functional groups of phenol compounds were presented in Table 27; Fig. 5. The UV results were supported the functional groups (Fig.6).

4.11. Gas Chromatography and Mass spectrometry analysis (GCMS) Gas chromatography Mass Spectrometry analysis of potential fungal extract of T. viride revealed the presence of four compounds by representing four prominent peaks.

The peaks with retention time of 2.092 min, corresponds to ethyl cis

13-docosenoate; 3.035 min. corresponds to olean-12-ene-1 beta, 3 beta 23-triol; 3.396 min corresponds to 1,3 dideoxy -1, 3-bis (N-methylacetamido)-myo-inositol 2,4,5,6 tetraacetate; 2.483 min corresponds to oleanolic acid. (Table 28; Fig. 3 and 4).

4.12. Molecular characterization of fungi 4.12.1.

Molecular characterization of 18S rRNA gene in T. viride by PCR

amplification The molecular characteristics of T. viride was evaluated by PCR amplification of 18S rRNA gene. The genomic DNA and amplified products were separated in agarose gel and shown in plate. (Plate XXII)

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4.12.2. 18S rRNA sequencing of T. viride The18S rRNA genes of T. viride isolated from chilli field soil was partially sequenced using specific 18S rRNA sequence primer (TCCGTAGGTGAACCTGCGG3‟ forward TCCTCCGCTTATTGATATGC-3‟ reverse primer).

4.12.3. Phylogenetic analysis of ITS gene in T. viride The 18S rRNA gene sequences of T. viride were compound with sequences obtained Gene Bank using BLAST and 100% similarity was assessed. The results revealed that the close relative to the isolates of T. viride and Trichoderma asperellum (100% similarity). Phylogenetic relatedness of the T. viride was analysed by neighbour joining method. (Fig. 7 )

4.12.4. 18S rRNA Secondary structure prediction of T. viride The secondary structure of T. viride was predicted by using Bio-informatic tools. The secondary structure of 18S rRNA of T. viride showed 23 stems, 16 bulge loops and 8 hairpin loops in their structure. The free energy structure of 18S rRNA secondary structure of T. viride were 130.1 KKal/mol respectively (Fig.8 ).

4.12.5. Restriction site analysis of 18S rRNA of T. viride Restriction site map in 18S rRNA of T. viride was analysed. The restriction sites found in the 18S rRNA are in 52 ambiguous sites. A large number of restriction enzyme sites was observed in the fungal isolate. The GC content of this species was 56% similarly the AT content of T. viride was 44% which were determined using NEB cutter program V 2.0 in online www.heb.com/NEBcutter2/indes.php. (Fig.9).

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4.13. Application of Trichoderma sp. 4.13.1. Seed treatment After sowing the seeds a maximum percentage of germination, shoot length and root length (cm) was observed in T. viride at the period of 10 days was about 99%, 5.9 and 4.8cm when compared to T. harzianum it showed about 97%, 5.2 and 4.5 cm. Then the minimum percentage of germination, shoot length and root length (cm) was observed in control plant was about 82% , 4.6 and 4.2cm respectively (Plate XXIII; Table 29).

4.13.2. Root dipping Chilli spice crops were selected to the introduction of Trichoderma viride, T. harzianum inoculants, to disease control, improvement of growth and yield traits. Among 10 antagonistic organisms, 2 were selected for pot culture experiments based on their biocontrol capacity. The following parameters such as shoot length, root length, yield were observed and tabulated (Plate XXIV; Table 29).

4.13.2.1. Shoot length After transplantation the maximum shoot length was observed in Trichoderma viride at the duration of 30th, 60th, 90th and 120th days was about 50.2, 67.0, 67.5 and 85cm when compared to T. harzianum it showed about 48.4, 62.1, 71.6 and 78cm. Then the minimum shoot length was observed in control plant is about 45.6, 56.7, 66.0 and 75.2cm respectively (Plate XXIV; Table 29).

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4.13.2.2. Root length The maximum root length was observed in T. viride at the duration of 30th, 60th, 90th and 120th days was about 14.5, 20.7, 23.5 and 24.3 cm when compared to T. harzianum it showed about 13.9, 18.9, 21.6 and 24.2cm. Then the minimum root length was observed in control plant was about 12.6, 18.7, 22.5 and 23.9 cm respectively (Plate XXIV; Table 29).

4.13.2.3. Weight of fruits The maximum fruits were recorded in T. viride at the duration of 30th, 60th, 90th and 120th days was about 720, 960, 1040 and 1250 gm, when compared to T. harzianum it showed about 695, 780, 980 and 1140gm. Then the minimum fruits were recorded in control plant about 655, 780, 960 and 1060 gm respectively (Plate XXIV; Table 29).

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5. DISCUSSION

Generally the top soil contains high organic matter, which is in the presence of adequate moisture supply, acted upon by the microorganisms to decompose the complex organic residues into simpler forms. Hence, microbial populations are generally higher in the surface soil layer (Shamir and Steinberger, 2007) as compared to the lower depths, However, the distribution of soil microbial population is determined by a number of environmental factors like pH, moisture content and soil organic matter (Kennedy et al., 2005), higher fungal population during rainy and autumn season supported the findings of other workers (Arunachalam et al., 1997), which perhaps due to prevailing favourable moisture and temperature setting during the period. Litter and other plant residues are decomposed faster during rainy season and sufficient soil organic and humus accumulates and may have enhanced the colonization of the soil microbes in the subsequent period.

