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Bacteria Diversity
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Posted By: Ashish Uniyal Member Level: Gold Points/Cash: 6
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This project is based on microbiolical subject. Its on bacteria diversity that is found in Himalayan location.
INTRODUCTION
Soil is generally a favorable habitat for the proliferation of microorganisms with micro colonies, developing on soil particles. Typically, 106 - 109 bacteria per gram are found in soil habitat. Microorganisms are ubiquitous in nature and form vital components of all known ecosystem on earth. Their ubiquity is attributed mainly to the small size, easy dispersal, ability to survive and multiply in diverse habitat , including anaerobic and other extreme condition their metabolic versality and flexibility to utilize with substrate as nutrient source. To provide food security to the ever-increasing population, greater agricultural production is a pressing need in the 21st century.
Microorganisms are essential for the maintenance of sustainable ecosystem microbial diversity, however, is often neglected. Microorganism including bacteria, actinomycetes and fungi occupy important niches in all ecosystem and are responsible for the recycling of elements in nature. These must be separated and identified for a wide variety of reasons such as determination of pathogens, developing inoculants for plant growth promotion and disease control selection and isolation of strains of fermentative microorganism useful in industries, comparison of biochemical activities for taxonomic purposes etc. (Cappuccino and Sherman 1996).
The rhizosphere (from the Greek, rhizos-meaning root) was first described in 1904 by Lorentz Hiltner as the region around the root, which is directly affected by the root system. Hiltner, (1904) also identified two major effects of rhizosphere on plants, along with areas to be intensely researched during the next decades. These areas were: (1) the relationship between the rhizosphere and plant nutrition, growth and development, and (2) the influence of rhizosphere phenomenon on pathogens and pathogenesis (cited in Curl and Truelove, 1986).
The rhizosphere is more than a zone of academic fascination. Plant growth and development are controlled largely by the soil environment in the root region, an environment which the plant itself help to create and where microbial activity constitutes a major influential force. Understanding the relation of exudates induced microbial activity in the rhizosphere to the plant health and vigor is essential to the development of better crop production system. The rhizosphere is the soil region that is influenced by plant root and is characterisezed by a high microbial activity (Hiltner 1904). The bacterial community composition in the rhizosphere is important for the performance of the plant, as bacterial species can have beneficial neutral or harmful relationship with the roots (Sylaia and Chellemi, 2001).
The rhizosphere is thus an environment created by the interaction between exudates and microorganisms that may either utilize the organic materials released as nutrient source or be inhibited by them. In general the microbes that inhabit the rhizosphere serve as an intermediary between the plant, which requires soluble inorganic nutrient, and the soil, which contains the necessary nutrients but mostly in complex and inaccessible forms.
The quantitative and qualitative nature of microbial population in the rhizosphere and rhizoplane are related either directly or indirectly to root exudates and thus will vary somewhat occurring to some environmental factors that influence exudation. The rhizosphere effect of an actively growing crop plant is most pronounced with bacteria, followed by actinomycetes estimates of population for rhizosphere (R) and non rhizosphere (S); expressed as R/S ratio, vary widely with different crop plant and with the sampling isolation and assessment procedures used. Population of soil fungi in general fall in third place below those of bacteria and actinomycetes, based on dilution plate counts
During last few decades, agricultural production has increased due to the use of high yielding varieties and enhanced consumption of chemicals, which are used both as fertilizers to provide nutrition and as protection agents to control the damage caused by phytopathogens. Although the use of chemicals has several advantage, such as ease of handling and yielding predictable results. Yet several problems related to the continuous export of fertility of the soil, yielding great amount of ecological disastrous-soil damage, health problems and high irrigation demand, etc., came into existence (Pandey and Kumar, 1989; Gloud, 1990; Harman, 1992)
Excessive use of chemicals and change in traditional cultivation practices has resulted in the deterioration of physical, chemical and biological health of the cultivable soil (Paroda, 1997). Therefore the productivity including production of a wide range of agricultural commodities, under conditions of shrinking land resources and diminution of both biological potential of soil and biological wealth need to be increased. There is no simple or single solution to these complex ecological, socio-economic and technological problems facing those engaged in promoting sustainable advances in agricultural biotechnology (Swaminathan, 1999). 2; Lynch, 1992; Nautiyal, 2000; Pandey et al., 2004).
The objective of agriculture in coming decades is to optimize soil productivity (inclusive of stressed soils) while preserving its capacity to function as a healthy system. In this context there is a strong case for using microorganisms for improved plant performance in integrated plant management systems. The use of soil microorganisms, which can stimulate plant growth, will be environmentally benign approach for nutrient management and ecosystem functions. This may ensure that the nature is not exploited in the production process but is, instead harmonized so that the entropy of environment decreases and sustainability in agricultural production is promoted (Purohit, 1995; Sinha, 1997).
Significantly implicated in the competitive struggle for substrate and survival in the rhizosphere are soil borne pathogens, particularly the fungi. The quantity and quality of root exudates along with sloughed epidermal cells directly or indirectly affect the growth and reproduction of these microorganism influencing inoculum density and disease potential. The interaction between the microbes and the roots of plants can be beneficial, harmful or neutral and this delicate balance is a consequence of both soil and plant type. The bacteria that provide benefit to the plant can be either symbiotic or free living in soil, but are found in abundance near roots. Such beneficial free-living bacteria have been termed as Plant Growth Promoting Rhizobacteria (PGPR) (Glick, 1995).
The concept of Plant Growth Promoting Rhizobacteria (PGPR) is now well established for both growth promotion as well as biocontrol; still the technology is not really commercially successful, mainly because of the lack of reproducibility between trials conducted under controlled conditions (laboratory or glasshouse or greenhouse) and the fields. This happens so as in most cases the microbial inoculants are usually taken from one environment and introduced in another. With a view of developing microbial inoculants suitable for field applications in the colder mountainous region, a systematic investigation was launched some ten years back in the Institute. These investigations indicated dominance of the species of Bacillus and Pseudomonas in soils of colder regions, and particularly three species namely Bacillus megaterium, B. subtilis and Pseudomonas corrugata were considered suitable for developing as potential inoculants for wide spread application in the mountains (Pandey et al., 2004)
Soil microbial populations are multispecific communities, which fluctuate according to the influence of environmental changes induced naturally or through human interference. Microorganisms, which share the same ecological, niche e.g., those within a habitat which have similar nutritional requirement and environmental optima are most likely to be in direct competition for materials that are in limited supply. Soil-plant interactions are complex and there are many ways in which the outcome can influence the plant health and productivity (Kennedy 1998). These interactions are sometimes beneficial and sometime neutral to the plants.
Primarily, two kinds of interaction occur in mixed populations.
(a) Those which promote growth of individual within the population (commensalisms and symbiosis).
(b) Those in which organism are inhibited (antagonism: competition, antibiosis, hyperparasitism and predation)
However, our focus should be to exploit the beneficial interaction of plants and microbes.
In the present study microorganisms were isolated from the rhizosphere of wheat plant which was already cultivated in the experimental nursery of G. B. Pnat Institute of Himalayan Environment and Development, Kosi – Katarmal, Almora. These isolates were characterized and enumerated for their various biotechnological applications, with following objectives,
1) study of microbial diversity of wheat rhizosphere
2) Characterization of bacterial isolates on the basis of their microscopic, biochemical and physiological characteristics
3) Qualitative estimation of the bacterial isolates for their phosphate solubilizing activity
4) Quantitative estimation of phosphate solubiloization of the selected isolates
5) Evaluation of selected isolates for antagonistic activity
REVIEW OF LITERATURE
Inoculation of plants with beneficial bacteria can be traced back for centuries. From experience, farmers knew that when they mixed soil taken from a previous legume crop with soil in which non-legumes were to be grown, yields often increased. By the end of the 19th century, the practice of mixing “naturally inoculated” soil with seeds became a recommended method for legume inoculation in USA (Smith, 1992). Scientific demonstrations of the value of legumes in contributing to the nitrogen nutrition of plants were conducted in the latter half of the 19th century by Boussingault, Hellriegel and Wilfarth (cited in Subba Rao, 1982). Nobbe and Hiltner, (1895) introduced a laboratory grown culture of rhizobia called “Nitragin” which was grown on solid medium containing extracts of leguminous plants, gelatin, sugar and asparagines. In this maiden attempt, 17 different inoculants for important leguminous crops were produced (cited in Burton, 1979).
