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Microbial association with selected medicinal plants in rhizosphere and their biodiversity.

Introduction

India is the richest source of medicinal and potential plants sources and varieties of chemical substances. The narrow region of soil is directly influenced by root exudates used in rhizosphere and associated soil microorganisms. Soil microorganisms constituted the world's largest reservoir of bioactive substance and their biodiversity are crucial in the functioning of terrestrial eco-system. The rhizosphere term was introduced to the scientific world by German scientist Hiltner (1994). The region of the rhizosphere in vicinity of roots can be distinguished into many micro habitats. The term is denoting the region of the soil which is subject to the influence of plant roots, and it is characterized by greater microbiological activity than the soil away from plant roots. Distance to which exudation from their root system can migrate depends on the intensity of activity, and it is characterized [1] to have three components [2]; rhizoplane, (surface of the root immediately formed), rhizosphere (the soil volume surrounding the rhizoplane that is immediately affected by root activity) and bulk soil (it is not directly affected by living roots). They are characterized by greater microbiological activity with various intensity, than the soil away from plant roots. Rhizosphere effect was the indicator of the overall influence of plant root on soil microorganism. It is influenced by different factors like soil type, moisture, pH, temperature, age and condition of plants. The metabolic activity of rhizosphere microorganism is different from those of the non-rhizosphere type. Rhizosphere zone is surrounding the roots of the plants in which complex relation exist among the plants. Biofilm associated with the plant roots can profoundly influence chemistry of soil including nitrogen transformation and pH. The rhizosphere encompasses in millimeters of soil surrounding a plant root were ecological and biological complex process occurs. This rhizosphere is important for the formation of soil and also includes the formation of earth's extremely weathered soils, ultisols and oxisols. [3] Hotspots of biological activities were gas exchange, moisture, respiration of nutrient and localized supplies of organic matters, [4] and the beneficial bacterial species were either harmful or natural relation of the roots [5], in which Actinomyces are aerobic, Gram positive bacteria, which is forming filaments used for the production of asexual spores and even of fruit bodies production. Streptomyces forms a genus of actinomyces, this organism has extensively branching secondary or aerial mycelium, and primary or substrate mycelium. It is used for production of largest antibiotic compounds such as antibacterial, antifungal and wide range of bioactive compounds. The root surface and the rhizophere area are the sites of high microbial activity. But, there is a lack of information on the role of microbes and their biodiversity in the selected medicinal plants of Thanjavur, Tamil Nadu region. The aim of this study is the enumeration of microbial population in rhizosphere samples from selected medicinal plants such as, Acalyph indica, Leucas aspera, Ocimum tenuiflorum L, Solanum nigrum and to identify their various activities.

Materials and Methods

The medicinal plants such as, Acalypha indica, Leucas aspera, Ocimum tenuiflorum L and Solanum nigrum were selected in and around Thanjavur, Tamil Nadu, India. These plants were authenticated by Dr. N. Ravichandran, CARISM, SASTRA University, Thanjavur. The media used were Actinomycetes Isolation Agar (AMA), Glycerol Aspergine Agar (GAA), Potato Dextrose Agar (PDA) and Nutrient Agar (NA), all procured from Himedia Laboratories Pvt. Ltd., Mumbai, India. The utilized carbon sources are, D-Glucose anhydrous, Lactose monohydrate, Maltose monohydrate, Sucrose, D (+) Manitol, D (+) Raffinose, and D (+) Xylose and, all were obtained from Nice Chemicals Pvt. Ltd., Cochin, India. All other reagents were analytical grade.

Collection of Sample:

Rhizosphere samples were collected by gently uprooting the plants using sterile shovel and then the unwanted soil particles were removed. The roots were covered with sterile polythene bags and the soil adjacent to five centimeters from the roots (non-rhizophere soil) was collected and then the samples were brought to laboratory, aseptically within 1-2 hours.