The abundance of microorganisms in soil varies spatially as well as temporarily and this pattern is related to temporal and spatial variations in the quantity and quality of nutrients (Nedwell and Gray, 1987; Wardle, 1992). Microorganisms respond to nitrogen (Wardle, 1992), organic matter (Hussey et al., 1985; Lynch and Whipps, 1990) and soil moisture (Bottner, 1985; Wardle, 1992).

Though the fungal species are cosmopolitan in distribution, their population in the particular habit varied due to fluctuation in the physico-chemical parameter. In the present study, the physico-chemical parameters of the soil samples like pH, electrical

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conductivity, orgnic carbon, organic matter, available nitrogen, phosphorus, potassium, zinc, copper, iron, manganese, soil fraction such as fine sand, coarse sand, silt, clay, cation exchange capacity, calcium, magnesium, sodium and potassium were analysed to find out the impact on fungal population.

Previously Madhanraj et al., (2010), reported that physico-chemical parameters such as temperature, humidity, soil, pH and organic matter present in soil with the growth of microbes in Meghamalai forest soils are found to be rich in cellulolytic organisms. Panda et al., (2009) also, reported that the surface layer possessed higher fungal population, more soil nutrients and less moisture.

Fungal diversity of any soil depends on a large number of factors of the soil such as pH, organic contents and moisture (Alexander, 1977; Rangaswami and Bagyaraj, 1998). In the present investigation, initially the survey was conducted to find out the fungal community isolated from the chilli field soils of Thiruvarur district, talukwise. The species diversity of fungi showed the existence of 46 species belonged to 18 genera. The members of the Deuteromycetes were represented as dominant group (39 species), followed by Phycomycetes (5 species), and Ascomycetes (2 species).

Danial Thomas et al., (2010) screened forty two species belonging to 11 genera in Shencottai (a small town in the South Western Tamilnadu bording Kerala State). The compositional differences were observed and saprophytic species predominating in the litter layer. Most of the genera detected belonged to the Ascomycetes with a fewer proportion belonging to form class Deuteromycetes. In the present study, among 46

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species, 39 species are Deuteromycetes and 2 species are Ascomycetes. Moreever, only a few species of Phycomycetes are noticed in seven different taluks of Thiruvarur district.

Evidently Madhanraj et al., (2010) reported that 45 soil samples were collected from eight different station along the entire Tamilnadu coast and examined by dilution plating method to access the fungal diversity and their population density. Totally 24 fungal species representing 12 genera are recorded. Aspergillus was constituted by more number of (9 species) followed by Penicillium (3 species) Fusarium and Monodictys (2 species) each.

Senthilkumar et al.,

(2009) collected 15 soil samples from three different

stations namely Koraiyar riverhead, Saradi and Xavier munai along the Muthupet mangroves in Tamilnadu and examined by dilution the plating method on PDA medium to access fungal diversity and population density. Out of 22 species screened, the Aspergillus and Penicillium were represented as dominant ones. Iram et al., (2011), isolated 30 microfungi from 6 sampling sites. Of these isolates 24 belongs to phylum Ascomycota, 3 to phylum zygomycota, 2 to phylum Basidiomycota and 1 to phylum for Deuteromycota. The most widespread genus were Aspergillus and the common species Aspergillus niger.

Similarly Nilima Wahegaonkar et al.,

(2011) reported that

Aspergillus was dominant in all the three types of soil samples of ecosystem.

Prince

et al., (2011) studied the seasonal variations of soil fungal populations in the sugarcane field. Among 49 species, the dominant ones were Aspergillus niger, A. flavus followed by Botrytis cinera, Trichoderma viride, T. harzianum, T. koeningii, T. glaucum,

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P. chrysogenum and P. citrinum. Kalaiselvi and Panneerselvam (2011) also studied the seasonal and depthwise variation of soil population in the paddy field of Thanjavur district.

In the present study, among 46 species, the Aspergillus were isolated most frequently (18 species) followed by Penicillium (5 species), Trichoderma (3 species), Fusarium (2 species), Curvularia (2 species), Cladosporium (2 species), Rhizopus (2 species).

All

other

genera

Absidia,

Alternaria,

Acrophilophora,

Botrytis,

Helminthosporium, Humicola, Masoniella, Pythium, Syncephalastrum and Torula were represented by one species in different taluks of Thiruvarur district.

Rani and Panneerselvam (2010) also reported that the diversity and distribution of different organisms in the marine environment are influenced by the physicochemical properties of both water and sediments. Point calimere includes many diverse habitats such as sandy, muddy shores and mangroves which have various physicochemical features. A total of 59 fungal species were isolated from all the stations. In the present study, a total of 46 soil fungi were isolated. However, a few fungal species were noticed in monthly variations of the year, across the sites.

The correlation co-efficient between physico-chemical character and the total number of species (TNS) were studied. In Thiruvarur taluk, correlation co-efficient between physico-chemical parameter and total number of species are statistically not significant at 0.05% level. In Nannilam taluk, positive correlation was observed between TNS and APO (Available potassium) 0.621≥0.05, TNS and available copper

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(AC) 0.630 ≥ 0.05, TNS and CEC (Cation Exchange capacity) 0.621≥0.05, TNS and magnesium (MG) 0.603≥0.05 followed by Kodavasal, positive correlation between TNS and AP (Available phosphorus) 0.683≥0.05, TNS and APO (Available potassium) 0.647≥0.05. In Valangaiman taluk, the positive correlation was observed between TNS and AN (Available nitrogen) 0.678≥0.05. In Needamangalam the positive correlation was observed between the total number of species (TNS) and clay (CL) 0.593≥0.05. Finally in Thiruthuraipoondi taluk, positive correlation was observed between the total number of species and the available potassium (APO) 0.630 ≥ 0.05, cation exchange capacity (CEC) 0.619 ≥0.05 and magnesium (MG) 0.672 ≥ 0.05 were statistically significant at 0.05% level.