The wheat rhizobacterial community structure is highly dynamic and influenced by different factors such as plant age, the fertilizers input and the type of bioinoculants. The community shifts during plant growth probably resulted modification of the root exudation pattern, which is different at maturity. The bioinoculant by PGPR, in the form of seed coating or in the soil close to the seed, may cause shift in the community composition of either small or high magnitude (Narcamulli et al; 1997, Kazdroj et al; 2004).With the discovery of free living bacterial species such as Azotobacter and Azospirillum (Beijerinck, 1925), interest developed in the potential application and management of these microorganisms to promote growth and vigor in non leguminous crops. Reports of significant benefits in terms of crop yield through seed inoculation by Azotobacter came from USSR early 20th century (Mishustin and Shilnikova, 1969)
Plant growth-promoting rhizobacteria
Plant growth –promoting rhizobacteria (PGPR) are free living, soil borne bacteria, isolates from rhizosphere, which, when applied to seed or crop, enhance the growth of plants or reduce the damage from soil borne plant pathogens (Kloepper et al, 1980) bacterial inoculants which help in groth ar generally considered to be of two types; (a) symbiotic (b) free living Kloepper et al, 1988; Frommel et al, 1991). Beneficial free living bacteria referred to as PGPR are found in the rhizosphere of the root of many different plants (Kloepper et al, 1989).
PGPR are able to exert a beneficial effect upon plant growth. N2-fixing and P-solublizing bacteria may be important for plant nutrition by increasing N and P uptake by the plant playing a significant role as PGPR in the biofertilization of crop. Trials with rhizosphere – associated plant growth promoting nitrogen fixation and P – solublization Bacillus spp.indicated yield increase in wheat (de Frcitas, 2000). The effect of PGPR on agricultural crop has been investigated and published by various authors in the last two decades with recent application on trees (Bashan and Holguin, 1998; Enebak et al). Bacterial strains that are isolated from rhizosphere field grown crops as identified as
Bacillus megatarium RCO7
Bacillus licheniformis RCO8
Paenibacillus polymyxa RCO5
Rhodobacter capsulatus RCO4
Psuedomonas putida RCO6
To provide food security to the ever-increasing population greater agricultural production is must. During the last 2-3 decades, agricultural production has increased due to use of high yielding varieties and enhanced consumption of chemicals, which are used both as fertilizers to provide nutrition and as protection agents to control the damage caused by phytopathogen. Although the use of chemicals offer several advantages, such ease of handling and yielding predictable result yet several social, economical and environment problems came in existence particularly after the green revolution which was primarily chemical-based.
MECHANISMS OF PLANT GROWTH PROMOTION
PGPR enhance plant growth by direct and indirect means, but the specific mechanism involved have not all been well-characterized (Glick B.R., 1995;Kloepper, J.W. 1993). There are several ways by which plant growth promoting bacteria can affect plant growth directly (Kloepper et al, 1989; Lynch, 1990; Glick, 1995), e.g., by fixation of atmospheric nitrogen, solubilization of minerals such as phosphorus, production of siderophores that solubilize and sequester iron, or by the production of plant growth regulators (hormones) that enhance plant growth at various stages of development. Indirect growth promotion occurs when PGPR promote plant growth by improving growth-restricting conditions (Glick et al., 1999). This can happen directly by the production of antagonistic substances (Thomashow and Weller, 1988; Weller, 1988; O’Sullivan and O’Gara, 1992), or indirectly by inducing resistance to pathogens (Van Peer et al., 1991; Glick, 1995; Leeman et al., 1996).
1. DIRECT EFFECTS OF PGPR
1.1. Phosphate solublization:
Phosphorus is an important plant nutrient, next only to nitrogen and classed alonged to nitrogen and potassium as a major plant nutrient element. Microorganisms are involved in a range of processes that effect the transformation of soil phosphorus (P) and are thus an integral component of soil P cycle. However, a large proportion of soluble inorganic phosphate added to the soil is rapidly fixed as insoluble forms soon after the application and become unavailable to the plants (Rodriguez and Fraga, 1999). The phenomenon of fixation and precipitation of P in soil is pH dependent; Al and Fe phosphate are formed in acidic soil while in calcareous soils high concentration of Ca results in Ph precipitation. Microorganisms are critical for the transfer of P from the poorly available soil pools.
Several soil bacteria particularly those belonging to genra bacillus and pseudomonas, posses the ability to change insoluble forms by secreting organic acids as Formic acid, acidic, propionic, lactic, glycolic, fumaric and succinic acid. Plants utilize only inorganic P, organic P compounds must first be hydrolyzed by the phosphatase enzyme, which mostly originates from plant roots, through the action of bacteria). . Since then it has been established that there are specific groups of soil microorganisms which increase the availability of phosphates to the plants, not only by mineralizing organic phosphorus compounds but also rendering inorganic phosphorus compounds more available to them (Gerretsen, 1948; Rose, 1957; Sperber, 1957; Louw and Webley, 1959; Sundara Rao, 1965; Taha et al., 1969; Jackson et al., 1972; Leyval and Berthelin, 1989; Peix et al., 2001). The efficacy of various phosphate solubilizing bacteria (PSB) in dissolving insoluble phosphates such as suspension, agar, soil, bone meal, hydroxyapatite, and rock phosphate has received considerable attention during last two decades (Goldstein, 1986; Goldstein and Liu, 1987; Gaur et al., 1990; Illmer and Schinner, 1992, Vanquez et al., 2000
Phosphate solubilizing microorganism (PSM) includes bacteria and fungi. Amongest bacteria most effeciant Phosphate solublizers belongs to genra Bacillus and Pseudomonas. Microorganisms directly affect the ability of the plants to acquire phosphorus from soil through a number of structural or process mediated mechanisms (Richardson, 2001). These include: (a) an increase in the surface area of the roots either an extension of existing root systems (e.g., mycorrhizal associations) or by enhancement of root branching and root hair development (i.e., growth stimulation through phytohormones), (b) by displacement of sorption equilibrium that results in increased net transfer of phosphate ions into soil solution or an increase in the mobility of organic forms of phosphorus, and (c) through the stimulation of metabolic processes that are effective in directly solubilizing and mineralizing phosphorus from the poorly available forms of inorganic and organic phosphorus
1.2. Nitrogen Fixation
Nitrogen (N) is essential element for growth in all the living organism. Although dinitrogen is the major component of air, most living forms, excepting certain microorganisms, fulfill their needs for nitrogen by using combined forms of N. Nitrogen is abundant on the earth and composes 80% of the atmosphere, but is unavailable to plants. It needs to be converted into ammonia, a form available to plants and other eukaryotes. Atmospheric nitrogen is converted into forms utilized by plants by three different processes
a) Conversion of atmospheric nitrogen into oxides of nitrogen in the atmosphere.
b) Industrial nitrogen fixation uses catalysts and high temperature ( 300-500°c) to convert nitrogen to ammonia and
c) Biological nitrogen fixation involves the conversion of nitrogen to ammonia by microorganisms using a complex enzyme system identified as nitrogenase ( Kim and Rees, 1994)
Biological nitrogen fixes about 60%of the earth’s available nitrogen and represents an economically beneficial and environmentally sound alternative to chemical fertilizers ( Ladha et al,1997)
PGPR that fixes nitrogen in non-leguminous plants are diazotrophs that form a non-obligate interaction with host (Glick et al, 1999). The process of nitrogen fixation is carried out by the nitrogenase enzyme coded by nif genes (Masepohl and Klipp, 1996; Kim and Rees, 1994). One of the best studied diazotrophs for nitrogen fixation is Azospirillium sp. Isolated from nitrogen poor soils by Beijernick in 1925( Holguin et al, 1999). Members of this bacterial genus are capable of fixing atmospheric nitrogen and of promoting plant growth.
In modern agriculture, the natural processes for replenishing nitrogen used up by crops are too slow to sustain the productivity needed. Major contributors of fixed N in the soil are the N fixing microbes and chemical fertilizers. Microbial system can siphon out appreciable amounts of N from the atmospheric reservoir and enrich soil with this important but scare nutrient (Okon, 1985; Pandey and Kumar, 1989). Microbial groups that affect plants by supplying combined nitrogen include: (a) symbiotic N2- fixing Rhizobium which are obligate symbionts of the legumes, and others like Azospirillum which colonize root zones and fix N in loose associations with the plants, (b) Actinomycetes in non-leguminous trees, and (c) free living N2-fixers as blue green algae, Acetobacter, Azotobacter, Bacillus, Klebsiella,and Pseudomonas (Subba Rao, 1982).
1.3. Production of Plant Growth Regulators (PGRs)/ Phytohormones:
One of the direct mechanism by which PGPR can promote plant growth is by production of plant growth regulators or phytohormones (Glick 1995).
Plant growth regulators ( PGRs) are the organic substances that influence physiologically processes of the plant at very low concentration. PGRs include bacterial metabolites that effect plant growth; examples of PGRs are hormones (phytohormones ) or their derivatives are produced by bacteria such as Azotobacter, Azospirillium, Bacillus and Psuedomonas . Beneficial effects of these PGRs includes the promotion and proliferation of root development, which results in efficient uptake of water and nutrients.