Estimation of Microbial Population in the Sample:

To estimate the total microbial population of the rhizosphere, the sample associated to roots were taken and the pour plate technique was followed. About one gram of rhizosphere and root associated soil were homogenized and was aseptically transferred to 9 ml of blank sterile water and then solutions were prepared to the dilution level of [10.sup.-1] to [10.sup.-4]. The aliquots of diluted solution were pipetted out into sterile petri dishes by using 1 ml sterile graduate pipettes. [6] The sterilized AMA, GAA, PDA and NA media were cooled to the temperature of 40[degrees]C and about 15-20 ml of the specific medium was poured into each petri dish. After the incubation for 24 hours the populations of bacteria were counted, and Streptomyces species samples were analyzed after incubation of 4-7 days from different medicinal plants. [6] The randomly selected bacterial colonies were sub-cultured on the same medium slants to obtain pure culture.

Rhizosphere effect:

The enhancement of the growth of a soil microorganism is influenced by physical and chemical alterations of the soil and the contribution of excretion and organic debris of roots within a rhizosphere. The rhizosphere effect [7] of the selected plants can be quantitative and calculated using the following formula,

R/S

R = Number of microorganism per gram of rhizosphere soil

S = Number of microorganism in a gram of non rhizosphere soil.

Identification of microorganism:

The isolates of bacteria was purified and identified to various genera by morphological and biochemical tests using well established procedures. [8,9] The morphological characterization of bacterial species was done by gram staining technique and identified. Micromonospora species was identified according to International Streptomyces Project (Shirling and Gotellieb 1966) [10].

Production and estimation of Indole Acetic Acid (IAA):

The nutrient broth with various pH ranges of 6, 7, 8 and 9 were prepared and supplemented with tryptophan (0.01g/100ml) and yeast extract (0.01g/100ml). The selected bacteria were inoculated and incubated and then few drops of salper's reagent (1ml of 0.5M ferric chloride in 50 ml of 35% per-choleric acid) were added. It indicates red color for the production of IAA. [11] both gram positive and gram negative rod shaped bacteria were inoculated in 100ml nutrient broth supplemented with tryptophan and yeast extract and incubated for 7 days in dark condition. After incubation the broth was centrifuged at 3000 rpm for 30 min and the supernatant was adjusted to pH 2.8 by using 1N hydrochloric acid, and extracted twice with equal volume of diethyl ether or ethyl acetate at 4[degrees]C. Again it was centrifuged at 1000 rpm for 10 min. The ether phase was dried overnight and dissolved in 2ml of methanol. IAA in methanol extract was estimated after adding 5ml of salper's reagent and kept aside for 25 min. The sample was analyzed by UV-Vis spectrophotometer (Model-106-Systronic, Mumbai, India) at 535 nm by using 2 ml methanol + 4 ml salper's reagent as blank. A change of color from pink to red indicates the production of IAA.

Oxidative-fermentative test:

The purpose of this test is to determine the effect of organism on sugar by fermentation and oxidation processes. Hugh and Leifsons basal medium [12] was sterilized in autoclave and cooled, then 1g of glucose was added and steamed it for 15 minutes. 3ml of media was transferred to the culture tubes and kept in slanting position for 24 hours. Gram negative rod culture was streaked and stabbed, and the culture tubes were kept for growth of Gram negative rod at room temperature for 24 hours. After incubation, the acid and gas production were observed visually with color and gas formations and the results were tabulated.

The color formation of Micromonospora species using different carbon sources: [13]

The color formation of Micromonospora species was carried by well established procedure of pour plate technique. The media such as PDA, NA, AMA, and GAA were sterilized and cooled, and then 1% concentration of the carbon sources like, sucrose, glucose, lactose, mannitol, xylose, maltose and raffinose were added in the above medium. The conical flasks containing media with carbon source were kept in water bath for 15 minutes and then transferred to sterilized petri plate and solidified. Then the Micromonospora culture was inoculated and incubated for two days at room temperature. The different colors production like red, yellow, green, pink, and white were observed visually.

Application of the microbes on the growth of selected seedling: [14]

In this experiment, polythene bags were disinfected with disinfectant and filled with soil which was previously sterilized. The Nutrient broth was used for growth of Gram positive rod, Gram negative rod, Micromonospora species and mixed inoculants (Azotobacter, Phosphobacteria, Gram positive, Gram negative rod and Micromonospora and then incubated for 5 days. After incubation, 5ml of broth culture was transferred into the model plant (Plectranthus amboinicus), and the plants were watered as and when required and allowed to grow for 10 days to compare with control plants. Differences between microbial inoculated and non-inoculated plants and the height, number of leaves were noted.