Evidently, Panda et al., (2009) reported that the surface layer of soil possessed higher fungal population.

Fungal population was positively correlated with total

organic carbon, moisture content and total soil respiration. Recently Madhanraj et al., (2010) studied the correlation analysis made between physico-chemical parameter and fungal population.

The study revealed no significant relationship among moisture

content, pH, organic carbon and available nitrogen contents of the soil. However water holding capacity (r=0.640; 0<0.2) and electrical conductivity r =0.338; P,0.17 showed positive correlation.

The saprophytic activity and the duration of survival of the pathogens in the soil are determined by the nature of the soil, available substrate, environmental factors, substrate utilizing ability of the pathogens and the background antagonism.

The

fluctuations in environmental parameters like soil moisture, soil reaction temperature

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and soil aeration influencing the saprophytic behaviour either directly acting upon the survival structures of pathogens or indirectly through antagonistic soil microorganisms. The saprophytic growth and activity of the pathogens vary depending upon the environmental conditions and soil.( Garrett, 1956, 1970).

Saprophytic survival of the test pathogen was carried out in the present study, and it was observed that pH 6 was favourable for the growth of Pythium debaryanum. The increase in temperature up to 40°C, decreased the percentage survival of pathogen. The soil was more stable than in the aerial environment, particularly in all respects, but also changed by many factors. Some changes in one or more factors may exert a profound effect on soil microorganisms (Baker and Cook, 1974).

The nature of survival of pathogens depends upon the interaction of numerous environmental conditions and organic elements. In the present study, it was found that the percentage survival of Pythium debaryanum was increased with an increase of the soil moisture up to 75% Moisture Holding Capacity (MHC).

Evidently Senthilkumar et al., (2011) reported that the saprophytic colonization and saprophytic suppression of the test organisms of Fusarium oxysporum was studied in relation to soil moisture, pH and temperature. The survival of the pathogen increased when the temperature increased. The percentage survival of F. oxysporum in the pre-colonized paddy straw bits buried in soil was recorded. The fungal population was increased up to 75 percent MHC.

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Plant pathogenic fungi is a widespread problem and the use of chemicals is hardly successful. However, the high cost associated with the use of fungicides to control the disease caused by soil borne fungi is a limiting factor in the profitability of crop production. Chemical fungicides to control disease could affect the beneficial microbes.

According to this study, biological control could be the best alternative and may helpful, especially against soil borne pathogens.

This is an integral part of the

integrated pest management philosophy, which entails the judicious use of biocontrol agents and reduce the amount of biocides, fungicides or other physical aspects (such as soil solarization).

Trichoderma sp. that are common saprophytic fungi found in almost any soil and rhizosphere mycoflora, have been investigated as potential biocontrol agents because of their ability to reduce the incidence of diseases caused by plant pathogenic fungi, particularly many soil borne pathogens (Papavizas, 1985; Sivan and Chet, 1986; Clavet et al., 1990; Elad et al., 1993; Spiegel and Chet, 1998; Freeman et al., 2004; Ashrafizadeh et al., 2005; Dubey et al., 2007), although some have been occasionally recorded as plant pathogens (Menzies, 1993).

Dennis and Webster, (1971) studied the in vitro efficacy of the fungal antagonists against Pythium aphanidermatum was tested by dual culture technique on PDA medium. Several microorganisms previously shown to have in vitro antagonistic activity against P. ultimum (Gravel et al., 2005) were tested in greenhouse assay to

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evaluate their effect on tomato root rot development as well as on plant growth and fruit yield.

Muthukumar et al., (2008) reported that four antagonists viz., Trichoderma viride, T. harzianum, T. hamatum and Trichoderma sp. showed their variation in the antagonism towards P. aphanidermatum.

Among them Trichoderma sp. showed

maximum inhibition with 43.33 percent reduction over control which was followed by Trichoderma viride with 37.78 percent over control.

It is reported that several

antibiotics like viridin, Gliotoxin, Trichodermin were produced by T. viride (Allen and Haenseler, 1935; Dennis and Webster, 1971; Papavizas and Lumsden, 1989).

In

addition some other mechanism like lysis, competition and mycoparasitism were also suggested by these authors for effective antagonism of T. viride. The antagonistic reaction of T. viride against several species of Pythium was reported by Dumitars and Sesan (1979).

Jun and Kim (2004) reported that the antifungal activity of T. virens and T. harzianum to Pythium sp. was stronger than that of T. koeningii and also Dharmaputra et al., (1994), tested two isolates of T. harzianum and one isolate of T. viride against three isolates of Ganoderma from oil palms. All the Trichoderma isolates inhibited the mycelial growth of the pathogen.

In the present study, antagonistic activity of Aspergillus, Penicillium and Trichoderma sp. against Pythium debaryanum was studied by in vitro in dual culture experiment. The maximum zone of inhibition were observed in T. viride (64.4%),

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followed by T. harzianum (62.2%), T. koeningii (60.0%), A. sulphureus (60.0%), A. niger (57.7%), Penicillium sp. (57.7%), A. sydowi (55.5%), A. flavus (55.5%), A. fumigatus (53.3%), respectively.