2. INDIRECT EFFECTS OF PGPR :
Phytopathogen can reduce crop yields from 25-100%, which is an enormous potential loss of productivity. Biological control of plant pathogenhave been identified as integral component of strategies to combat the phytopathogens. Biological PGPR has been found among the most abundant plant associated microbial genera such as Bacillus, Burkholderia, Psuedomonas, Streptomyces and Tricoderma. Such microorganism may limit the damage caused by various phytopathogen by producing antibiotics, siderophores, HCN, ammonia, volatile metabolities and enzymes etc. these can also function as competitors of pathogen for colonization sitesand nutrient or as
inducers of systematic resistance.
2. 1. Biological Control
Biological control as defined by Garret, (1965) involves red). Three classical categories of antagonism are: antibiosis, competition and exploitation (Park, 1960). Antibiosis refers to the production of metabolic agents by the organisms, which have harmful effects on the others (Jackson, 1965). Competition is the active demand in excess of immediate.
Supply of material or condition (Clark, 1965), and exploitation is either predation or direct parasitism (Boosalis, 1964). Clarifications of causal mechanisms gave new impulses to find ways for the use of biological means to protect plant roots against pathogens (Lynch and Ebben, 1986). uction in disease through the agency of one or more living organisms other than the host.
2.2. Production of siderophores
Many bacteria show the production of siderophores (Leong and Neilands, 1982). One of the major mechanisms by which biological control organisms exert their effects is the production of siderophores to deny iron to plant pathogens. Siderophore bind most of the Fe+3 that is available in the rhizosphere, and as a result effectively prevent any fungal pathogens in this immediate vicinity from proliferation because of lack of iron (O’ Sullivan and O’ Gara, 1992)
3. Carrier Based Formulations
The inoculant is the means of bacterial transport from the factory to the living plant (Bashan, 1998) and the carrier is regarded as the delivery vehicle of live microorganisms from the factory to the field; however, no universal carrier or formulation is presently available for the release of microorganisms into the soil (Trevors et al., 1992). The maintenance of cell survival and activity in both rhizosphere and non rhizosphere soils is an important consideration for success of any inoculation protocol refers to a formulation containing one or more beneficial strains (or species) in an easy-to-use and economical carrier material, organic, inorganic, or synthesized from defined molecules.
In the starting phase of the inoculation technology agar or liquid suspension based bacterial preparations were used. Although effective for Rhizobium (Smith, 1992), the problems relating to the inability of such formulations to provide a protective environment, difficult handling, poor survival during storage and after addition to seeds or plants has led to the use of alternative carriers (Subba Rao, 1982; Tilak, 1993; Bashan, 1998; Van Dyke and Prosser, 2000). Peat has been a material of choice to be used as carrier to formulate inoculants of various PGPRs (Burton, 1967; Bezdicek, 1978; Smith, 1987; Williams, 1984). Sedge peat in USA (Burton, 1967) and milled dry peat in Australia (Date,1974) have been used for formulating rhizobia inoculant.
Future Prospects of PGPR in Himalayan Regions :
The success of these products will depend on our ability to manage rhizosphere to enhance survival and competitiveness of these beneficial microorganisms. Genetic enhancement of PGPR strains to enhance colonization and effectiveness may involve addition of one or more traits associated with plant growth promotion. However, regulatory issues and public acceptance of genetically engineered microorganism may delay their commercialization. The use of multi-strain inoculum of PGPR with known function is of interest as these formulations may increase consistency in the field. A large number of bacteria were isolated from a number of soil samples collected from various temperate/alpine (up to 3600 m above mean sea level) locations (Pandey and Palni, 1997; 1998a; 1998b). The investigations indicated dominance of the species of Bacillus and Pseudomonas in soils of colder regions. Besides a number of biochemical growth parameters, these isolates were also characterized for various “beneficial” properties, e.g., production of antifungal compounds, effective against a wide range of phytopathogenic fungi, and ability to solubilize tri-calcium phosphate, etc. (Pandey et al., 1997; 2001; 2002a; Chaurasia, 2005).
PGPR offer an environmentally sustainable approach to increase crop production and health. The application of molecular tool is enhanching our ability to understand and manage the rhizosphereand will lead to new productwithimproved ffectiveness.
MATERIALS AND METHODS
Study Site
The soil samples were collected from rhizosphere of wheat plant cultivated at our institute in G.B.Pant Institute of Himalayan Environment and Development, Kosi Katarmal Almora, Uttarakhand.
1. Collection of soil samples:
Soil samples were collected directly from the field of wheat crops. Soil samples were aseptically placed in the paper bags respectively, and transported immediately to the laboratory for analysis. The soil samples were taken 6-10 cm depth.
2. Isolation and Subculturing:
2.1 Isolation of microorganisms from soil and water
1gm of soil was taken in 9ml of sterile distilled water and shaken vigorously. Decimal dilution were prepared in sterile diluents and 1ml of appropriate diluted suspension was pipetted in the petridish and mixed with molar media (Serial dilution techniques: Johnson and curl, 1972) .The czapek Malt Agar (Hi Media Mo33), Jensen’s Medium (HiMediaM710) and Actinomycetes Isolation Agar (Hi Media M490) were used for the isolation of bacteria, fungi and actinomycetes, respectively. Plates were incubated at 28°C in BOD incubator for 48hr for bacteria and 120 hr for actinomycetes and fungi. Colonies appeared on Agar plates were counted as colony forming units per g (cfu/g) of sample.
2.2 Sub-culturing and maintenance
Isolated colonies of microorganism were checked for purity by repeat sub-culturing and streaked on slants of their respective media on which they developed during isolation.
3.3 Preliminary Identification of bacterial Isolates
(Cowan and steel 1974; Collins and lyne, 1980; Shermau and cappuccino, 1996).Representative colonies were pricked up from the agar plates and subjected to the preliminary identification of the selected bacteria was done on the basis of their cultural & growth characteristics, morphological and biochemical properties.
3.1.1 Growth in broth:
A loopful of culture was inoculated in the respective broth. After incubation, observations were recorded on oxygen (aerobic/anerobic/facultative), distribution of growth, and other properties like pigmentation, etc.
3.1.2 Growth on agar plates:
Cultures were grown on the respective agar media. After required incubation observation were recorded on size shape (raised, Flat, domed, irregular, rizhoid, serated, umbonate etc), nature (dry, rough, mucoid, slimy etc) and pigmentation
3.1.4 Gram’s staining:
Differential staining is used for categorizing the bacteria into two group- Gram positive and gram negative. Hi Media K001 gram’s staining Kit was used for Gram staining.1) Thin Smears of isolates were prepared in sterilized water and the smear’s were dried and fixed (slightly heat).
2) Fixed smears were flooded with crystal violet for one minute and washed with distilled water.
3) Smear were flooded with iodine solution for one minute and washed with distilled water.
4) Now smears were subjected to decolorizing agent for not more than five seconds and washed with distilled water.
5) Finally, smears were subjected to the counter stain saffranin for ten seconds.
6) Washed stained slides and left for air dry.
7) Observed under light microscope.
3.2 Biochemical activities of bacterial isolates.
The biochemical reactions occur both outside and inside the cell are precisely controlled by some governing factors, the enzymes. Exoenzymes, which are few in number, are released from the cell and act on the substrate. Endoenzymes are mainly responsible for synthesis of new protoplasmic requirements and production of cellular energy from assimilated materials. As a result of these metabolic processes, metabolic products are formed and excreted by the cell into the environment. Assay of these end products not only aids in identification
3.2.1 Starch hydrolysis
Starch is degrade by the extracellular enzyme amylase and hydrolyzed into disaccharide namely dextrin and ultimately into maltose molecules. Starch in the presence of the iodine will impart a blue-black colour to the medium, indicating the absence of starch splitting enzymes and respectively the negative result. If the starch has been hydrolyzed, a clear zone of hydrolysis will surround the growth of the organism, representing a positive result.
Starch agar (Hi Media M107) plates were prepared under aseptic conditions. Plates were inoculated with test isolates by mean of single line streak. After inoculation, the plates were incubated in inverted position at 28°C for 48hr. The starch plates with culture were examined for the presence and absence of a blue-black surrounding the growth.
3.2.3 Gelatin hydrolysis
Gelatin below temperature 25ºC will maintain its gel property and exists as a semi-solid; gelatin in liquid at temperature above 25°C.Some microorganism is capable of hydrolyzing the gelatin. Once this degradation occurs, even very low temperature of 4ºC will not restore the gel characteristic.
Nutrient gelatin medium (Hi media M060) were prepared in deep tubes and autoclaved. Tubes were incubated with test isolates through single line stabbing. After inoculation, all deep tubes were incubated at 28°C for 48 h.After incubation the gelatin agar deep tubes cultures were placed into a refrigerator at 4ºC for 30 minutes. The cultures were observed to determine whether the medium was solid or liquid.
3.2.4 Catalase test
During aerobic respiration, microorganisms produce hydrogen peroxide and in some cases, an extremely toxic superoxide. Organisms capable of producing catalase rapidly degrade hydrogen peroxide.
