In general in agro-ecosystems, soil receives considerable carbon inputs from a variety of sources including leaf fall, stubbles, roots and root exudates as well as through external sources including farm yard manure and compost. The semi-dry system of rice cultivation is mainly confined to tracts that depend on rains and have no supplementary irrigation facilities.  In this semi-dry system part of the rice crop’s life cycle passes under aerobic conditions and part under anaerobic conditions. In the conventional rice cultivation practiced in irrigated areas, rice crops’ life cycle passes completely under anaerobic condition.  The amount and quality of organic carbon are crucial factors influencing soil productivity.  The endemic deficiency of organic matter in tropical sandy soils particularly those under the influence of arid and semi arid climates are a major factor contributing to their low productivity. To improve the soil organic carbon   content, organic system of nutrient management is essential to meet the nutrient requirement of the crops as well as to improve the soil organic carbon status. Soil organic matter in soils with tropical upland conditions is more difficult than in soils used for lowland rice conditions.(Sahrawat,2005).Kaboneka et al (1997) found that wheat straw,corn stover or soybean stubble were mineralized during 30 days incubation. Mineralization of green manure is slow in semidry condition hence compost or animal manures can be used as an organic source for semidry rice. Experiments were conducted to explore the possibility of recycling a common weed in the study area, Ipomea cornea as an organic source for semi dry rice.

Materials and Methods

The study area, located in east coastal area of southern peninsular India at longitude (E) 78010’- 79027’ and latitude (N) 9005’- 9056’. The green leaves of Ipomea cornea harvested from wastelands near the experimental site were chopped and mixed with cattle / poultry manures and wetted with deionized water to bring the mix to 60 per cent moisture content.  Under laboratory conditions, 5 kg of the green leaves of Ipomea cornea was composted with cattle manure and poultry manure @ 0.625, 1.25, 1.88 and 2.50 kg anaerobically for 30 days.  The matured compost was obtained at the end of composting period (30 days).  The nutrient contents of the organic materials composted are furnished in Table 1. The moisture content was maintained at 60 per cent.  Since the composting was done under anaerobic condition, the mix was not turned.  The ‘mix’ was subsequently maintained at this anaerobic condition.  A total of nine treatments were replicated for five times.  The CO2 –C evolution was measured at weekly intervals. (Bundy and Bremner, 1972). Separate containers were kept for each of the 5 sampling intervals so that once opened for CO2 –C measurement, the container could be discarded.    

 Field experiments were conducted in coastal sandy tracts with rice-rice cropping sequence under semi-dry condition.  The experimental soil (Typic Tropaquept) was alkaline in soil reaction (soil: water ratio 1:2) (pH 8.7), low in
N (Subbiah and Asija, 1956) (90 kg ha-), P (Olsen et al., 1954) (4.2 kg ha-1) and high in available K (Stanford and English, 1949)(324 kg ha-1 ) .The initial soil organic carbon content was 1.2 g kg-1.  The Ipomea cornea compost obtained from another batch of composting was applied basally (10 kg / plot) as per the treatment schedule.  The experimental plot size was 5 x 4 m2.  The design of the experiment was a randomized block design with three replicates.  The oxidizable soil organic carbon content was measured (Walkely and Black, 1934) in various growth stages of rice, tillering, panicle initiation, flowering and harvest stages.  At harvest stage, rice grain and straw yields and soil temperature were recorded.

Result and Discussion

CO2 -C   evolution

     Faster mineralization followed by a steady decline in the rate of mineralization with time.  Initially, the mineralization was faster; with increase in the period of composting, there was a steady decline in the mineralization rate.  The exponential nature of carbon mineralization from soil organic matter and added plant residues was previously reported by Vanlauwe et al., (1994).  At all sampling intervals, the lowest amount of C was mineralized from poultry manure and the highest from cattle manure.  The pattern of C mineralization from Ipomea cornea compost was similar to that of the control soil from fourth week after incubation onwards; indicating that most of the C added through compost had been mineralized within four weeks of incubation (Figure1). High rates of CO2-C evolution from the Ipomea cornea –cattle manure compost immediately after incubation was noticed. This could be due to the presence of easily decomposable organic compounds in the cattle manure as compared to less easily decomposable organic compounds in the poultry manure.  Poultry manure contains large amounts of CaCO3, struvite and other basic compounds (Bril and Solomons, 1990).  Low level of decomposition in Ipomea cornea-poultry manure compost could be attributed to high concentration of Ca and neutralization of organic acids and H+ by Ca and buffering reactions (Mahimairaja et al., 1995).

Field Experiment

 Oxidizable soil organic carbon content

  At all stages of crop growth, significant improvements in oxidizable soil organic carbon content were observed in the Ipomea cornea-poultry manure compost treatments as compared to in the control and Ipomea cornea-cattle manure compost treatments. Highest oxidizable soil organic carbon content (4.30 g C kg-1) was recorded for the Ipomea cornea-poultry manure (50% RD) compost treatment (Table 3).  Many studies have revealed a direct linear relationship between soil organic carbon storage and gross annual C input to soil (Halvin et al., 1990; Paustian et al., (1992).   With increase in the level of Poultry manure (50% RD) used in the compost, Oxidizable soil organic carbon content was increased. 