Evidently Ha (2010) report on laboratory and field trials has also proved that Trichoderma species had the ability to suppress the growth of fungal plant pathogens and enhance plant growth and development. Experiments conducted on several crops such as peanut, tomato, cucumber and durian indicate that selected Trichoderma strains could reduce significant diseases caused by fungal pathogens including Phytophthora sp, Rhizoctonia solani, Fusarium sp. Sclerotium rolfsii and Pythium sp. Siameto et al., (2010) reported that sixteen selected isolates of T.harzianum from different land use types in Embu, Kenya were tested for antagonism against five soil borne phytopathogenic fungi (Rhizoctonia solani, Pythium sp., Fusarium graminearum, F. oxysporum f.sp., Phaseoli and F. oxysporum f.sp., Lycopersici) using dual culture assay.

All T. harzianum isolates had considerable antagonistic effect on mycelial

growth of the pathogens in dual cultures compared to the controls.

Maximum

inhibitions occurred in Pythium sp. 055E interactions (73%) compared to other pathogens.

Mishra, (2010), previously reported that, Trichoderma species were screened against Pythium aphanidermatum by dual culture method. Among the strains tested, T. viride was found to be the most effective against P. aphanidermatum. Amin et al., (2010) tested for seven fungal plant pathogens against Trichoderma species. Among the strains, T. viride was the most effective in reducing the mycelial growth of seven

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fungal pathogens. Volatile metabolites from T. viride causing maximum reduction in mycelial growth of Colletotrichum capsici and Alternaria brassicicola was recorded with T. viride whereas, in Helminthosporium oryzae, T. harzianum accounted for maximum reduction in mycelial growth. Mahalingam et al., (2011), reported antagonistic potentiality of some soil fungi against Ceratocystis paradoxa a pathogen causing pine apple disease in sugarcane field, using dual culture method.

Dung and Loi, (1991) reported that microorganisms and medicinal plants are rich source of secondary metabolites which are potential sources of useful drugs and other useful bioactive products. The biosynthesis of these metabolites is controlled genetically and affected strongly by environmental influences that may be biotic or abiotic (Fowler, 1980; Hamill et al., 1987). As a result, there are fluctuations of the concentrations and quantities of these secondary metabolites such as alkaloids, glycosides, volatile oils and steroids etc. (Deans and Swoboda, 1990).

Harman and Hadar, (1983) understanding the mechanisms involved in the antagonistic effect of Trichoderma sp. against plant pathogen are important in the selection of suitable biocontrol agent for effective and safe utilization. Different isolates of Trichoderma have various effects of fungal antagonism and on the plant health. The possible mechanism of antagonism employed by Trichoderma sp. realized so far includes competitions, antibiosis by producing non volatile, volatile antibiotics and exploitation.

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Madhanraj et al., (2010) reported that ethyl acetate culture filtrate of T. koeningii was subjected to GCMS analysis to find out the compounds such as dodecanonic acid, tetradecanoic acid, pentadecanoic acid, 9-hexadecanoic acid, n-hexadecanoic acid, oleic acid, 1,2-benzenedicarboxylic acid, diisoocytyl ester and squalene. In the present study, the extract of T. viride was subjected to GCMS analysis to find out the components produced by the fungus. It yielded four prominent peaks with retention time, 2.092, 3.035, 3.3.96 and 2.480 min.

Senthilkumar et al., (2011) also studied that Gas Chromatography Mass Specrtrum analysis of acetonitrile extract of the filtrate of T. harzianum revealed the presence of six compounds representing six major peaks. The peaks correspond to diethylphthalate, tetradecanoic acid, 12-octadecadienoic acid (z,z), oleic acid, 1,2benezene dicarboxylic acid, disooctyl ester and squalene.

In the present study, the GCMS analysis of T. viride revealed the presence of four compounds representing four major peaks. The peaks correspond to ethylcis – 13docosenoate,

Olean-12-ene-lbeta,

3

beta,

23-triol,

1,3-dideoxy-1,

3-bis

(N-methylacetamido)- myo-inositol 2,4,5,6 – tetraacetate oleanolic acid. However, the minimum number of antimicrobial compounds was identified.

Six species of Trichoderma were tested for their ability to produce volatile metabolites against seven fungal plant pathogens viz., Fusarium oxysporum, Rhizoctonia solani, Sclerotium rolfsii, Sclerotina sclerotiorum, Colletotrichum capsici, Helminthosporium oryzae, Alternaria brassicicola. This study indicated that T. viride

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(TV-1) was the most effective in reducing the mycelial growth of F. oxysporum (41.88%) whereas, in the case of R. solani, T. viride (TV -2) has accounted for maximum reduction in mycelial growth (30.58%) and sclerotial production (65.65%). Volatile metabolites from T. viride (TV-1) caused maximum inhibition of mycelial growth and sclerotial production in S. rolfsii and S. sclerotiorum. Maximum inhibition of mycelial growth of C. capsici and A. brassicicola was recorded with T. viride (TV-1), where in H. oryzae, T. harzianum (Th-1) accounted for maximum reduction in mycelial growth (37.16%) (Amin et al., 2010).

Species of Trichoderma have been demonstrated in vitro to act against fungal plant pathogens by producing diffusible volatile antibiotics. Claydon et al., (1987) reported antifungal properties of volatile compounds (Alkyl Pyrones) produced by T. harzianum. Similarly, Rathore et al., (1992) reported volatile activity of T. viride against F. solani which vocuolated most hyphae of pathogen and that the hyphae of pathogen were comparatively thin as compared to control. Michrina et al., (1995) and Pandey and Uapadhyay (1997) have also reported the effectivenesss of diffusible volatile compounds by T. viride and T. harzianum in vitro.