Anti-microbial activity:

Anti-microbial activity was carried by agar disc diffusion method. This method was used to determine the anti-microbial activities of the Micromonospora. Disc assay was found to be simple, cheap and reproducible practical methods. [15] The two human pathogens of Vibrio cholera and Klebshilla pneumonia was spread on the PDA media, and 10 microliter of Micromonospora sample containing disc was placed in the plates and incubated at 37[degrees]C for 24-48 hours.

Lyophilization of Micromonospora species:

100ml of potato dextrose broth was prepared and sterilized; then Micromonospora sample were inoculated and kept for 20 days in room temperature. After 20 days the mat formation was observed. The sample mixed properly and stored at -4[degrees]C for two days and transferred to lyophilizer.

Surface Morphology:

Scanning Electron Microscope (SEM) [16] (Jeol, JSM-6360, Japan) is used to study surface morphology of the freeze dried solid content. It was mounted on screw shaped stubs using double--sided carbon adhesive tape. The samples were coated with platinum in an argon atmosphere under vacuum condition by using sputter chamber and they were examined at 15000v accelerating voltage.

Chromatographic analysis:

The paper chromatography technique [17] was used to analyze proteins present in streptomyces sample. This technique was carried out in the 7 cm x 4 cm whatman filter paper1 (Whatman international Ltd. Maidston, England). The Micromonospora sample standard proteins (arginine and cystenine) were dissolved in 95% absolute alcohol (Hayman Ltd., England) which was spotted and dried and then placed in previously prepared and saturated solvent system (neutral and acidic solvent system) and identified the amino acids by using ninhydrin solution as a detecting agent.

Results:

Total heterotrophic bacteria, fungi, and Micromonospora species were enumerated from the rhizosphere and non-rhizosphere soil samples of 4 selected medicinal plants like Acalypha indica, (Kupaimani), Leucas aspera (Dumbi), Ocimum tenuiflorum L (Tulasi) and Solanum nigrum (Manathakali). The results indicated the occurrence of bacteria, fungi and Streptomyces by using 4 different media.

Estimation of microbial population in selected sample:

The rhizosphere root samples were found to have the maximum bacteria of 507 x [10.sup.1] CFU/g in Aclypha indica root and minimum of 32 x [10.sup.1] CFU/g in Leucas aspera root sample. The maximum Micromonospora population was found as 8X101 CFU/g in Solanum nigrum root sample in PDA media and the minimum was found to be 5 x [10.sup.1] CFU/g in Leucas aspera root sample in PDA media (Table-1, Figure-1(a), (b)). Micromonospora in aerial mycelium white color and substrate mycelium pink color were formed in PDA media.

Identification of selected bacteria was carried out in the non-rhizosphere root associated soil sample and the maximum heterotrophic bacteria was 247X[10.sup.1]CFU/g in Leucas aspera and the minimum was found in 83X[10.sup.1]CFU/g in Acalypha indica soil,.(Table1). Bacterial and Micromonospora colonies was sub cultured in the respective media.

Identification method:

The gram staining technique was carried out for the bacterial colonies isolated from 4 different medicinal plants (Acalypha indica, Leucas aspera, Ocimum tenuiflorum L and Solanum nigrum). They show Gram positive and Gram negative stains (Table-2)

Oxidative fermentative test:

Gram negative rod shaped bacterial colonies from Leucas aspera root sample and soil sample and from Ocimum tenuiflorum L root sample were stabbed and streaked in the oxidative fermentative medium and the change of pink to yellow color was observed for indication of the acid production. Bacteria isolated from Ocimum tenuiflorum L root sample showed lifting of the media which indicated the gas formation and air bubble production in the media (Figure-2).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Color characterization of Micromonospora sample:

The characterization of Micromonospora species was studied by the method recommended by International Streptomyces Project (Shirling and Gottlieb, 1966) [10]. Characterization of Micromonospora done by color formation in both aerial and subtrate mycelia. It provides certain nutrients for triggering of genes for the conversion as well as expression of others metabolic products which, in turn, leads to different aerial and substrate mycelial coloration. This coloration differences may be due to the primary and secondary metabolites production provided by the media, were observed and shown in the table and figure (Table.5, figure-3(a),(b)). The aerial mycelial color expressed was white, green, yellowish and white series and substrate mycelia color for each strain were totally different like white, red, green, pink etc.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