2H2O2 CATALASE 2H2O + O2
HYDROGEN PEROXIDE (3%) WATER FREE OXYGEN
Nutrient agar slants were prepared and autoclaved. A loop full of test organism was inoculated into the slants by mean of single line streak. Uninoculated tubes served as control. The cultures were incubated at 28 ºC for 48 h. After incubation, three or four drops of 3% H2O2 were allowed to flow over the entire surface of each slant culture. Each culture was examined for presence or absence of bubbling.
3.2.5 Oxidase test
Cytochrome oxidase catalyses the oxidation of a reduced cytochrome by molecular oxygen, resulting in the formation of H2O or H2O2. The ability of bacteria to produce this enzyme can be determined by discs containing redox dye (tetramethyl-p-phenylene-diamine). The dye is reduced to a deep purple colour.
Nutrient agar plates were inoculated with test organisms by mean of single line streak, under aseptic conditions. All the plates were incubated at 28 ºC for 48 h. After incubation, oxidase discs were moistened with distilled water in petriplate. Wet disc was touched to the colony. A positive reaction was indicated that by a change in colour within 10 seconds as appearance of deep purple colour, a delayed positive reaction appears in 10 – 60 seconds.
3.2.6 Urease test
Urease is a hydrolytic enzyme. The presence of this enzyme is detectable when the organisms are grown in urea broth medium containing indicator phenol red. Bacteria decompose urea by means of the enzyme urease, creating alkaline environment and turns indicator to deep pink colour.
Urea broth (Hi Media M111) tubes were prepared and inoculated with test organisms by mean of inoculation loop, under aseptic conditions. The tubes were incubated at 28 ºC for 48 h. after incubation; urea broth was examined for development of colour.
Test for identification of Enterobacteriaceace (IMViC Test)
Indole Production:
Indole nitrate medium tubes were inoculated with test isolates. After inoculation, all the tubes were incubated at 28 °C for 48 h. Uninoculated tubes served as control. 1.0 ml of Kovac’s reagent was then added to each tube including control. In each tube, the colour of the reagent layer was examined. Formation of a cherry – red reagent layer indicated positive reaction.
Methyl Red (MR):
MR – VP medium tubes were inoculated with test isolates. After inoculation, the tubes were incubated at 28 °C for 48 h. Uninoculated tubes served as control. 5 drops of methyl red indicator was then added to each tube including control. Tubes were observed for the change in colour of methyl red indicator. Broth retaining red colour was taken as positive reaction.
Voges Proskauer (VP):
MR – VP medium tubes were inoculated with test isolates and were incubated at 28 °C for 48 h. Uninoculated tubes served as control. 10 drops of Barritt’s reagent A were added and cultures were shaken thoroughly. After this 10 drops of Barritt’s reagent B were added in each tube including control. The cultures were examined after 15 min, for the development of colour. Formation of a pink complex was indicative of positive reaction.
Citrate Utilization:
Citrate agar medium slants were inoculated with test isolates and incubated at 28 °C for 48 h. after incubation all slant cultures were examined for the presence or absence of growth and colouration of media. Change in colour of the medium from green to blue along the bacterial growth indicated positive result reaction.
Fermentation of carbohydrates by different bacterial isolates.
Bromocresol Purple broth tubes were prepared and inoculated with test isolates under aseptic conditions. In each tube a coated disc of known sugar was added and medium was covered with paraffin oil. Uninoculated tubes served as control. All the tubes were incubated at 28 °C for 48 h. after incubation tubes were examined for change in colour and appearance of bubbles. The Bromocresol Purple broth contains bromocresol’s indicator, which is light pink or straw colored at neutral pH that turns to deep pink at low pH and yellow at high pH (positive reaction).
Characterization of bacterial isolates for plant growth promotion properties:
The six bacterial isolates were tested for their plant growth promotion activities, viz. their ability to solubilize insoluble phosphate and for their biocontrol properties.
Phosphate solubilization properties of bacterial isolates
The six bacterial isolates were assayed for their phosphate solubilizing activity by plate based assay using Pikovskaya (Pikovskaya, 1948) medium. One strain per plate was streaked in the center with the help of sterile toothpicks. The halo and the colony diameter were measured after incubation of the plates for 6-7 days at 28oC Halo size was calculated by subtracting colony diameter from the total diameter.
Determination of soluble phosphorus (µg) in the growth medium. (Chlorostannus reduced molybdo-phosphoric acid blue method.)
Take the broth of bacterial isolates and centrifuge it and for actinomycetes filter it.After centrifugation or filtration, 7.0 ml aliquots of each 6 cultures was transferred to test tube. To this 2.0 ml of ammonium molybdate solution was added and left for 10 min. for colour development. Then added 2.0 ml of freshly prepared stannous chloride solution was added. Absorbance of the final solution was measured at 700nm.
Calculations
c = I-C
c = µg phosphorus solubilization.
I = µg phosphorus obtained
C= µg phosphorus obtained from graph in control.
Using the above formula the amount of phosphorus solubilized was calculated.
Preparation of Reagent:
1. Phosphorus standards: for stock solution 0.4393g of dry KH2PO4 was dissolved in 1L distilled water to obtain 100 microgram/ml of phosphorus in the solution.
2. Ammonium molybedate-sulphuric acid reagent 6.25g of (NH4)6NO7O24.4H2O was dissolved in 50.0ml distilled water slightly heated to dissolve, 70.0ml conc. HSO4 was added to this carefully(with mixing and cooling). The final volume was made to 250ml in volumetric flask. The reagent was stored in amber coloured bottle in a cool & dark place.
3. Stannous chloride reagent: in 0.5g SnCl2 2.0 ml Conc. HCL was added and heated slightly. The final volume was made up to 250ml with distilled water. This was always prepared fresh just before use.
Diffusible antifungal activity in bacterial isolates:
The bacteria were tested for the production of antagonistic compounds by dual culture technique. To test the ability of the bacteria to inhibit the pathogen, a mycelial disc of the test fungus (6.0 mm in diameter) from a 5 day-old PDA culture was placed off-center on each Potato carrot agar (PCA) plate. After 24 h incubation at 28oC, a filter paper disc (0.5 mm in dia) dipped in bacterial suspension containing 106 cfu ml-1 grown in TY broth for 24 h at 28oC, was placed 2.5-3.0 cm away from the respective test fungal colony. Petri plates were incubated in the dark at 28oC and antagonistic effect was recorded every 24 h for 5 days by measuring the percentage inhibition. Percent growth inhibition was calculated by the following formula: 100 x (R2-R1)/R2 (where R2 represents the radius of the colony of the test fungus in the direction with no bacterial colony and R1 is the radius of the fungal colony in the direction of the bacterial colony).
RESULTS
MICROBIAL POPULATION IN SOIL:
Isolations were carried out on three selective and three non selective media at three temperatures. Moderate populations of bacteria, fungi and actinomycetes (x 104 and x 105) were recorded on Czapek Malt Agar, Sabouroud Dextrose Agar, Trypton Yeast Extract Agar and Actinomycetes Isolation Agar plates Table 1:
The growth of pure cultures of isolates was observed on five different media and observations were recorded for various morphological and cultural characteristics (Table 1.1, 1.2, l.3; Fig 1 A&B). Trptone Yeast Extract Agar (TYA) was found to be the best media for the growth of all the isolation of bacteria followed by Jensen’s Media (JM) & Actinomycetes Isolation Agar (AIA) was best for the growth of Actinomycetes and fungi grow best on Sabouraud Dextrose Agar (SDA) and Czapek Malt Agar (CMA). The size of colonies ranged from 8mm to 15mm on TYA; 9mm to 15mm on AIA and 5 to 10mm on SDA and CZP.