There are many reports of successful use of antifungal metabolite extracted from Trichoderma sp. to control disease causing fungi such as S. rolfsii causing disease on vegetables (Maiti et al.,

1991), P. aphanidermatum causing wilt of cotton and

watermelon (Ordentlich et al., 1992) and damping-off of cucumber (Intana, 2003) and Phytophthora sp. causing various plant diseases (Wilcox et al., 1992). This research indicated an additional successful use of antifungal metabolites from T. viride wild type

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and mutant strains in controlling damping off of disease in chilli caused by P. debaryanum.

T. koeningii is capable of producing many compounds that are produced by many other fungal species. The antimicrobial activity of the 1,2-Benzenedicarboxylic acid and diisocotyl ester have already been reported by Ushadevi (2008) from the marine isolates of Penicillium lividum and Trichoderma lignorum. Likewise there are reports on the occurrence of tetradecanoic acid, dodecanoic acid and n-hexadecanoic acid in the extract of heads space of Aspergillus versicolor, dodecanoic acid and tetradecanoic acid from P. chrysogenum (Griffith et al., 2007); pentadecanoic acid and oleic acid from Mortierella alpine

(Wang et al.,

2005) and oleic acid from

Phytophthora cinnamonis (Zaki et al., 1983).

The bioactive compound analysis of T. viride was also studied using the thin layer chromatography. The results revealed the presence of phenol, sterol, Flavonoids, saponin and Tannins. In the present study, it was found that phenol showed antifungal activity. Hence relatively high antifungal activity of T. viride could be attributed to the presence of the following functional groups of phenol such as Amines, N-H stretching vibrations secondary free, one band imines (= N-H); One band amine salts, unsaturated nitrogen compounds, C≡N stretching vibrations-Isocyanides, unsaturated nitrogen compounds

C≡N stretching vibrations α, β – unsaturated compounds and Halogen

compounds C-X stretching vibrations, C-CL.

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In general, medicinal plants are the effective sources of both traditional and modern medicine and are genuinely useful in primary healthcare. Many plant extracts have been used as the source of medicinal agents to cure several diseases in human and animals (Vijayakumar et al., 2010).

Several studies have been conducted in the past three decades that focused on the antimicrobial properties of herbs, spices and their derivatives such as essential oils, extracts and decoctions (Kivanc and Akgul, 1986; Dorman and Deans, 2000; Hsieh et al., 2001; Alma et al., 2003).

Aswar et al., (2009) reported that different extracts of Vitex negundo leaves were investigated for its antimicrobial and antifungal activity. Water and ethanolic (50:50) extract showed maximum antimicrobial and antifungal activity against all tested fungal species.

Prince and Prabakaran (2011) stated that antifungal activity of eight different medicinal plants namely Aloe vera, Ocimum sanctum, Centella asiatica, Piper betle, Calotropis gigantea, Vitex negundo, Ocimum basilicum and Azadirachta indica was tested against plant pathogenic fungus Colletotrichum falcatum by agar well diffusion method. Among the different plants tested, all the three solvents of the Vitex negundo showed maximum antifungal activity against plant pathogen tested, whereas the other plant extracts were showed moderate to minimum antifungal activity. In the present study antifungal activity by using different medicinal plants such as Aloe vera, Alternanthera sessilis, Lawsonia inermis, Murraya koeningii, Mimosa pudica,

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Pithecolobium dulce, Phyllanthus niruri, Tephrosia purpurea was assayed by agar well diffusion method. Among all the plant extracts, n-butanol and methanolic extracts of Vitex negundo exhibited maximum antifungal activity (15 and 30 mm) followed by Lawsonia inermis (15 and 25 mm), Phyllanthus niruri (15 and 20 mm), Murraya koeningii (15 and 10mm) against Pythium debaryanum whereas other plant extracts showed moderate to minimum antifungal activity. The antibiotic sensitivity test using standard antibiotics viz., (Amphotericin B, Griseofulvin and Fluconazole) were tested against Pythium debaryanum.

All the antibiotics used were exhibited antifungal

activity. The results confirmed that the solvent extracts of all medicinal plants exhibited a higher antifungal activity against Pythium debaryanum when compared to the standard antibiotics. Antifungal effect of methanol, n-butanol and aqueous revealed no activity against pathogenic fungi.

Saadabi (2007) reported that leaf samples of Lawsonia inermis effectively inhibit the growth of 6 human pathogenic fungi. The growth of all pathogens was inhibited to varying degrees by increasing the concentration of the extract. These results confirm the antifungal activity of henna leaves and support the traditional use of the plant in bacterial and fungal infections. Similarly Chaudhary et al., (2010) reported that the plants have analgesic, antibacterial, antimicrobial, antifungal, antiviral and anticancer properties.

Murugesan et al., (2011) reported that antifungal activity of eleven different medicinal plants namely Aloe vera, Alpinia calcarata, Acalypha indica, Carum copticum, Leucas aspera, Ocimum sanctum, Piper betle, Phyllanthus niruri, Solanum

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trilobatum, Memycelon umbellatum and Tridox procumbens was tested against Fusarium oxysporum by agar well diffusion method. Kaur et al., (2005) also made antifungal activity against six species of fungi viz., Alternaria, Aspergillus, Fusarium, Penicillium, Phytophthora and Pythium observed in petroleum ether and chloroform root extracts. Petroleum ether extract of shoot showed antifungal effect against five fungal species viz., Alternaria, Aspergillus, Fusarium, Phytophthora and Pythium. Methanol root extracts also showed antifungal activity against Aspergillus and Pythium species only. This study justifies the uses of this herb in the traditional system of medicine to treat various diseases.