IAA Production:

The isolates of bacteria were checked for IAA production. Isolates of Gram positive and Gram negative bacterial strains produced IAA. The degree of IAA production by bacteria isolates was estimated spectrophotometrically. Among them Leucas aspera root species produced maximum IAA in alkaline pH 9.0 (1.030) The bacteria isolated from the rhizosphere soil were able to produce IAA in the presence of tryptophan supplement when compared to bacterial strains in Gram negative and Gram positive bacteria. In addition to higher plants, numerous bacteria have the ability to synthesize plant growth regulators such as indole acetic acid and other indole related compounds. Generally microorganism isolated from the rhizosphere and rhizoplane of various crops have more potential for producing auxins than those from the root free soil. The bacterial inoculation enhanced the root growth which varied with the different combinations used (table-4, figure-5, 6(a), (b)). The combination of Gram negative rod, Gram positive rod, Micromonospora species. Azotobacter beijerinckii, phosphobacteria and mixed cultures enhanced the average length of primary roots after the inoculation of 5ml culture in seedling.

[FIGURE 5 OMITTED]

Application of the microbes on the growth of selected seedling:

The average number of stem per seedling was almost similar (Table.7). The bacterial treatment significantly enhanced the shoot height (Table.8). The leaf area was also found to be increased in different inoculated culture. The success rate was found to be 90% when bacterial inoculation was coupled with application of intermediate levels of fertilizers (Nitrogen, Phosphorous and Potassium). Hence such combined bacterial inoculation will reduce 40-50% use of fertilizers in agricultural crops.

[FIGURE 6 OMITTED]

Antimicrobial activity:

The samples D and E showed the maximum inhibition of 1.6 cm in both Vibrio cholerae, Klebsiella pneumonia and the sample N showed the minimum inhibition of 0.2 cm in Klebsiella pneumonia. The zone of inhibition was also shown in other samples also (table-6, figure-7).

[FIGURE 7 OMITTED]

Surface Morphology:

The images of Scanning Electron Microscope (SEM) were observed at various magnifications (x2000, x5000, x5000, x2000) and the images showed the smooth and thread like surface structure attached on the branch like structures (Figure-8).

[FIGURE 8 OMITTED]

Rhizosphere effect:

The rhizosphere effect was quantified for the selected plants and was found to be the maximum 4.22 x [10.sup.4] for gram negative bacteria observed in Acalypha indica and the minimum 0.18 x [10.sup.4] for gram negative bacteria effect was seen in Leucas aspera. The Ocimum tenuiflorum L and solanum nigram showed the effect of 0.48 x [10.sup.4] and 0.20 x [10.sup.4] respectively.

Chromatography analysis:

The Rf values are shown by paper chromatography technique which revealed the presence of amino acids in Micromonospora sample containing various carbon sources. The data were compared with standard amino acid sample like cystenine and arginine (Table-6).

Discussion:

The bacterial population was higher in the entire root zone of medicinal plants followed by fungal and Micromonospora populations. Similarly the number of microorganism was more in the rhizosphere soil than in the non-rhizosphere soil. The results can be related with the reports [13] that found the maximum rhizosphere population of 8.92X[10.sup.8] CFU/g and minimum rhizosphere count of 2.35 X108 CFU/g in Carea arenaria. The maximum non-rhizosphere population was 6.61X[10.sup.6]CFU/g and the minimum bacterial number was 1.01X[10.sup.6]CFU/g. This was reported (18) that the bacterial count in the rhizosphere was 7.45X[10.sup.7]CFU/g in Pisum satirum. The actinomycetes population was more in the rhizosphere soil than in the non-rhizosphere soil in all the experimental plants. But Oza et.al.(2002) (19) reported that the population was remarkably high in the nonrhizosphere as compared to rhizospohere soil in the dominant plant species in the semiarid soil of Rajkot.