Table 1. Colonies of various media [Rhizospheric and Non- Rhizospheric soil sample];
Media Microbial population ( cfu = colony forming units) x 10 5
Bacteria Fungus
R1 NR1 R2 NR2 R3 NR3 R1 NR1 R2 NR2 R3 NR3
Czapek Malt 9 11 - 70 6 70 6 * * - * *
30 18 14 16 26 10 2 2 * - * -
22 19 28 14 9 33 4 2 2 - * -
Sabouroud Dextrose Agar 36 9 49 16 12 13 2 - 6 - 1 -
35 31 4 22 39 21 - - 1 2 4 *
35 16 44 18 17 26 4 1 * - 1 -
Trypton Yeast Extract Agar 38 28 73 28 49 39 2 - 2 - 2 -
51 24 129 24 82 50 - - - - 2 2
62 42 162 42 162 41 - 1 * 2 3 *
*= Full plate growth of fungus
Media Microbial population ( cfu = colony forming units) x 10 5
Bacteria Fungus
R1 NR1 R2 NR2 R3 NR3 R1 NR1 R2 NR2 R3 NR3
Jensen’s media 95 11 - 70 6 70 6 * * - * *
105 18 14 16 26 10 2 2 * - * -
48 19 28 14 9 33 4 2 2 - * -
Actinomycetes Isolation Agar 36 9 49 16 12 13 2 - 6 - 1 -
35 31 4 22 39 21 - - 1 2 4 *
35 16 44 18 17 26 4 1 * - 1 -
Table 2. Cultural and Morphological characteristic of Bacterial isolates
Isolate
No. Colony Colour Size
(mm) configuration Margin Elevation
B-1
White 15 Irregular and spreading Smooth Crateriform
B-2 White 14 Concentric Lobate Crateriform
B-3 White 15 Filamentous Lobate Hilly
B-5 White 10 Irregular and spreading Wavy Hilly
B-6 White 10 Wrinkled Wavy
Umbonate
B-7 Cream 9 Wrinkled Smooth Raised
B-8 White 10 Round Entire Raised
B-9 Orange 11 Round Smooth Convex
B-11 Off white 8 Round Entire Convex
B-12 White 10 Round Entire Raised
B-14 Off white 12 Round with raised margin Smooth Flat
B-15 Milky 9 Rhizoid Wooly Crateriform
B-15(a) White 8 Filamentous Branching Ingrowing into medium
B-17 Creamy white 10 Irregular and spreading Wavy Flat
B-19 yellow 7 Round Ciliate Umbonate
B-24 White 9 Wrinkled Lobate Hilly
B-24(a) pinkish 5 Round with scalloped margin Lobate Drop like
B-28 White 10 Round with raised margin Smooth Raised
B-28(a) white 13 Concentric Smooth Raised
B-28(b) off white 14 Round Entire Hilly
B-29 white 11 Round with raised margin Smooth Raised
B-32 Creamy 9 Round with scalloped margins Wavy Umbonate
B-33
Orange 12 Round Entire Convex
B-34 White 11 Round Wavy Flat
B-35 Off white 14 Round Wavy Raised
B-35(a) White 13 Round with raised margin Wavy Flat
B-36 White 9 Round Smooth Convex
B-37 White 10 Concentric Lobate Raised
B-38 White 6 Wrinkled Wavy Umbonate
B-39 White 11 Concentric Wavy Raised
B-40 yellow 10 Round with scalloped margin Entire Raised
B-41 Creamy white 9 Irregular and spreading Irregular (erose) Ingrowing into medium
B-43 yellow 12 Round Irregular Convex
B-44 White 8 Round Irregular Convex
B-45
pinkish 7 Round
Lobate Ingrowing into medium
B-46 White 13 Irregular and spreading Wavy Ingrowing into medium
B-47 White 12 Round Entire convex
B-48 Brown 9 Round Lobate Raised
B-51 off white 10 Round Lobate Ingrowing into medium
B-52 Creamy 12 Round Smooth Ingrowing into medium
B-53 white 11 Round Smooth Ingrowing into medium
B-55 orange 10 Round Entire convex
B-56 Orange 10 Round Entire convex
B-57 White 7 Round Entire Raised
B-58 Off white 9 Irregular and spreading Wavy Raised
B-59 White 5 Round Smooth Raised
B-60 White 7 Round Smooth Raised
B-62 White 9 Round with scalloped margin Lobate Raised
B-67 White 7 Wrinkled Wooly Raised
B-68 yellow 13 Round Entire Convex
B-69 White 11 Round Smooth Ingrowing into medium
B-70 Off white 9 Round with scalloped margin Wooly Ingrowing into medium
B-71 White 10 Round Smooth Raised
B-72 White 13 Round Smooth Ingrowing in medium
B-73 white 14 Round Smooth Drop like
B-74 Off white 9 Round smooth Drop like
B-75 White 7 Round with scalloped margin Wavy Raised
B-75(a) White 8 Round Smooth Drop like
B-76 White 10 Round with scalloped margin Wavy In growing into medium
B-77 Brown 6 Round with scalloped margin Ciliate Raised
B-78 Yellow 11 Round with scalloped margin Wavy Convex
Outs of these 60 bacterial strains only 25 strains are selected for further studies, this is due to the reason that maximum of bacteria and actinomycete are repeated.
Microscopic characterizations
All the bacterial species were observed for their microscopic characters and records are presented in table 2 and plate C. All the strain showed typical characters of fungi. The colonial characters were considered to be more important for identification of the species. Some of the isolates showed spore bearing cells (Phialides).
Table 3. Microscopic characterizations of Bacterial isolates
S no Bacterial isolates Gram reaction Size in ? Cell morphology Shape
Length Width
1 B-1 Gram +ve 5 1 Singles Rod
2 B-3 Gram+ve 4 0.5-1 Chain , Single Rod
3 B-5 Gram +ve 4-5 1 Singles Rod
4 B-6 Gram+ve 6-9 2-2.5 Chain , Single , Doubles Rod
5 B-7 Gram +ve 2.5-3 2.0 Clusters Round
6 B-8 Gram+ve 2.5-3 2.0-2.5 Clusters Round
7 B-11 Gram +ve 0.5-1 0.25-0.5 Single , Doubles Short Rod
8 B-14 Gram+ve 4-5 2.0 Clusters, Doubles Rod
9 B-15(a) Gram+ve 6-8 1-1.5 Clusters , Chains Rod
10 B-17 Gram +ve 3-5 1.0 Clusters , Chains Rod
11 B-19 Gram+ve 0.5-1 0.25-0.5 Single , Doubles Very short Rod
12 B-24 Gram +ve 4-7 1.5-2.0 Clusters, Doubles Rod
13 B-24(a)? Gram+ve 35-80 0.5-1 Filamentous Branched
14 B-28 Gram +ve 2-3 2-3 Clusters , Chains Round
15 B-28(b) Gram+ve 4-5 1-1.5 Clusters , Chains Round
16 B-33 Gram -ve 1-1.5 0.25-0.5 Singles Very Small Rod
17 B-35(a) Gram+ve 5-7 1-1.5 Doubles , Chains Rod
18 B-43 Gram +ve 3-4 2-3 Clusters Round
19 B-43(a) Gram+ve 2-4 0.5-1 Singles Rod
20 B-48? Gram +ve 45-85 1-1.5 Filamentous Branched
21 B-58 Gram+ve 3-4 0.5-1.0 Chains Rod
22 B-72 Gram +ve 3-4 2-3 Clusters , Chains Round
23 B-76(a) Gram+ve 3-3.5 2-3 Chains Round
24 B-77? Gram +ve 70-85 1.0-1.5 Filamentous Branched
25 B-79 Gram+ve 3-4 1.0-1.5 Clusters Rod
* = Actinomycetes
Extracellular Enzymatic activities of bacterial isolates:
Result on hydrolysis of starch and gelatin is presented in table 3. Out of 25 isolates 23 were capable of hydrolyzing and 11 hydrolyzed gelatin
Table 4.showing Extracellular enzymatic activity
S. No. Bacterial Isolate No. Starch Hydrolysis
Gelatin Hydrolysi
1 B-1 + _
2 B-3 + +
3 B-5 + +
4 B-6 + +
5 B-7 _
+
6 B-8 _ +
7 B-11 _ +
8 B-14 + +
9 B-15(a) + +
10 B-17 + +
11 B-19 _ +
12 B-24 + +
13 B-24(a) _ +
14 B-28 + +
15 B-28(b) _ +
16 B-33 + +
17 B-35(a) _ +
18 B-43 _ +
19 B-43(a) _ +
20 B-48 _ _
21 B-58 + +
22 B-72 + +
23 B-76(a) _ +
24 B-77 + _
25 B-79 _ _
Intracellular enzymatic activities of Bacterial isolates:
(Reaction with catalase, oxidase and urease)
The reaction of bacterial isolates against catalase, oxidase and urease are presented in table 4, bacterial isolates were positive for catalase. All isolates were positive for oxidase and none are positive for urease.
Table 5. Showing intracellular enzymatic activity
S. No. Bacterial Isolate No. Catalase
Oxidase Urease
1 B-1 + + _
2 B-3 + + _
3 B-5 + + _
4 B-6 _ + _
5 B-7 _
+ _
6 B-8 _ + _
7 B-11 _ + _
8 B-14 _ + _
9 B-15(a) + + _
10 B-17 + + _
11 B-19 _ + _
12 B-24 _ + _
13 B-24(a) _ + _
14 B-28 _ + _
15 B-28(b) _ + _
16 B-33 + + _
17 B-35(a) + + _
18 B-43 _ + _
19 B-43(a) + + _
20 B-48 _ + _
21 B-58 _ + _
22 B-72 _ + _
23 B-76(a) _ + _
24 B-77 _ + _
25 B-79 _ + _
Test for identification of Enterobacteriaceace (IMViC Test)
Test for characterizing the properties of family Enterobacteriaceae were performed for all the isolates and the results are presented in the table 5. 10 bacterial isolates produced Indole. 4 of the isolates were positive for Methyl Red reaction. 15 were positive for Voges-Proskauer and 15 for citrate utilization.