 Yield of rice

 Application of Ipomea cornea-poultry manure compost (37.5%RD) recorded higher grain (3550 kg ha-1) and straw yields (4260 kg ha-1) which was on par with the application of Ipomea cornea-poultry manure compost (50% RD) (Table 4).  This could be due to the higher amount of CaCO3 in the poultry manure.  Calcium in poultry manure exchange with Na in the soil exchange complex, thereby reduce the ill effects of Na on soil and plant.  The experimental site was alkaline in soil reaction. Despite a higher nutrient content in the poultry manure as compared to cattle manure the presence of CaCO3 in poultry manure could have favourable effect on the experimental soil. Low yield in Ipomea cornea-cattle manure compost applied plots could be due to the lesser amounts of nutrients added through cattle manure.  

Soil Temperature

 At harvest stage a negative linear correlation between soil temperature and soil organic matter status was observed (Figure 2).  As soil organic matter status increased, decrease in soil temperature was noticed.

 Conclusions

 Ipomea cornea   is one of the most rapidly spreading weeds in southern peninsular India. It is fast encroaching on cultivated lands, water reservoirs and waste lands. Significant amount of time, effort and money has been used for its eradication. Recycling of this weed Ipomea cornea could serve dual purpose of its eradication and serving as a better organic material. Ipomea cornea   could be composted with animal manures and used as manure for semidry rice cultivation. Between cattle manure and poultry manure, Ipomea cornea   composted with poultry manure recorded lower CO2 evolution,wider C:N ratio and higher rice yield and organic carbon status

 REFERENCES

Bril and Solomons, 1990. Chemical composition of animal manure: A modeling   approach.  Neth.J. Agric. Sci, 38, 333-351.    

Bundy, L.G., and Bremner, J.M. 1972. A simple titrimetric method for the determination of inorganic carbon in soils. Soil Sci. Soc. Am. Proc. 36, 273-275.

Havlin, J.L., Kissel, D.E., Maddux, L.d., Classen, M.M and Long, J.H. 1990.  Crop         Rotation and  tillage  effects  on  soil  organic  carbon  and  nitrogen .Soil       Sci. Soc. Am. J, 54, 448-456.

Kaboneka,S.,Sabbe ,W.E.,and Mauromaustakos,A.1997.Carbon decomposition kinetics and N mineralization from corn,soyabean and wheat residues.Commun.Soil Sci.Plant Anal.28(15&16):1359-1373.

Mahimairaja, S., Bolan, N.S and Hedley. M.J. 1995.Dissolution of phosphate rock       during the composting of poultry manure: An incubation experiment. Fert. Res, 40, 93-104.

Olsen, S.R., Cole, C.L., Watanabe, F.S., and Dean, D.A. 1954.  Estimation of available phosphorus in soils by the extraction with sodium bicarbonate, U.S.D.A., Circ. 939.

Paustian, K., Parton , W. J. and Persson, J. 1992. Modeling soil organic matter in   organic amended and N fertilized long term plots. Soil Sci. Soc. Am. J, 56, 476-478.

Stanford, S and English L. 1949.  Use of flame photo meter in rapid soil test of K and Ca.  Agron J., 41 : 446-447.

Subbiah, B.V. and Asija, G.L. 1956.  A rapid procedure for the estimation of available N in soils.  Curr. Sci., 25 : 259-260.

Vanlauwe, B., Dendooven, L. and Merckx, R.1994. Residue fractionation and      Decomposition: the significance of the active fraction .Pl. Soil, 158, 263-274.

Walkley, A and C.A. Black. 1935. An examination of methods for determining organic carbon and N in soils. J. agric. Sci, 25, 598-609.

Table 1.  Nutrient contents of manures (mg g-1 of dry matter) used in the

                Study (Mean values)

Nutrients

Cattle manure

Poultry manure

Ipomea cornea

N

32.5

45.0

11.6

P

7.0

16.5

3.8

K

16.0

18.5

3.1

Ca

6.5

43.0

1.2

Mg

6.5

5.5

3.8

S

3.5

5.5

2.7

Organic carbon

112

238

601

Organic matter

193

410

1036

Table 2.  Estimated quantity (kg ha-1) of nutrients added to the soil through the

                manures evaluated in this study

Treatments

Amount of cattle/poultry manure added

(tha-1)

Nutrients added through manures (kg ha-1)

N

P

K

Cattle manure

12.5% of RD*

0.625

20.31

4.38

10.00

25.0 % of RD

1.250

40.63

8.75

20.00

37.5% of RD

1.875

60.94

13.13

30.00

50.0% of RD

2.500

81.25

17.50

40.00

Poultry manure

12.5% of RD*

0.625

28.13

10.31

11.56

25.0 % of RD

1.250

56.25

20.63

23.13

37.5% of RD

1.875

84.38

30.94

34.69

50.0% of RD

2.500

112.50

41.25

46.25

(*RD -Recommended dose-5t ha-1)