The total count of fungus in all selected medicinal plants of the present study was higher in the rhizosphere soil than in the non-rhizosphere soil. The results of the study are similar to that of Hissy et. al. (1980) [20] who studied the presence of more number of fungus in the rhizosphere soil than in the non-rhizosphere soil of fine plants in Egypt. The varying degrees of population observed in the roots of the plants is due to the effect of the chemical composition of root exudates of the individual plants such as Acalypha indica, Leucas aspera, Ocimum tenuiflorum L and Solanum nigrum on the microorganism. Many of the environmental factors such as temperature, light (Hodge et. al., 1997) [21] and atmospheric C[O.sub.2] concentration are known to influence microbes in the rhizosphere. It is not yet known to what extent plants can select a constant rhizosphere community from highly contrasting reservoirs of bulk soil population (Duine et. al., 2005) [14].

The rhizosphere effect was higher in Acalypha indica and minimum in Leucas aspera. The greater the rhizospheric effect the higher will be microorganisms number. Greater rhizosphere effect is seen with bacteria than with Micromonospora, Streptomyces and fungi and only negligible changes were noted with regard to protozoa and algae (Subbarao, 2000) [7]. The rhizosphere effect greatly decreases as when moved away from the root (Curl and Turelove, 1986) [4]. The varying types and quantities of rhizodeposits have been postulated to act as key factors influencing the density and diversity of the rhizospheric microorganism (Grayston and Campbell, 1996) [22].

The presence of Micromonospora might have been a driving force in controlling the air and soil borne pathogens asperour observation. Thus, Micromonospora might have created a conducive atmosphere for the proliferation of the seedling. The biofertilizer effect of these bacteria and Micromonospora is more pronounced in combination than in individual inoculants. The relationship between microbes and plant growth may be direct or indirect. However saprophytic microorganism provides the necessary nutrients to plant through their activity. The involvement of antagonistic Micromonospora in the growing seedling is an advantage because it inhibits the plant pathogens and the synthesis of IAA to enhance the growth of plants. It as been attempted for the first time and has encouraging results.

The mechanism of activation of growth promoting IAA by microorganisms seems to vary according to their physiological properties and to the conditions of the environment in which they live. It is also presumed that microorganisms act on seedlings both through IAA production and through nutrient absorption. Release of IAA and free enzymes and their subsequent participation in the promotion of growth and nutrient regeneration would assess the potential fertility of the environment. Maximum zone of inhibition was observed in sucrose containing AMA media of Solanum nigrum plant soil than other samples and the least was with maltose and raffinose containing AMA and NA media with root of Solanum nigrum and leucas aspera plant respectively. According to Rengasamy and Bakiyaraj, (2002) [23] scanning electron microscope images of our strain looks like Micromonospora [21]. It is understood that there is diversity and is unique in medicinal plant. The occurrence and antagonistic property of Micromonospora in the medicinal plant is a new record.

Acknowledgement

Authors are grateful to the authorities of the management of SASTRA University, Thanjavur for providing facilities and encouragements through out the course of study.

References

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G. Ramesh, B.N. Vedha Hari and K. Dhevendaran

School of Chemical and Biotechnology, SASTRA University, Thanjavur-613401. Tamil Nadu, India.

Corresponding Author: K. Dhevendaran, Ph.D., D.Sc., Professor, School of Chemical and Biotechnology, SASTRA University, Thirumalaisamuthiram, Thanjavur- 613 401. Tamil Nadu, India.

Email: dheven.k@gmail.com
Table 1: Enumeration of Microbial Population From Selected
Medicinal Plants (Acalypha Indica, Leucas aspera, Ocimum
tenuiflorum L, Solanum Nigrum)

                                                         Number of
                                                         colonies
                                                      cfux[10.sup.1]g
                                                        dry weight
Date of
collection     Medicinal plants     Sample   Media   Bacteria   Fungi

5.01.2011      Acalypha indica       Root     NA       108        --
                Leucas aspera        Root     PDA       32        --
             Ocimum tenuiflorum L    Root     AMA       48        --
                Solanum nigrum       Root     GAA       --        --
18.01.2011     Acalypha indica       Soil     AMA       83         3
                Leucas aspera        Soil     NA       173        --
             Ocimum tenuiflorum L    Soil     GAA       98        --
                Solanum nigrum       Soil     PDA       --        --
24.01.2011     Acalypha indica       Root     NA       507        --
                                     Soil     GAA      120        --
                Leucas aspera        Root     PDA       --        --
                                     Soil     AMA      247        --
             Ocimum tenuiflorum L    Root     NA        80        --
                                     Soil     AMA      247        --
                Solanum nigrum       Root     PDA       --        --
                                     Soil     GAA      123        --