Table 6.showing IMViC test
S. No. Bacterial Isolate No. Indole Production
Methyl Red Voges Proskeur Voges Proskeur
1 B-1 _ _ + _
2 B-3 _ _ + +
3 B-5 _ _ + +
4 B-6 + _ _ _
5 B-7 _
_ _ +
6 B-8 + _ _ +
7 B-11 _ _ + +
8 B-14 + _ + _
9 B-15(a) _ + + +
10 B-17 _ _ + +
11 B-19 + _ + _
12 B-24 _ _ + +
13 B-24(a) + _ _ _
14 B-28 + _ + _
15 B-28(b) _ _ + +
16 B-33 _ _ + _
17 B-35(a) + _ _ +
18 B-43 _ _ + +
19 B-43(a) _ _ _ +
20 B-48 _ _ _ _
21 B-58 + + _ _
22 B-72 _ + + +
23 B-76(a) _ + + +
24 B-77 + _ _ _
25 B-79 + _ _ +
Fermentation of carbohydrates by bacterial isolates.
All the bacterial isolates were tested for their ability to utilize various carbon sources and and results obtained in table 6.(III) and 6(IV).Total numbers of sugars under various categories were tested
Phosphorus solubilization
Qualitative estimation of phosphate solubilization:
The exent of growth and phosphate solubilization of all the bacterial species has been tabulated in the table 7.Out of 25 species, 6 solubilized phosphate at 28°C temperature. The colony diameters and zone of solubilization were measured for all the bacteria after 10 days of incubation. It was found that 28°C was the optimum temperature for most of them for solubilization. 6 strains showing maximum solubilization selected for estimation of solubilization of tricalcium phosphate quantitatively.
Plate bases assay for phosphate solubilization by bacterial isolates:
Table 7. Plate bases assay for phosphorus solubilization by Bacterial Isolates.
S.No Bacterial isolates Phosphorus solubilization
1. B-1 +
2. B-3 -
3. B-5 +
4. B-6 -
5. B-7 -
6. B-8 -
7. B-11 -
8. B-14 -
9. B-15a -
10. B-17 -
11. B-19 -
12. B-24 -
13. B24a -
14. B-28 -
15. B-28b -
16. B-33 -
17. B35a -
18. B-43 +
19. B-43a +
20. B-48 +
21 B-58 -
22 B-72 -
23 B-76a -
24 B-77 +
25 B-79 -
Quantitative estimation of tri- calcium phosphate solublization.
Quantitative estimation of phosphate solubilization by the selected penicillium isolates in broth base assay is recorded in table 9. It was found that isolates number 4 has solubilized maximum phosphate on day of incubation. After reaching the maximum value the phosphate solubilization activity decreased sharply irrespective of the isolate selected. A uniform decreases in Ph with a few exception was also recorded (table).
Table 8(I). Quantitative estimation of tri calcium phosphate solublization.
Bacterial isolates 7th day p- solublization (µg/ ml)
O.D. I C O.D. I C Mean
B-1 2.275 85 79 2.185 90 84 69.0
B-5 1.304 50 44 1.495 53 47 45.5
B-43 1.697 60 54 2.305 90 84 69.0
B-43(a) 2.469 88 82 2.701 106 100 94.5
B-48 0.968 13.7 7.7 1.181 41 35 25.2
B-77 2.758 106 100 2.679 100 94 97.0
OD of control : 6 µg/ ml
Bacterial isolates 14th day p- solublization (µg/ ml)
O.D. I C O.D. I C Mean
B-1 1.171 42 36 1.006 39 33 39.5
B-5 1.762 65 59 1.698 62 56 57.5
B-43 1.872 70 64 1.912 74 68 66.0
B-43(a) 1.393 50 44 1.532 57 51 47.5
B-48 1.872 39 33 1.249 45 39 36.0
B-77 1.042 74 68 2.042 77 71 69.5
OD of control : 6 µg/ ml
Table 8 (II). Quantitative estimation of tri calcium phosphate solublization.
The quantitative estimations of phosphate solubilization of bacteria six isolates are selected and their spectrophotometry was taken in duplicate and their mean is calculated. These values indicate the amount of phosphate solubilized.
Phosphatase activity:
There was no any bacterial isolate, which shows phosphatase enzyme activity in the experiment.
Table 9. Changes in pH due to tricalcium phosphate solubilization of selected bacteria on 7th day
Bacterial strains
Changes in pH due to phosphate solubilization on 7th day by selected bacterial isolates in duplicate
(1) (2)
B-1 6.6 6.6
B-5 6.1 6.1
B-43 6.0 6.3
B-43(a) 6.1 5.9
B-48 7.2 7.4
B-77 5.5 5.4
Table 10. Changes in pH due to tri-calcium phosphate solubilization of selected bacteria on 14th day
Bacterial strains
Changes in pH to phosphate solubilization on 14th by selected isolates bacterial isolates in duplicate
(1) (2)
B-1 6.6 6.6
B-5 6.1 6.1
B-43 6.0 6.3
B-43(a) 6.1 5.9
B-48 7.2 7.4
B-77 5.5 5.4
Antagonism (antifungal activity):
There are 6 bacterial stains for which antagonism was performed and among those six few bacteria shows positive antagonism.
11. Table of Antagonism shown by different pathogenic strain of fungi
Bacterial isolates cladosporium Fusarium A.alternate Wheat pathogen
B-1 - - - -
B-5 + + + +
B-43 - - - -
B-43(a) - - + -
B-48 - + + +
B-77 - - - -
Diffusible antifungal effect of bacterial isolates:
Out of 6 bacteria only 3 were showing antagonistic effect to fungiwhere B-5 showed antifungal effect against all 4 pathogenic fungi.B-48 showed antifungal effect against 3 fungi namely A.alternate, Fusarium and wheat pathogen.while B-48 showed the effect only against A.alternate.
Zone of inhibition:
Zone of inhibition is calculated by the formula
100 x (R2-R1)/R2
On putting the measured values in the formula, we get the zone of inhibition of the isolated bacteria
B-5:
(i) A. alternate R1= 1.4, R2=3.5
zone of inhibition = 54.8
(ii) Fusarium R1=1.5, R2=2.6
zone of inhibition = 42.3
(iii)Wheat pathogen R1=1.2, R2=2.3
zone of inhibition = 47.8
(iv) Cladosporium R1=0.7, R2=1.7
zone of inhibition = 58.8
B-43:
(i) A. alternate R1= 1.8, R2=2.9
zone of inhibition = 37.9
B-48:
(i) A. alternate R1= 1.3, R2=2.7
zone of inhibition = 58.4
(ii) Fusarium R1=1.9, R2=3.0
zone of inhibition = 36.6
(iii)Wheat pathogen R1=1.2, R2=2.8
zone of inhibition = 57.1
4.9. Field Experiment
The data showing the influence of bacterial inoculations on growth and yield of wheat are presented in Table 11 The treatments resulted in improvement in biomass, in terms of root; shoot and grain weight both on per plant and unit area (m2) basis. In this experiment also, B. subtilis gave the best performance, with an increase of 1.40 and 1.55 fold for total biomass and grain yield, respectively, on per plant basis. For P. corrugata treatment the increase was 1.26 and 1.36 fold and for B. megaterium it was 1.17 and 1.25 fold, respectively. The harvest index per unit area also showed an increase in all the treatments as compared to control. There was a positive increase in root length; it was 12.34, 15.60, 16.80 and 15.83 cm in control, B. megaterium, B. subtilis and P. corrugata treatments, respectively. The shoot height was also positively influenced by bacterial treatments in the order: B. subtilis > P. corrugata > B. megaterium > control.
DISCUSSION
Indirect and indirect effect of plant species and soil properties on the composition and activities of microorganism in the rhizosphere are numerous and difficult to resolve (Shaw and Burns 2005). Growth promotion by PGPR is depending upon root exudation partially in the areas of root where deleterious organism becomes active (Bakker and Schippers, 1987). The bacterial inoculation were seen to positively influenced the measured growth parameters viz, increment in shoot length and stem girth while in the treated plant in general appeared healthy and showed better growth. The inoculation resulted in better growth. The inoculated plants have been transferred to the plantation site for further monitoring.
In the present study, 60 bacterial strains were isolated from the rhizosphere of wheat plant. Out of which 25 bacterial strains were taken for further study, among these 25 there were three actinomycetes. Six soil samples were taken from different plots, in which R1, R2 and R3 were rhizosphere soil and NR1, NR2 and NR3 were nonrhizosphere soil. In rhizosphere, R1 was treated as control while R2 and R3 were inoculated with PGPR. Rhizospheric soil contained more microorganisms than nonrhizospheric soil because rhizospheric soil contains root exudates which help in providing nutrition to the microorganisms (Curl and Truelove1986).The rhizospheric soil treatments namely T1R1, T2R2 and T3R3 were taken for experiment for serial dilution at the dilution factor 10-5. Among all bacterial strains 25 strains were showing different morphological characters. At 280C, these bacteria were showing good growth. On the basis of their characters these were screened. On different media microorganisms show different characters. The first step of identification of bacteria is Gram Staining. This is useful in differentiate bacteria into Gram positive and Gram negative bacteria. Out of 25 strains there was only one Gram negative strain while all others were Gram positive. After the microscopic study in the Phase Contrast Microscope it was found that there were maximum Bacillus species of bacteria present in the soil samples, some were coccus and three strains were Actinomycetes.