Table 3 . Oxidizable soil organic carbon in crop growing period (g kg-1 soil)

Treatments

Tillering

Panicle Initiation

Flowering

Harvest

Cattle manure

12.5% of RD

1.4

1.6

1.7

1.9

25.0 % of RD

1.7

2.0

2.2

2.5

37.5% of RD

1.9

2.3

2.6

3.0

50.0% of RD

2.3

2.5

2.8

3.1

Poultry manure

12.5% of RD

1.6

2.0

2.2

2.5

25.0 % of RD

2.1

2.3

2.6

3.2

37.5% of RD

2.5

2.7

2.9

3.4

50.0% of RD

2.8

3.3

3.6

4.3

Table  4  .  Yield (Kg ha-1) as influenced by the incorporation of organics

Treatments

Grain

Straw

Cattle manure

12.5% of RD

2320

2784

25.0 % of RD

2574

3063

37.5% of RD

3265

3918

50.0% of RD

3097

3685

Poultry manure

12.5% of RD

2725

3270

25.0 % of RD

3287

3912

37.5% of RD

3550

4260

50.0% of RD

3425

4110

SEd

137

164

CD(P:0.05)

325

389

Table.5 Cumulative CO2-C mineralization (mg kg-1) in the compost

(i) Ipomea cornea – cattle manure compost

Treatments

Incubation intervals (weeks)

IC-CM

1

2

3

4

5

12.5% RD

300

650

810

790

720

25.0 % RD

345

687

835

797

754

37.5%  RD

373

692

869

804

805

50.0%  RD

410

724

925

910

831

SEd

3.86

5.09

2.69

2.39

1.61

CD(P=0.05)

9.45

12.47

6.58

5.85

3.94

Soil

1.0

1.2

1.9

1.6

1.5

  (ii) Ipomea cornea – poultry manure compost

Treatments

Incubation intervals (weeks)

IC-PM

1

2

3

4

5

12.5%  RD

200

390

454

442

420

25.0 % RD

227

415

475

457

443

37.5%  RD

254

452

517

489

481

50.0%  RD

273

469

534

510

528

SEd

0.95

0.93

0.81

1.99

3.45

CD(P=0.05)

2.33

2.28

1.99

4.88

8.45

Soil

1.00

1.20

1.90

1.60

1.50

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1. Department of biotechnology Dr. G.R.D.C.S, Coimbatore.

2. Department of biotechnology, Alagappa University, karaikudi.

ABSTRACT

Bacterial biofilms are sessile communities with high cell density that are ubiquitous in natural, medical, and engineering environments; they are fascinating since they are primitive tissues with an advanced chemical communications network. Currently, there is an explosive amount of biofilm research, most of it with the ultimate aims of biofilm prevention, control, or eradication.The present investigation was aimed to study the cell signaling principle among seaweed epibiotic bacterial organisms , totally 54 bacterial isolates were made out of 20 seaweed species. Among 54 bacterial isolates 17 of them producer, another 17 of them inducer strains rest of 20 isolates were normal that have not showing any signs of activity. The 17 producers and 17 inducer strains were subjected to the cross species induction by quorum sensing principle and found that only 3 of them have responded to the cross species induction in production of antibacterial compound against the respective inducer strains. All the 3 producers and inducer were identified by biochemical analysis and surprisingly that all the 3 producer strain belongs to Genus Pseudomonas and inducer strains were belongs to Genus Vibrio. The supernatant obtain from mixed culture of Pseudomonas & Vibrio shows the antibiotic activity against the various Vibrio species which were isolated from other sources. Such as Vibrio from primary film, Vibrio from sediment Vibrio from seaweeds epibiotics. The obtain results clearly shows the particular Pseudomonas from seaweed epibionts has the capability of producing potential antibiotic compound against wide range of Vibrio species through quorum sensing.