Alam et al., (1999) reported that the antifungal effects of leaf and root extract of Vinca rosea and leaf, root and seed extracts of Azardirachta indica against chilli fruit rot pathogen such as Alternaria tenuis. Aloe vera gel is also said to promote wound healing due to the presence of some components like anthraquinones and homones (Davis, 1997) which possess antibacterial, antifungal activities. However, most of the constituents are found in the gel and not in leaf. Hence the gel is likely to be more active than leaf. In the present study also Vinca rosea and Aloe vera medicinal plants showed antifungal activity against Pythium debaryanum causing damping off of disease in chilli. Generally, Kindermann et al., (1998) attempted a first phylogenetic analysis of the genus Trichoderma, using the sequence analysis of the ITS 1 region of the rDNA. Neverthless, the use of phylogenesis based on single gene sequence is now normally discredited, especially as regards the use of ITS1 and ITS2, as some fungi and plants have been shown to contain paralogous copies (Lieckfeldt and Seifert, 2000).

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Taylor et al., (1999), proposed phylogenetic species concepts between five or more gene trees.

These results interrelation among species and while combined with

phenotypic characters, can lead to a reliable taxonomy, that is reflective of phylogenetic relationships. Druzhinina et al., (2004), was able to identify 70 out of a total of 77 investigated genus of Trichoderma. Kubicek et al., (2003) demonstrated that seventyeight isolates of Trichoderma, 37 strains were positively identified as T. harzianum, others were

T. virens (16 strains), T. spirale (8 strains), T. koeningii (3 strains),

T. aureoviride (3 strains), T. asperellum (4 strains), Hypocrea jecorina (2 strains), T. viride (2 strains), T. hamatum

(1 strain) and T. ghanense (1 strain). Ospina –

Giraldo et al., (1999) showed that phylogenetic analyses were closely related to an isolate of T. harzianum compared to other Trichoderma isolate.

The purpose of the present study was to establish a species of T. viride gene sequence in chilli field isolates based on the sequence analysis of ITS-1 and ITS-2 regions of the rRNA gene. Recently Chakraborty et al., (2011) reported Genomic DNA of Macrophomina phaseolina isolated from mandarin rhizosphere, purified and PCR amplification of 18S rRNA was done using genus specific ITS-1 and ITS 4 primers. Amplified product (550 bp) was sequenced and aligned against ex-type strain sequences of M. phaseolina from NCB1 Gene Bank using BLAST and phylogenetic analysis was obtained using MEGA 4 software.

In the present study, genomic DNA of T. viride isolated from chilli field soil was purified and PCR amplification of 18S rRNA was done using genus specific ITS1 and ITS4 primers. Amplified product (544 bp) was sequenced and aligned against extype

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strain sequences of T. viride from NCB1 GenBank using BLAST and phylogenetic analysis was obtained using MEGA 4 software.

The genus Trichoderma possesses a major challenge for systematics because the phylogenetic relationships of many of its members still now are unclear. So the concepts of “species aggregate“ and “section” introduced by Bissett (1991) and Rifai (1969) have helped to clarify placement of conflicting species such as T. harzianum, T. viride and T. atroviride within the genus. However, the influences of environmental conditions on morphological and physiological characteristics have made accurate identification very difficult (Lieckfeldt et al., 1998).

In the present and previous work (Rehner and Samuels, 1994) the sequence of the ITS1 region the most accepted species of Trichoderma has been examined. The studies provided more useful information for assessing the Trichoderma taxonomy. ITS-1 region sequences are used as reference sequence for further study, involving the identification of T.viride. Amplification of ITS1 region of the rDNA has showed potential as a rapid technique for identifying the fungi in all the cases.

A phylogenetic tree was also constructed by Neighbour – joining method. The present investigation concludes that the culture of T. viride closely related to Trichoderma asperellum (at 100% level) is based on nucleotide homology and phylogenetic analysis. The secondary structure of T. viride 18S rRNA showed 23 stems, 16 bulge loops and 8 hairpin loops in their structure.

A large number of

restriction enzyme sites were observed in the fungal isolate. However the cleavage sites

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and the nature of restriction enzyme of T. viride is in 52 ambiguous sites. The GC and AT content of this species was determined to be 56% and 44% using NEB cutter programme.

Thangaraj and Meenupriya (2011) reported phylogenetic relationship in four Aspergillus species internal transcribed spacer region 1 (ITS1). The secondary structure predicted could be used for the identification of fungi at genetic level. In the present study phylogenetic tree is also used for the identification of fungal species of T. viride.

Biological control of soil borne plant pathogens can be achieved by seed treatment with antagonists.

Harman et al., (1980) reported the biocontrol of

Rhizoctonia solani and Pythium sp. by coating radish and pea seed with T. harmatum (Bain). Hadar et al., (1979) and Elad et al., (1980) also investigated that the application of wheat bran colonized by T. harzianum to soils infested by Rhizoctonia solani and Sclerotium rolfsii reduced the incidence of disease caused by these pathogens in beans.

Jayalakshmi et al., (2003) has also observed that the seed treatment with Trichoderma viride followed by T. harzianum was found to be effective in reducing the wilt disease incidence in coriander. Similarly, Nakkeeran and Devi (1997) observed that Alternaria alternata causing blight disease in Pigeon pea was most effectively reduced by seed treatment with T. harzianum. Ghosh et al., (2002) also revealed that Trichoderma viride, T. hamatum and A. awamori inhibited the growth of Alternaria alternata. Trichoderma sp. are well documented as effective biological control agents

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of plant diseases caused by soil borne fungi (Sivan et al., 1984; Coley-Smith et al., 1991).