After that these bacterial strains were differentiated with the help of biochemical tests. For extra-cellular enzymatic activity there were two tests which were and Gelatin Starch Hydrolysis, when extra-cellular enzymatic activities of bacterial isolates were done, it was found that there was very much difference. In the Starch hydrolysis there were 25 bacterial strains which hydrolysis starch and forms a zone around the bacterial colony while 2 bacterial strains showed negative result. Now in the case of Gelatin Hydrolysis there were 11 bacterial strains which liquidify gelatin and 14 bacterial strains which were showing negative result. But in previous study on the bacterial strains isolated from soil samples it was noted that maximum strains of bacteria were hydrolyzing gelatin. Two strains of actinomycetes B-48 and B-24(a) were showing negative result while B-77 showed positive result. In Intra cellular enzymatic activity of bacterial isolates, three biochemical tests were performed for catalase, oxidase and urease.
Most of the bacterial isolates could ferment only simple sugar, in complex sugar as polysaccharides polyhydric and trisaccharide, there was no positive result. It was found that actionomycetes B -24(a), B-48 and B – 77 had very less ability to ferment sugar. This test is mainly done for study fermentation reaction in members of enterobacteriaceae. B-6 was the only bacterial strain which fermented maximum number of sugars and isolate number B-19 also fermented same number of sugars. Fructose is only sugar which was fermented by maximum sugars. For fermentation tests bromocresol purple media was used because it acts as pH indicator which turns into higher and complex sugars. Adonitol Dulcitiol and Inositol were the sugar which ware not fermented by any number of bacterial strain. For the test of identification of enterobacteericaece IMViC test was performed which were the group of four tests, Indole Production, Methyl red, Voges-Proskauer and Citrate Utilization. For Indole production out of 25 bacterial isolates only 10 give positive result remaining 15 were not producing indole. Then for methyl red there was only 5 bacterial strains which were showing positive results other 20 were negative. For Voges-Proskauer there were 15 positive results while remaining 10 are negative. For citrate utilization again 15 bacterial isolates showed positive results. Among these two isolates B-24(a) and B-77 (actinomycetes) were showing positive results for only Indole production, while B-48 was negative for all the tests. In this study no isolate was found which showed positive result for all the tests.
Out of 25 bacterial isolates from the rhizospheric soil, 6 isolates were found to be phosphate solubilzing. Bacteria were cultured in Pikovskaya broth and then centrifuged. While for actinomycetes, filtration of bacteria was required. All the six cultures showed positive results. On 7th day the least value was calculated in isolate No. B-48 (0.9688). The same procedure was performed on 14th day, but it was observed that on 14th day the value became less than the value of 7th day.
Phosphatase is activated when there is low P-availability in soils, but the presence of organic phosphate causes feedback inhibition of this enzyme. Yadav and Tarafdar (2003), showed that the secretion of phosphatase were maximum at phosphate deficient conditions compared to phosphate sufficient condition. Since there was phosphoric acid production, but not any organic acid production was shown. Other than organic acid production there was many other method which was mentioned by Illmer and Schinner (1992), they have shown that some phosphate solubilizing calcium phosphate (Ca- P) without producing organic acids. They showed that no direct contact between microorganism and Ca-Ps was necessary for effective solubilization. In their opinion the most probable reason for solubilization without acid production is the release of protons accompanying respiration or NH4+ assimilation. It was shown that solubilization of Ca- P with two different solubility products may lead to a short term increase of the amount of at least one Ca-Ps. Precipitation and subsequent resolubilization of different organic or inorganic phosphates result in hardly predictable p- concentration in culture solution.
Wani et al (1979) found that for maximum solubilization of tricalcium phosphate in Pikobskaya medium the best pH was around 6.0 for fungi and 7.0 to 8.0 for bacteria (Pseudomonas & Bacllius) because the bacteria and actinomycetes are uncommon in acid habitats the fungal dominates the microbial community in areas of low pH. Bacterial and actinomycete isolates on 7th day show pH from 5.4 -7.4 and there was no much difference on 14th day pH, it ranged from 5.2 to 6.9.
In the present study, these parameters utilized for determining the antifungal nature of bacterial isolates. However observation recorded from the petriplate indicated that the stability of inhibition zone and other properties should also be given due emphasis while examining antagonistic behavior. For example, the bacterial isolate producing a smaller inhibition zone which does not show any contamination on further incubation may prove to be a better biocontrol agent then isolate producing a large but unstable inhibition zone.
Isolate No. B-48 produced a very strong and stable inhibition zone against Alternaria alternata. Isolate no. B-5 showed inhibition zone against all four pathogens but the strong zone was found in case of Fusarium. But there were many other bacterial strains which were not showing antagonistic effect against any of the four pathogens. Isolate no. B-43(a) also showed strong inhibition zone against Alternaria alternata.
The bacterial antagonist selected in this study was found to be of broad spectrum utility. The use of broad spectrum antagonist may be useful for suppressing the major pathogen while a potential biocontrol agent should possess specific antagonistic activity. As the bacterial inoculants have less impact on the environment than chemical control of root disease is very promising (Nakas Hogedorn 1990). Understanding of the mechanism through which the biocontrol of plant diseases occurs is critical in the eventual improvement and wider use of biocontrol methods. These mechanisms are generally classified as competition and antibiosis (Weller 1998). The fate of such microbial inoculation depends on three major factors namely microbial strain, plant cultivar and environmental condition.
CONCLUSION
Normally soil contains approximately 106 bacteria in per gram of soil while it was found that rhizospheric soil contain about 109 bacteria in per gram of soil. It may be due to the reason that the plant roots contain secretion of root exudates. These root exudates may be growth factors, antibiotics, vitamins, enzymes, etc., which promotes the growth of microorganisms.
From this study it is concluded that-
1. Rhizosphere of wheat plant has large number of bacteria which help to provide the good growth to the crop. The PGPR provided good yield to the crop which was indicated in bio-mass of the wheat seeds.
2. PGPRs are helpful not only to provide nutrients to the plant, but also act as bio-control agents (BCAs). These BCAs helps the crop against phyto-pathogen. As it is studied that there are many bacteria which shows antagonism against pathogenic fungi, which can causes diseases to the plant.
3. There were also presences of some phosphate solubilizing bacteria which solubilize inorganic phosphate into organic phosphate. This makes easy to uptake phosphorus in simple form and provides nutrients to the plants.
REFERENCES
Annelies S., Gorge A. et al 2005 Rhizosphere bacterial community composition in natural stands of Carex arenaria Is determined by bulk soil community composition, Soil biology and Biochemistry vol 37, no 2
Chaurasia B, Pandey A, Palni LMS, Trivedi P, Kumar B, Colvin N. 2005. Diffusible and volatile compounds produced by antagonistic Bacillus subtilis strain cause structural deformities in pathogenic fungi in vitro. Microbiological Research 160, 75-81.
Chen YX, Mei RH, Lu S, Liu L, Kloepper JM. 1996. The use of yield increasing bacteria (YIB) as plant growth promoting rhizobacteria in Chinese agriculture. In Management of Soil Borne Diseases Eds. Utkhede RS, Gupta VK. Kalyani Pres
Davies KG, Whitbread R. 1989. Factors effecting the colonization of a root system by fluorescent Pseudomonas: The effects of water, temperature and soil microflora. Plant and Soil 116, 247-256.
De Boer W, Gunnewiek JAK, Lafeber P, Janse JD, Spit BE, Woldendrop JW. 1998. Anti-fungal properties of chitinolytic dune soil bacteria. Soil Biology and Biochemistry 30, 193-203.
De Freitas JR, Germida JJ. 1991. Pseudomonas cepacia and Pseudomonas putida as winter wheat inoculants for biocontrol of Rhizoctonia solani. Canadian Journal of Microbiology 37, 780-784.
Elroy Curl, Bryan Truelove; The rhizosphere 1986; 57 figure , page 6-7
Harmen GE. 1992. Development and benefits of rhizosphere competent fungi for biological control of plant pathogens. Journal of Plant Nutrition 15, 835-843.
Harris JM, Lucas JA, Davey MR, Lethbridge G, Powell KA. 1989. Establishment of Azospirillum inoculation in the rhizosphere of winter wheat. Soil Biology and Biochemistry 21, 59-64.
Kloepper JW, Schroth MN. 1981. Plant growth-promoting rhizobacteria and plant growth under gnotobiotic conditions. Phytopathology 71, 642-644.