*Corresponding author e-mail: vijay10bas@yahoo.co.in

INTRODUCTION

We are living in a microbial planet. About 71 % of the surface of this planet is covered by sea water. A typical milliliter of seawater contains 103 fungal cells, 106 bacteria, and 107 viruses, including pathogens that cause widespread mortalities and microbes that initiate fouling of host surfaces (Rheinheimer, 1992). Thus, marine plants and animals are continually exposed to high concentrations of potentially harmful microbes. These microorganisms in nature exists as free living planktonic mode of life in sea water or it may exist as epibiotic organisms in various living and nonliving surfaces. Among living organisms, seaweeds and invertebrates act as suitable substrate for the establishment of epibiotic organisms Seaweeds are known to release a large amount of organic carbon into the surrounding environment providing a nutrient rich habitat for microorganisms like bacteria. Bacteria are generally considered to be independent unicellular organisms. One cell accomplishes all of the tasks of feeding, locomotion, ‘reproduction, respiration and all other processes necessary to keep an organism alive. There are several classes of bacteria such as primary film forming bacteria, sediment bacteria, symbiotic bacteria, and epibiotic bacteria in various aquatic organisms. The marine surface environment is a site of intense composition for living space by a wide variety of organisms. Bacteria are generally recognized as primary colonizers of this habitat and are able to form biofilm on marine surface such as invertebrates and algae (Bryers, et al., 1982). Bacteria may also be abundant on the surfaces of some algae as an important epibiotic organism. In many cases, the bacterial population found to be specific, with changes occurring throughout the year or life span of the algal surface. This algal-bacterial relationship is symbiotic in most cases; the epibiotic bacteria in seaweed play a protective role by releasing secondary metabolites into the surrounding seawater that help preventing extensive fouling of the surface. Epibiotic bacteria are therefore attracting attention as a source of new natural products. Bacteria from the larvae of some crustaceans protect them from fungal infection by the production of simple antimicrobial compounds. Bacteria isolated from the surface of a tunicate prevented the settlement of barnacle and tunicate larvae exposed to the bacteria as biofilm in petridishes (Evelyn et al., 2001).

Seaweeds itself secretes secondary metabolites to prevent fouling and grazing. In addition to that epibiotic bacteria on macro algae can also produce antifouling compounds that work in concert with the seaweed derived compounds to protect the seaweed surface. Recent studies have highlighted important roles of epibiotic bacteria colonizing the surface of seaweeds and releasing antifouling compounds. For the past 50 years antibiotics have revolutionized medicine by providing cures for formerly life threatening diseases. However, strains of bacteria have recently emerged that are virtually unresponsive to antibiotics such multidrug resistance, arising mainly through antibiotic misuse, is now recognized as a global health problem. In this situation, it is clear that new classes of antibiotics are urgently needed. Many marine bacteria have been shown to produce secondary metabolites that display antibacterial properties. The first antibiotic from a marine bacterium was identified and characterized in 1966. In addition, bacteria in biofilm on the surface of marine organisms have been documented to contain a higher proportion of antibiotic producing bacteria than some other marine environments (Burgess, et al., 1999). Marine epibiotic bacteria, associated with nutrient-rich algal surfaces have also been shown to produce antibacterial secondary metabolites which inhibit the settlement of potential competitors. Recently a lot new novel antibiotics such as Phenazine, thiomarinol, phenazine-1-carboxylic acid, 1-hydroxyphenazine 2-n-heptylquinol-4-one, 2-n-nonylquinol-4-one pyolipic, loloatins, agrochelin, sesbanimides, pelagiomicins, indomycione and indomycione have been identified from various marine epibiotic bacterial organisms. In particular, some species of the genus Pseudomonas produce both antibiotics and several other bioactive substances. For example, Pseudoalteromonas rubra and Pseudoalteromonas aurantia have been reported to be antibiotic producing bacteria. The phenomenon of higher organisms utilizing their associated microflora for the production of beneficial secondary metabolites is common in the marine environment (Yotsu, et al., 1987). A study of bacteria isolated from marine algae surfaces indicated that the incidence of antibiotic producing strains from this habitat was 20% whereas that from sea water was only a few percent. In addition, some bacteria that previously did not produce any active compounds have been found to be producing such metabolites when they are exposed to other bacterial species or extra cellular chemical from other bacteria. Bacteria may also produce antimicrobial compounds when they sense the presence of competing organisms. However, few attempts have been made to study such chemical communication between different bacterial species or how this might affect. The secretion of antimicrobial compounds (Mearns-Spragg, et al., 1998). Bacterial communication by the chemical signals for specific function is simply known as Quorum sensing. In which a bacterial population receives input from the environment and elicits an appropriate response (Hiroaki and Kristina. 2003). The term “quorum sensing” describes the ability of a microorganism to perceive and response to diffusible signal molecules. Bacterial cells sense their population density through a sophisticated cell to cell communication system and trigger expression of particular genes. Tne first system of density-dependent regulation was studied in detail with the luminescence of Photobacterium fischeri (formerly known as Vibrio fischeri) by Bassler et al., 1997. Eventually, they discovered that 3-oxo-N-(tetrahydro-2-oxo-3-furanyl) hexanamid or N-3-(oxohexanoyl) homoserine lactone (OHHL) was responsible the agent in the broth that induced luminescence. Followed by this many researchers have confirmed that in Gram negative bacteria acyl-homoserine lactone is responsible for the cell to cell communication system.

In gram positive bacteria peptide and derivative peptide based signaling molecules seem to be the predominant mode of communication. During high cell density the marine bacteria can produce enzymes, surfactants, toxins, and antibiotics by the chemical signal communication. Marine epibiotic bacteria are also known to produce compounds active against drug resistant hospital pathogen by the cross species induction method. Building on assays described by Austin (Billaud and Austin 1990) a screening procedure has been developed in which marine bacteria are challenged by exposing them to terrestrial bacteria prior to assay of antimicrobial compounds. Hence in this present investigation it is proposed to find out the ability of sea weed epibiotic bacterial organism to produce antibacterial compounds through quorum sensing.