                                               Number of colonies
                                               cfux[10.sup.1]g
                                               dry weight
Date of
collection     Medicinal plants     Sample     Micromonospora

5.01.2011      Acalypha indica       Root      --
                Leucas aspera        Root      --
             Ocimum tenuiflorum L    Root      --
                Solanum nigrum       Root      --
18.01.2011     Acalypha indica       Soil      --
                Leucas aspera        Soil      --
             Ocimum tenuiflorum L    Soil      --
                Solanum nigrum       Soil       6
24.01.2011     Acalypha indica       Root      --
                                     Soil      --
                Leucas aspera        Root       5
                                     Soil      --
             Ocimum tenuiflorum L    Root      --
                                     Soil      --
                Solanum nigrum       Root       8
                                     Soil      --

                                               Number of colonies
                                               cfux[10.sup.1]g
                                               dry weight
Date of
collection     Medicinal plants     Sample     Dry weight(g)

5.01.2011      Acalypha indica       Root      0.79
                Leucas aspera        Root      1.40
             Ocimum tenuiflorum L    Root      1.70
                Solanum nigrum       Root      1.09
18.01.2011     Acalypha indica       Soil      0.50
                Leucas aspera        Soil      0.75
             Ocimum tenuiflorum L    Soil      0.48
                Solanum nigrum       Soil      0.73
24.01.2011     Acalypha indica       Root      1.10
                                     Soil      1.03
                Leucas aspera        Root      1.01
                                     Soil      0.90
             Ocimum tenuiflorum L    Root      1.17
                                     Soil      1.08
                Solanum nigrum       Root      0.93
                                     Soil      1.04

NA-Nutrient Agar, GAA-Glycerol Aspergine Agar, PDA-Potato
Dextrose Agar, AMA-Actinomycetes isolation Agar, cfu- colony forming
unit.

Table 2: Characteristics of Selected Bacteria.

                                                   Strain

                                 Microbial     Gram       Gram
Medicinal plants        Sample     Media     positive   negative

Acalypha indica          Root       NA          +          -
Leucas aspera            Root       PDA         -          +
Ocimum tenuiflorum L     Root       AMA         -          +
Solanum nigrum           Root       GAA         +          -
Acalypha indica          Soil       AMA         -          +
Leucas aspera            Soil       NA          -          +
Ocimum tenuiflorum L     Soil       GAA         +          -
Acalypha indica          Root       NA          +          -
                         Soil       GAA         -          +
Leucas aspera            Soil       AMA         -          +
Ocimum tenuiflorum L     Root       NA          -          +
                         Soil       AMA         +          -
Solanum nigrum           Soil       GAA         -          +

                                       Appearance

Medicinal plants        Sample      Rod         Cocci

Acalypha indica          Root        +            -
Leucas aspera            Root        +            -
Ocimum tenuiflorum L     Root        -            +
Solanum nigrum           Root        +            -
Acalypha indica          Soil        -            +
Leucas aspera            Soil        +            -
Ocimum tenuiflorum L     Soil        +            -
Acalypha indica          Root        -            +
                         Soil        -            +
Leucas aspera            Soil        +            -
Ocimum tenuiflorum L     Root        +            -
                         Soil        +            -
Solanum nigrum           Soil        -            +

AMA- Actinomyces Isolation Agar, NA- Nutrient Agar, GAA- Glycerol
Aspergine Agar, PDA- Potato Dextrose Agar, (+ve) presence of
strain, (-ve) absence of strain.

Table 3: Effect of microbes on the Growth of the Seedling
(Plectranthus amboinicus).

                                     Height of plant    Quantity
                                     (in Cm)           (in number)

Treatments                           Initial   Final   Stem   Leaves

Control                                3.5      4.0    3.0      1
Gram Positive Rod                      4.0      5.0    4.0      3
Gram Negative Rod                      3.4      4.0    5.0      2
Gram Negative Rod                      4.0      4.8    2.0      2
Micromonospora                         3.5      4.2    3.0      1
Phosphobacteria+azotobacter+GGram      3.0      4.0    4.0      4
  Negative Rod+Gram Positive Rod+
  Micromonospora

Cm--Centimeter

Table 4: Effect of pH on the growth and Indole Acidic Acid (IAA)
Production from Bacterial strains (Gram positive and Gram negative).