In previous report, Trichoderma species, either added to the soil or applied as seed treatments, grow readily along with the developing root system of the treated plants (Ahmed and Baker, 1987; Harman, 2000; Harman, 2001; Howell, 2003) Papavizas et al., (1989) also reported that seed treatment with T. harzianum reduced Pythium seed rot of pea and Rhizoctonia, damping off of cotton. The application of Trichoderma harzianum to bean roots resulted in a 25 to 100% reduction in the severity of the foliar disease, gray mould, caused by Botrytis cinera (De Meyer, et al., 1998). Muthukumar et al., (2008) reported that chilli seeds treated with biocontrol agent like Trichoderma sp. (NI) at 4g/kg P. fluorescens -1(NI) at 10g/kg was found to reduce the population of P. aphanidermatum up to 30 days after sowing.

In the present study, the result of seed treatment, the maximum percentage of germination, shoot length and root length were observed in T. viride. At the period of 10 days about 99%, 5.9 and 4.8 cm when compared to T. harzianum showed about 97%, 5.2 and 4.5cm. Then the minimum percentage of germination, shoot length and root length (cm) observed in control plant is about 82%, 4.6 and 4.2cm respectively.

Seed treatment with biocontrol agent was effective in controlling many fungal diseases. For example, Mao et al., (1997) showed that seed treatment with Gliocladium virens, Trichoderma viride isolates increased seedling stand, plant height and fresh weight and decreased root rot severity in corn, giving results comparable to those of treating with a fungicide (captan). Verticillium wilt of cotton was effectively controlled

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by seed treatment with Trichoderma virens while T. viride and some bacterial species were effective against anthracnose of cowpea (Hanson, 2000; Adebanjo and Bankole, 2004). T. harzianum also effectively controlled Sclerotium rolfsi on blue lupines, of tomato and peanuts (Wells et al., 1972).

In root dipping, after transplantation the maximum shoot length was observed in T. viride at the duration of 30, 60, 90 and 120 days and found to be about 50.2, 67.0 and 85 cm when compared to T. harzianum, which showed about 48.4, 62.1, 71.6 and 78 cm and the minimum shoot length observed in control plant is about 45.6, 56.7, 66.0 and 75.2 respectively. The maximum root length was observed in T. viride at the duration of 30, 60, 90 and 120 days and found to be about 14.5, 20.7, 23.5 and 24.3 cm when compared to T. harzianum, which showed about 13.9, 18.9, 21.6 and 24.2 cm and the minimum root length observed in control plant is about 12.6, 18.7, 22.5 and 23.9 cm respectively.

The maximum weight of fruits were recorded in T. viride at the duration of 30, 60, 90 and 120 days and found to be about 720, 960, 1040 and 1250gm, when compared to T. harzianum, which showed about 695, 780, 980 and 1140gm and the minimum fruits recorded in control plant is about 655, 780, 960 and 1060gm respectively. The formulation of Trichoderma species was tested for their ability to control pre-emergence and post-emergence damping-off caused by Pythium ultimum in green house grown Echinacea angustifolia seedlings.

A wettable powder formulation of T.harzianum

isolate, significantly reduce the pre and post emergence damping-off caused by P. ultimum.. Yang et al., (2004).

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6. SUMMARY

The thesis is entitled as Studies on soil mycoflora in chilli field of Thiruvarur District and Biological control of Pythium debaryanum. It deals with the (i) Monthly variations of soil mycoflora and physico-chemical parameters in chilli field soil of Thiruvarur district each taluk wise. (ii) Correlation co-efficient between physicochemical parameters and the total number of species and colonies. (iii) Among 45 species Pythium debaryanum is a causal organism causing damping off of disease in chilli and pathogenicity was confirmed by adopting Koch‟s postulation and also the effects of physico-chemical factors on saprophytic survival were studied.

(iv)

Identification of potential antagonistic species against P. debaryanum a soil borne pathogenic fungi and also antifungal activity was made by using various medicinal plants. (v) Bioactive compounds were analysed in potential antagonistic fungi by using TLC, GCMS and FTIR & UV. (vi) Molecular characterization and gene sequencing studies were used to confirm the correctness of potential antagonistic microorganisms with reference to 18S rRNA. (vii) Application of Trichoderma species considered relatively novel biological control agents are applied to the soil as seed treatment, root dipping of seedlings which can help farmers to reduce plant diseases and increase plant growth.

Forty six species belonging to 18 genera were isolated and identified in chilli field soil.

Among 46 species, most of the genera screened belonged to the

Deuteromycetes (39 species) with a fewer proportion belonging to Ascomycetes (2 species) and Phycomycetes (5 species). The species of Aspergillus were isolated most Studies on the soil mycoflora in chilli field of Thiruvarur district and biological control of Pythium debaryanum Hesse

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frequently, (18 species) followed by Penicillium (5 species), Trichoderma (3 species), Fusarium (2 species), Chaetomium (2 species) Curvularia (2 species), Cladosporium (2 species) and Rhizopus (2 species). Acrophilophora,

Botrytis,

All other genera Absidia, Alternaria,

Helminthosporium,

Humicola,

Masoniella,

Pythium,

Syncephalastrum and Torula were represented by one species.