MATERIALS AND METHODS:

SAMPLE COLLECTION:

Seaweed samples were collected from Gulf of Mannar Marine Biosphere Reserve and identified up to species level by using CMFRI bulletin (14) as follows:

Table 1. List of Seaweeds species collected for the present study

SPECIES NAME FAMILY

Halimeda gracilis Chlorophyceae

Ulva lactuca Chlorophyceae

Microdictyon tenunis Chlorophyceae

Chondrococcus hornemonii Chlorophyceae

Enteromorpha intestinalis Chlorophyceae

Caulerpa cupressoides Chlorophyceae

Caulerpa racemosa Chlorophyceae

Dictyota dichotoma Phaeophyceae

Turbinaria ornata Phaeophyceae

Padina gymnospora Phaeophyceae

Sargassum cinearifolium Phaeophyceae

Dictyota batryensis Phaeophyceae

Sargassum sps Phaeophyceae

Hypnea musciformis Rhodophyceae

Acanthophora dendroides Rhodophyceae

Jania rubens Rhodophyceae

Hypnea valentiae Rhodophyceae

Hypnea pannose Rhodophyceae

Hypnea esperi Rhodophyceae

Acanthophora spicifera Rhodophyceae

ISOLATION OF EPIPHYTIC BACTERIA

The collected seaweed samples were thoroughly washed with sterile seawater to removes the loosely attached bacteria/particles. Seaweed fronds were scrubbed with sterile cotton swabs to obtain epiphytic bacteria. Epiphytic bacterial organism in the swab were inoculated in sterile peptone broth (50% sea water) and incubated at 28°C in an incubated shaker (220 rpm / min) for overnight. After the incubation period the enriched cultures were serially diluted up to 10-8 concentration and 200 microlitre of each diluted samples were transferred into the nutrient agar plate (50% sea water). The plates were incubated at 28°C for 5 days and the plates with crowded colonies were selected. In the crowded plates those colonies, which showed the sign of inhibition zone around its margin to the neighboring colony, were selected and considered as producer strain. The neighboring sensitive colonies were treated as inducer strain. Both producer and inducer strains were streaked repeatedly until to get pure culture. The pure culture were properly labeled and subjected to the quorum sensing analysis.

QUORUM SENSING

EXPERIMENT NUMBER 1

In this present study, the producer and inducer strains were cross reacted to find out the production of antibiotic compound through quorum sensing. Totally three set of cultures were maintained as follows (along with one as control).

A. Live cells of producer and inducer strains

B. Live cells of producer strain alone

C. Live cells of inducer strain alone

In culture system A 200ul of 16 hours old broth culture of both producer and inducer strains were added to the 15 ml of nutrient broth.

In culture system B 200ul of 16 hours old producer strain alone was inoculated.

In culture system C 200ul of 16 hours old inducer strain alone was inoculated.

All the cultures were incubated at 28°C for 5 days. After the incubation period the cultures were centrifuged at 10,000 rpm for 15mins. The supernatant was collected and subjected to antibacterial assay with respective inducer strain.

EXPERIMENT NUMBER 2

In this experiment, culture supernatant was obtained as per the procedure given in the experiment 1. 50ml of supernatant was mixed with equal volume of 80% methanol and 1% acetic acid mixture and it was shaked thoroughly in a separating funnel. Finally the methanol and acetic acid fractions were collected and concentrated by evaporation using water bath at 55°C. The viscous colloidal residues were resuspended in 600 microlitre of 50% methanol and it was used for antibacterial assay against different test organism.

TEST ORGANISMS:

1. Epiphytic Vibrio from seaweeds

2. Vibrio from primary film

3. Vibrio from Sediments

4. Pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella sp. and Proteus sp

The test organisms Vibrio species were isolated from seaweed as epiphyles, biofilm, sediment and puffer fish by using TCBS medium (Hi media) The pathogenic bacteria were collected from clinical laboratories.

ANTIBIOTIC ASSAYS

Antibiotic activity was performed in duplicate using a standard paper disc diffusion method as well as well assay. In well assay 10mm in diameter wells were made in marine agar plates and the plates were swabbed with 16 hours old inducer strain. To these wells 200ul of cell free supernatant were added to each well. In paper disc assay the Watmann no.1 filter paper discs (6mm in diameter) were saturated with 200ul of cell free supernatant. The impregnant discs were Dlaced in the centre of the plates swabbed with test organisms. The plates were Incubated at 37°C overnight and observed for inhibition zone. The zone of inhibition was measured as the distance from the border of paper disc to the edge of the clear zone and expressed in mm.

BACTERIAL IDENTIFICATION

The organisms responded to the quorum sensing process alone were identified by the following biochemical analysis.