                                                             IAA
                                              Growth        (OD at
Medicinal plants       Sample rods   pH   (OD at 600 nm)    535 nm)

Ocimum tenuiflorum L   Root (-ve)    6        1.333         0.639
Solanum nigrum         Soil (-ve)    7        1.239         0.491
Leucas aspera          Soil (-ve)
Ocimum tenuiflorum L   Root (-ve)
Acalypha indica        Soil (-ve)    8        1.273         0.553
Leucas aspera          Root (-ve)
Leucas aspera          Soil (-ve)    9        1.302         1.030
Acalypha indica        Root (+ve)    6        1.719         0.235
Solanum nigrum         Root (+ve)    7        1.418         0.156
Leucas aspera          Root (+ve)
Leucas aspera          Soil (+ve)    8        1.307         0.160
Ocimum sactum          Root (+ve)    9        1.148         0.019
Ocimum tenuiflorum L   Soil (+ve)

OD--Optical Density, IAA--Indole Acetic Acid, (+ve) Gram positive,
(-ve) Gram negative.

Table 5: Mycelial Coloration of Selected Micromonospora
and Streptomyces Species.

Sample    Carbon
coding    source          Media   Medicinal plants   Sample


A         Manitol         PDA     Solanum nigrum     Root
B         Manitol         NA      Solanum nigrum     Soil
C         Xylose          PDA     Leucas aspera      Root
D         Xylose          PDA     Solanum nigrum     Root
E         Sucrose         AMA     Solanum nigrum     Soil
F         Sucrose         PDA     Solanum nigrum     Soil
G         Raffinose       AMA     Solanum nigrum     Root
H         Raffinose       PDA     Solanum nigrum     Root
I         Maltose         AMA     Solanum nigrum     Root
J         Maltose         NA      Leucas aspera      Root
K         Lactose         AMA     Leucas aspera      Root
L         Lactose         PDA     Leucas aspera      Root
M         Glucose         PDA     Leucas aspera      Root
N         Glucose         NA      Solanum nigrum     Soil

Sample
coding    Aerial mycellium   Substrate mycelium

A         Green              Whitish pink
B         Yellowish white    White
C         Greenish white     Pink
D         Green              Whitish pink
E         Yellowish green    White
F         Whitsh green       White
G         Whitish yellow     White
H         Green              Pink
I         Whitish pink       Pink
J         Green              White
K         Yellowish white    White
L         Whitish green      Pink
M         Greenish white     Pink
N         Yellowish white    White

AMA- Actinomyces Isolation Agar, NA- Nutrient Agar, GAA- Glycerol
Aspergine Agar, PDA- Potato Dextrose Agar,

Table 6: Selected Micromonospora and Streptomyces Used Zone of
Inhibition and Rf Value of the Sample.

                      Rf value          Zone of inhibition(cm)

                                         Vibrio     Klebsiella
Sample coding   Solvent 1   Solvent 2   choleraie   pneumonia

A                 0.25        0.40         1.5         1.4
B                 0.32        0.30         NI          0.7
C                 0.32        0.14         NI          0.7
D                 0.30        0.21         1.6         1.5
E                 0.26        0.34         NI          1.6
F                 0.30        0.20         NI          0.6
G                 0.24        0.50         1.2         1.5
H                 0.30        0.22         NI          0.3
I                 0.30        0.10         NI          0.5
J                 0.40        0.30         0.3         1.3
K                 0.40        0.35         NI          0.5
L                 0.35        0.32         0.6         0.7
M                 0.22        0.10         NI          0.6
N                 0.13        0.14         NI          0.2
Arginine          1.50        0.40
Cystenine         0.43        0.52

Cm--Centimeter, NI--No Inhibition.
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Title Annotation:Original Article
Author:Ramesh, G.; Hari, B.N. Vedha; Dhevendaran, K.
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Geographic Code:9INDI
Date:Apr 1, 2012
Words:5064
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