The soil characteristics such as pH (7.1 to 8.2) electrical conductivity (0.2 to 1.36 dsm-1) cation exchange capacity (6.9 to 27.6 mol proton+ /kg), organic carbon (0.12 to 0.58%), organic matter (0.31 to 0.99%), available nitrogen (82.2 to 152.3 kg/ac), available phosphorus (3.01 to 5.36 kg/ac), available potassium (102 to 189 kg/ac), fine sand (40.29 to 48.62%), coarse sand (17.67 to 26.54%), silt (15.16 to 24.65%), clay (5.52 to 22.58%), available zinc (0.29 to 1.56 ppm), iron (0.45 to 9.64 ppm), copper (0.38 to 1.58 ppm), manganese (1.24 to 3.93 ppm), calcium (5.8 to 16.6 mg/kg), magnesium (5.5 to 13.5 mg/kg), sodium (0.15 to 2.98 mg/kg), potassium (0.48 to 0.11 mg/kg) were determined. The variation of physico-chemical parameter between the samples collected from the Taluks in Thiruvarur district during the period of June 2009 to May 2010 was also recorded.

The correlation co-efficient between physico chemical parameters and the total number of species in chilli field soil in the Taluks of of Thiruvarur district such as Nannilam, Kodavasal, Valangaiman, Needamangalam and Thiruthuraipoondi stations as statistically significant at 0.05% level.

In Thiruvarur and Mannargudi stations are

statistically not significant at 0.05% level.

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Saprophytic survival of P. debaryanum were studied in relation to soil moisture, pH and temperature. The percentage survival of P. debaryanum in the precolonized chilli straw bits buried in soil was recorded maximum (62%) in the soil with moisture content 75% MHC. The fungal population was increased up to 75% MHC. The pH of the saprophytic survival of P. debaryanum was more favoured by alkaline range than the acidic range. The percentage survival of the pathogen was 41, 58 and 65 in the precolonized substrates recovered from the soil incubated at 15 ± 2, and 42 ± 2ºC respectively. S50 values decreased with an increase in temperature. Among 46 species, 9 species was made for antagonistic activity against

P. debaryanum, a known soil

borne fungal pathogen.

From several studies, it has been confirmed that Trichoderma sp. has antagonistic and biologically control activity in which T. viride inhibited the pathogenic fungus for the maximum in dual culture technique. It showed the maximum inhibition of T. viride (64.4%), followed by T. harzianum (62.2%),

T. koeningii (60.0%),

A. sulphureus (60.0%), A. niger (57.7%), Penicillium sp. (57.7%), A. sydowi (55.5%), A. flavus (55.5%), A. fumigatus (53.3%), respectively.

Antifungal activity of ten medicinal plants extract was assayed by agar well diffusion method. The result revealed that the extract of ten medicinal plants showed a significant reduction in the growth of P. debaryanum. Among all the ten plants extract the n-butanol and methanol extract of Vitex negundo exhibited maximum antifungal activity (15 and 30 mm) followed by Lawsonia inermis (15 and 10mm), Phyllanthus niruri (15 and 20mm) and Murraya koeningii (15 and 10mm). The n-butanol and

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methanol extract of Tephrosia purpurea (10 and 15 mm), Aloe vera (10 and 15mm) showed moderate activity against P. debaryanum. The methanol extract of Mimosa pudica (20mm), Pithecolobium dulce (15mm), Alternanthera sessilis (10mm), Vinca rosea (10mm) exhibited least activity against P. debaryanum. The results of antifungal effect of aqueous extract of all tested ten plants showed no activity against P. debaryanum. The antibiotic sensitivity test using standard antibiotics viz., (Amphotericin B, Griseofulvin and Fluconazole) were tested against Pythium debaryanum. All the antibiotics used were exhibited antifungal activity. The results confirmed that the solvent extracts of all medicinal plants exhibited a higher antifungal activity against Pythium debaryanum when compared to the standard antibiotics. Antifungal effect of methanol, n-butanol and aqueous revealed no activity against pathogenic fungi.

TLC fractionation of the mycelial extract of T. viride grown in PDA broth yielded saponin, flavonoid, sterol, tannin and phenol.

The phenol showed antifungal activity, hence relatively high antimicrobial activity of T. viride could be attributed to the presence of the following functional groups of phenol such as amines, N,H stretching vibrations – secondary, free, one band imines (= N-H); one band amine salts, unsaturated nitrogen compounds, C≡N stretching vibrations isocyanides, unsatured nitrogen compounds

C≡N stretching vibrations-α,β-

unsaturated compounds and halogen compounds C-X stretching vibrations, C-CL.

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Gas Chromatography Mass Spectrum analysis of potential fungal extract of T. viride revealed the presence of four compounds representing four prominent peaks. The peaks correspond to ethylcis – 13-docosenoate, Olean-12-ene-lbeta, 3 beta, 23-triol, 1,3-dideoxy-1, 3-bis (N-methylacetamido)- myo-inositol 2,4,5,6 – tetraacetate oleanolic acid.

The Genomic DNA was isolated from T. viride and PCR amplification was performed.

The 18S rRNA gene sequencing was made by using ITS (Internal

transcribed spacer) method.

Seed treatment and application of the antagonist in root dipping are alternative methods for chemical control which could improve the establishment and colonization ability in the plant rhizosphere. This will enhance the effectiveness of disease control with the added advantage of using small volumes of biocontrol preparations.

In seed treatment and root dipping of chilli seedlings the transplanting to the pot after 30th, 60th, 90th and 120th days, the maximum percentage of germination, shoot length, root length and yield of fruits were observed in the treatment of T. viride and T. harzianum when compared to control.

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