Colony morphology, Gram staining, Motility test, Oxidase test, Catalase test, Indole Production, Methyl red test, Voges Proskauer test, Citrate Utilization test, Triple sugar Iron test, Nitrate reduction test, Lactose fermentation, Urease test

Starch hydrolysis test, Protein hydrolysis test, Lipid hydrolysis test, Oxidative / Fermentative test, Salt concentration (0%, 3%, 5%, 7%, 10%), TCBS, Growth in Temperature, 42°C and 47°C

All the above mentioned biochemical tests were performed by following standard methodology given in the Microbiological Laboratory Manual by James 3.Cappuccino (1999).

RESULTS AND DISCUSSION:

QUORUM SENSING/CROSS SPECIES INDUCTION ANALYSIS

In the present investigation totally 54 isolates were collected out of seaweed species. Among 54 isolates, 17 of them are producer strain, another 17 are the inducer strain rest of 20 isolates is normal and not showing any signs of activity (Table.2).

a) Among these 17 producers strain 6 strains were isolated from Hypnea musiformis. 6 from Gracillaria edulis, 4 from Ulva lactuca & 1 from Sediment.

b) Among these 17-inducer strain 6 strains were isolated from Hypnea musiformis, 6 from Gracillaria edulis, 4 from Ulva lactuca & 1 from sediment.

All the 17 strains were named as

PRODUCERS STAINS

BrA+, BrB+, BrC+, BrD+, BrE+, BrF+ Hypnea musiformis

GcA+, GcB+, GcC+, GcD+, GcE+, GcF+ Gracillaria edulis

U1+, U2+, U3+, U4+ Ulva lactuca

SA+ Sediment

INDUCER STRAIN

BrA-, BrB-, BrC-, BrD-, BrE-, BrF- Hypnea musiformis

GcA-, GcB-, GcC-, GcD- GcE-, GcF- Gracillaria edulis

U1-, U2-, U3-, U4- Ulva lactuca

SA- Sediment

In this experiment among 17 Producer and Inducer strains only 3 of them have responded to the quorum sensing principle. (BrB+/BrB-), (GcC+/GcC) and (SA+/SA-)

Table 2: The results of Quorum Sensing analysis of epibiotic bacterial isolates from seaweeds.

Seaweed sample Producer organism Inducer organism Cross-species producer with inducer Cross-species supernatant test with inducer Zone of clearance (mm)

Hypnea musiformis

1. BrA+

2. BrB+

3. BrC+

4. BrD+

5. BrE+

6. BrF+ BrA-

BrB-

BrC-

BrD-

BrE-

BrF- Br A+ /Br A-

Br B+ /Br B-

Br C+/Br C-

Br D+/Br D-

Br E+/Br E-

Br F+/Br F- BrA-

Br B-

BrC-

BrD-

BrE-

BrF- NIL

39

NIL

NIL

NIL

NIL

Gracillaria

edulis 7. GcA+

8. GcB+

9. GcC+

10. GcD+

11. GcE+

12. GcF+ GcA-

GcB-

GcC-

GcD-

GcE-

GcF- Gc A+/GcA-

Gc B+/GcB-

Gc C+/GcC-

Gc D+/GcD-

Gc E+ /GcE-

Gc F+/GcF- GcA-

GcB-

GcC-

GcD-

GcE-

GcF- NIL

NIL

26

NIL

NIL

NIL

Ulva lactuca

13. U1+

14: U2+

15. U3+

16. U4+ U1-

U2-

U3-

U4- U1+/U1-

U2+/U2-

U3+/U3-

U4+/U4- U1-

U2-

U3-

U4- NIL

NIL

NIL

NIL

Sediment 17. SA+ SA- SA+/SA- SA- 28

c) The normal 20 bacterial strains isolated from 20 algal species were crossed with terrestrial bacteria such as E-coli, Staphylococcus aureus separately

This experiment does not showed any inhibition zones

Bacterial identification

The 3 producer and 3 inducer strains which were responded the quorum sensing principles alone were subjected to biochemical analysis for identification. The obtained results revealed that all the producer strains showed sings of Pseudomonas sps and the inducer strains showed signs of Vibrio sps. So, based on the obtained result all the producer strains seems to be a Pseudomonas sps where as all the Inducer strain belongs to the genus vibrio.

In the present investigation, it was aimed to produce the antibiotics from the seaweed epibionts through quorum sensing principle. The bacterial isolates of seaweed epibionts were identified as species of Pseudomonas and Vibrio from seaweeds Hypnea musiformis and Gracillaria edulis. In this study the Pseudomonas acts as a producer strain and Vibrio as inducer strain. The recent finding says that the seaweed epibionts having potential to control the metabolic activity of competitor organisms. Allison et al., 1998 reported that many bacterial strains up on attaching to a surface produce exopolysaccharides or exopolypeptides. In addition, it has been postulated that exopolysaccharides could mediate the attachment of the bacteria to the surface and induce metabolic changes.