Kloepper JW. 1993. Plant growth-promoting rhizobacteria as biological control agents. In Soil Microbial Ecology: Applications in Agriculture and Environmental Management Ed. Metting FB Jr . Marcel Dekker NY. pp. 255-274.
Kundu BS, Gaur AC. 1980. Effect of nitrogen fixing and phosphate solubilizing microorganisms as sngle and composite inoculants on cotton. Indian Journal of Microbiology 20, 225-229.
Line OMA, Dragar C. 1993. Isolation of bacteria antagonistic to a range of plant pathogenic fungi. Soil Biology and Biochemistry 25, 247-250.
Liz J Shaw, Rhichard G. Burns 2005 Rhizodeposit of Trifolium Pratenseand lotium , Soil biology and Biochemistry, vol; 37 no. 5 995-1002
Nautiyal CS. 2000. Plant beneficial rhizosphere competent bacteria. Proceedings of National Academy of Science of India 70(B) II, 107-123.
Newman EI, Bowen HJ. 1974. Patterns of distribution of bacteria on root surfaces. Soil Biology and Biochemistry 6, 205-209.
O’Sullivan JD, O’Gara F. 1992. Traits of fluorescent Pseudomonas spp. involved in suppression of plant root pathogen. Microbiology Review 56, 662-676.
Ocampo JA, Barea JM, Montoya E. 1975. Interactions between Azotobacter and "phosphobacteria" and their establishment in the rhizosphere as affected by soil fertility. Canadian Journal of Microbiology 21, 1160-1165.
Pandey A, Palni LMS. 1998b. Microbes in Himalayan Soils: Biodiversity and Potential Applications. Journal of Scientific and Industrial Research 57, 668-673.
Pandey A, Shende ST. 1991. Effect of Azotobactor chroocoocum inoculation on yield and post harvest seed quality of wheat. Zentralblatt für Mikrobiologie 146, 489-494.
Paroda RS. 1997. Forward. In Biotechnological Approaches in Soil Microorganisms for Sustainable Crop Production Ed. Dhdarwal KR. Scientific Publishers, Jodhpur. p. 351.
Patten CL, Glick BR. 1996. Bacterial biosynthesis of indole-3-acetic acid. Canadian Journal of Microbiology 42, 207-220.
Peix A, Rivas-Boyero AA, Mateos PF, Rodriguez-Barrueco C, Martinez-Molina E, Velazquez E. 2001. Growth promotion of chickpea and barley by a phosphate solubilizing strain of Mesorhizobium mediterraneum under growth chamber conditions. Soil Biology and Biochemistry 33, 103-110.
Podile AR, Prakash AP. 1996. Lysis and biological control of Aspergillus niger by Bacillus subtilis AF1. Canadian Journal of Microbiology 42, 533-538.
Press CM, Wilson M, Tuzun S, Kloepper JW. 1997. Salicylic acid produced by Serratia marcescens 90-166 is not the primary determinant of induced systemic resistance in cucumber or tobacco. Molecular Plant-Microbe Interactions 10, 761-768.
Promod KC, Dhevendaran K. 1987. Studies on phosphobacteria in Cochin backwater. Journal of Marine Biological Association of India 29, 297-305.
Purohit AN. 1995. The Murmuring Man: Man in Search of Environmentally Sound Development. Bisen Singh Mahendra Pal Singh Publishers, Dehradun, India. p. 80.
Trivedi P, Kumar B, Pandey A, Palni LMS. 2005a. Growth promotion of rice by phosphate solubilizing bioinoculants in a Himalayan location. In Recent Advances in Microbial Phosphate Solubilization Eds. Velazquez E, Rodriguez-Barrueco C. Kluwer, Dordecht, The Netherlands (In press).
Trivedi P, Pandey A, Palni LMS, Bag N, Tamang MB. 2005c. Colonization of rhizosphere of tea by growth promoting bacteria. International Journal of Tea Science 4, 19-25.
Trivedi P, Pandey A, Palni LMS. 2005b. Carrier based formulations of plant growth promoting bacteria suitable for use in the colder regions. World Journal of Microbiology and Biotechnology (accepted).
Van Dyke MI, Prosser JI. 2000. Enhanced survival of Pseudomonas fluorescens in soil following establishment of inoculum in a sterile soil carrier. Soil Biology and Biochemistry 32, 1377-1382.
Vining, L.C. 1990. Functions of secondary metabolites. Annual Review of Microbiology 44, 395-427.
Viswanathan R, Samiyappan R. 2001. Antifungal activity of chitinase produced by some fluorescent pseudomonads against Colletotrichum falcatum Went causing red rot disease in sugarcane. Microbiological Research 155, 309-314.
DM. 1988. Biological control of soil borne plant pathogens in the rhizosphere with bacteria. Annual Review of Phytopathology 26, 379-407.
Whipps, J.M. 2001 Microbial interactions and biocontrol in the rhizosphere. Journal of Experimental Botany 52, 487-511.
Yuen GY, Schroth MN. 1986. Interactions of Pseudomonas fluorescens strain E6 with ornamental plants and its effect on the composition of root-colonizing microflora. Phytopathology 76, 176-180.
APPENDIX
MEDIA COMPOSITION:
Actinomycetes Isolation Agar;
Ingredients Grams / litre
Sodium caseinate 2.0
Aspargine 0.1
Sodium propionate 4.0
Dipotassium phosphate 0.5
Magnesium sulphate 0.1
Ferrous sulphate 0.001
Agar 15.0
Bromo cresol purple broth;
Ingredients Grams / litre
Peptic digest of animal tissue 10.0
Sodium chloride 5.00
Beef extract 3.00
Bromocresol purple 0.04
Citrate agar;
Ingredients Grams / litre
Magnesium sulphate 0.2 gm
Sodium chloride 5.0 gm
Ammonium dihydrogen phosphate 1.0 gm
Dipotassium hydrogen phosphate 1.0 gm
Citric acid / Sodium citrate 2.0 gm
Bromthymol blue 0.08 gm
Agar 20.0 gm
Czapek Malt Agar;
Ingredients Grams / litre
Malt extract 40.0
Sucrose 30.0
Sodiumnitrate 2.0
Potassium chloride 0.05
Magnesium sulphate 0.50
Ferrous sulphate 0.01
Dipotassium phosphate 1.00
Agar 20.00
Jensen’s Medium;
Ingredients Grams / litre
Sucrose 20.0
Dipotas phosphate 1.0
Magnesium sulphate 0.05
Sodium chloride 0.50
Ferrous sulphate 0.10
Sodium molybdate 0.005
Calcium carbonate 2.0
Agar 15.0
Nutrient agar;
Ingredients Grams / litre
Peptic digest of animal tissue 1.0
Meat extract 1.0
Sodium chloride 5.0
Agar 15.0
Nutrient gelatin;
Ingredients Grams / litre
Peptic digest of animal tissue 5.0
Beef extract 3.0
Gelatin 120.0
Potato Carrot Agar;
Ingredients Grams / litre
Carrot infusion 200.0
Potato infusion 250.0
Agar 15.0
Potato Dextrose Agar;
Ingredients Grams / litre
Potato infusion 200.0
Dextrose 20.0
Agar 16.0
Pikovskaya’s Agar Medium;
Ingredients Grams / litre
Yeast extract 0.50
Dextrose 10.0
Calcium phosphate 5.0
Ammonium sulphate 0.50
Potassium chloride 0.20
Magnesium sulphate 0.10
Manganese sulphate 0.0001
Ferrous sulphate 0.0001
Sabouroud Dextrose Agar;
Ingredients Grams / litre
Special peptone 100.0
Dextrose 20.0
Agar 4
Trypton Yeast Extract Agar;
Ingredients Grams / litre
Casein enzymic hydrolysate 5.0
Yeast extract 3.00
Agar 4.0
Urea broth base;
Ingredients Grams / litre
Monopotassium phosphate 9.10
Dipotassium phosphate 9.50
Yeast extract 0.10
Phenol red 0.01
ABBREVATIONS
amsl : above mean sea level
kg : kilogram
g : gram
oC : degree celcius
mg : milligram
mm : millimeter
ug : microgram
hr : hours
l : litre
ml : milliliter
ul : microlitre
nm : nanometer
H2SO4 : sulphuric acid
HCl : hydrochloric acid
MgCl2 : magnesium chloride
NaCl : Sodium Chloride
P : phosphorus
(NH4)6MO7.H2O : ammonium molybedate
SnCl2 : stannous chloride
KH2PO4 : potassium dihydrogen phosphate
Conc. : concentration
M : molar
mM : millimolar
uM : micromolar
Na2CO3 : sodium carbonate
NaHCO3 : sodium bicarbonate
Ph : hydrogen ion concentration
NaOH : sodium hydroxide
p-nitrophenol : paranitrophenol
V/V : volume by volume
W/V : weight by volume
/ : per
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