The results of Vanderivere and Kirchman 1993 suggest that the addition of increased surface by adding sand will induce the exopolymer synthesis through the high cell density dependent system. In the same way the bacterial organisms attached to the surface of seaweed shows alteration of genes expression may be due to the response to the high competitive environment. When cell density increases the competition for space and nutrients are also increased. So the existing bacteria were forced to protect themselves in this competitive environment. Normally in this condition the bacteria will be activated to induce the expression of certain hidden genes in genetic material through quorum sensing. The quorum sensing is principles were active compounds (autoinducer) from bacterial cell will promote the expression of a particular hidden gene of other bacterial organism in a stressed condition.

Quorum sensing usually focused on the bacteria growing in homogeneous environment. However few studies have attempted to a study this principle in heterogeneous environment also. In this present investigation we have attempted to study both homogeneous as well as heterogeneous environment. In former one we have isolated producer strain in seaweed eipbionts and it shows inhibitory activity against the inducer organism at the same seaweed epibionts. Later producer strains from seaweed epibionts, were treated with various Vibrio organisms from different environment. The obtain result of this study shows that the producer strain are capable of secreting antibiotic compounds not only to their natural competitors in its own habitate but also to the pathogen inhabiting in a distant related environment.

In the gram negative bacteria AHSL is an active principle of quorum sensing. Our producer strain is also been identified as Pseudomonas sps. So in these organisms also active principle must falls under the AHSL. The cell-cell signaling mechanism can either require import of the signal and subsequent interaction with intracellular effectors or a two-component signaling system that transducers the information across the membrane. In V. harveyi genetic analysis of the density sensing apparatus has two independent density-sensing systems, and each is composed of a sensor-auto inducer pair; system one is composed of sensor I and Al -1, and system two is composed of sensor 2 and AI-2. The two densities – sensing system are redundant, because a null mutation in either system alone results in expression of hidden genes (Bassler, et al.,1999.).

The earlier genetic analysis in Pseudomonas reveals the Pseudomonas consist of two quorum sensing systems as Las R1-I and Rh1R-l and have linked with R and I genes, in addition recently a third Lux R homolog that is advanced to a cluster of quorum sensing – controlled (qsc) genes were detected. Las R is a transcriptional regulator that responses primarily to the Las I – generated signal and Rh1R is a transcriptional regularly that responses best to the Rhl -generated signal. In Pseudomonas auriginosa, at low population densities Las I produce a basel level of 3-O-C12-HSL. As density increases, 3-0-C12-HSL builds to a critical concentration, at which point interacts with LasR. This Las R -3-0-012-HSL complex that activates transcription of a number of genes [Whileley, et al., 1999].

We suggest that the above said mechanisms in Pseudomonas with quorum sensing principle might have occurred in the present study also. This induces the bacteria Pseudomonas in epibiotic seaweeds to secrete certain active compound against to the competitor Vibrio species.

In this present work the totally 54 isolates were screened from 20 different seaweed species out of which 34 species were showed the signs of quorum sensing i.e. 17 producer and 17 inducer strains, but when these organisms where subjected to quorum sensing principle in mixed culture only 3 them have responded. So the present study reveals around 17% of bacterial species isolated from seaweeds and sediment were responded to quorum sensing. According to the results of bacteria isolated from marine algae surfaces indicated that the incidence of antibiotic producing strains from this habitat was 20% where as that from sea water was only a few percent. In the present study also reveals more or less the same ratio in Pseudomonas spp. was observed. Our results also reveals a results of Kell et al., 1995; Stead et al., 1996; they have said the culture supernatant of Pseudomonas sps known to contain AHLS which induces the production of phenazine antibiotics. In this investigation due to time constraint, It was not attempted to identify the active compound secreted by Pseudomonas through quorum sensing, which may leave the space for the further intensive research in future.

In concluding this discussion, the quorum sensing is wider spread among bacterial population then was previously thought, (In Gram positive, Gram negative bacterial communication). Current assays for antimicrobial activities are inadequate because some antibiotic producing bacteria may require the presence of another bacterial species. These findings have important implication for the discovery of novel antimicrobial compounds from marine bacteria and may allow the development of new methods for screening novel compounds active against multidrug resistant bacteria.

CONCLUSION:

The present investigation was aimed to study the quorum sensing principle among seaweed epibiotic bacterial organisms. In the past few decades there was no findings of new novel antibacterial class compounds were identified. But, the pathogenic microorganisms show much higher rate of resistant development even to the potential antibiotics. So, there is an urgent need to discover new novel antibiotic compounds. The marine inhabitants such as microorganisms, seaweeds, invertebrates, etc., act as an undepleted source of wide range of natural products among which the seaweeds act as a potential source of antibiotic compounds. Currently the cross species induction / quorum sensing attracts the total attention of researchers in finding new novel drugs against multidrug resistant pathogenic microorganisms. So, the present study aim to find out the capability of seaweed epibiotic bacterial organisms to produce novel drugs against the animal and plant pathogens

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Ali Parsaeimehr1

1.PhD student at the field of plant Biotechnology at the National Academy Of science institute of G.S.Davtyan Institute of Hydroponics Problems NAS RA and Academician of young researcher organization.

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Abstract: it is clear that Plants, as a renewable source with low energy consumption that can offer complex biochemical syntheses even in medicinal purposes , between the synthesis compounds of plants alkaloids play an important but dilemma role at these aims? , alkaloids for plants known as a secondary metabolite which could by? essential for plants ecological surviving but it s a question remaining are these compound?s as a source for medical uses that it s going to world spread are vital for animals cells or not ?, well through this question a disquisition have been done for clearing some of the answers , we know that cells in both kingdom of plants or animals regulate intra from extra environment well these adjustment? and regulations applies by use of? ions channels? (Na+ ,K? , Ca? , Cl?) or the activity of? Na?/K? pump well we can safely assume that? behavior and the mobility? of ions? guarantied the cells vital. Some of secondary metabolites such as alkaloids in plants, known as the inhibitors of Na+, K?, ATPas or some of plant alkaloids like Harmaline, Nitidine, Capsaicin, Soleonopsin could disrupt these act , the molecular? structure of plant alkaloid with some vital compound?s in cells? and their similarity of theme could be the reason of their serious potential of act as an inhibitor or resonancer in animals cells vitality well Amino acids like Phe, Tyr, Try have Many physiologically active, and have limited distributions in the plant kingdom for example the strychnine alkaloids from the dried seeds of Strychnos vux vomica could be play a deadly way in the vitality of animals Kingdome and according to this acaealepsy we can safely assume there are too many targets for alkaloids acts such as: plasma membrane , ribosome?s or even DNA or RNA in animals cells ,? The other location for alkaloids effects are the neurotransmitters? which closely coupled and band with ion channels (Na+ ?K? , Ca? ? Cl?) as a fact the receptors or ion channels both are incipit for nervous signals ?and as a consequences affected? one of them? by inheritors or resonators? could occasion the vitality of animal cells. It?s amazing that the plant alkaloids even could show allergic sign in the animals cell? which its refers to the immunological responds to the? plants alkaloids? for instance alkaloids such as coumarins, furanocoumarine could act in this importance, the Mechanism of Allelochimical Activities in Antiviral, Antimicrobial and Allelopathic Interactions of alkaloids are the other mysterious role of these kind of secondary metabolites in animals vitality There are circumstantial evidence that some alkaloids such as Quinolizidine alkaloids protect the producing plants against viruses, bacteria , fungi, and other plants , relative to alkaloidesanimal interaction , these modes of action have been less well or hardly at all, a number of antimicrobial alkaloids such as sanguinarine, quinine , or berberine intercalate with viral and microbial DNA to bind it well these compounds may thus inhibit process such as DNA replication and RNA transcription which are vital for the microorganisms or animal cells, Well all in all alkaloids augment which is far from complete shows that many plant alkaloids inhibiter or over stimulate central process at the cellular or organ level. In this completion only a limited of structures have been discussed.

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?Key word: plants Alkaloids, animals cell vitality, antimicrobial effects

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1.Introduction: In toadies world use of medicines by the base of plants between the nations and countries have been wide spread and the effect of these kinds of drugs are undeniable but of course it?s better that we show the effect of secondary metabolites compounds and between them ,the alkaloids which synthesizes in plants cells and

shows their dilemmas effect of these kind of secondary metabolites, well according to cells biology and their? metabolites , cell s adjust their intra ?from? extra environment in a caution way , these adjustment? and regulations applies by use of? ions channels? (Na+ ,K? , Ca? , Cl?) or the activity of? Na?/K? pump well we can safely assume that ?behavior and the mobility? of ions ?guarantied the cells vital(Alberts et al.1993). Some of secondary metabolites such as cardiac glycoside in plants, insects and a kind of toads from the family of (Bufonidae) have been discovered, these kinds of secondary metabolites known as the inhibitors of Na+, K?, ATPas or some of plant alkaloids like Harmaline, Nitidine, Capsaicin, Soleonopsin which act as the same way and affect at Na+, K?, ?ATPas or ion channels and as it clearly shown these ions plays a vital role in order to cells vitality or even at the animals nervous system (Mann, J. 1992).in animals cells most of the cells activities such as endo- exocytose, cells division? is by base of microtubules or microfilaments activities in these suit some of Alkaloids such as ??Colchicines, Maytansin, Taxol, Vinca Alkaloids ?have the potential of coupling? with theme and inhibiter their activities due the cells progress , these expanded domain of activity of plants alkaloids in animals cell and the reason of it should by search in their molecular structure.

2. Discussion & Deliberation of Alkaloids potential: alkaloids have been extracted from and found in ~20% of vascular plants, their Compounds usually basic (alkaline) ~40,000 compounds currently described and Structurally the most diverse class of secondary metabolites and most of them use for medicinal usage like Morphine (painkiller),Vincristine (anticancer agent) , Cocaine (anesthetic, drug of abuse), Caffeine (stimulant) , well its seems that the best describe of this kind of secondary metabolites have been done at 1963 through their? chemotaxonomy and by this base they remain in 3 group such as:

Proto alkaloids .3??????? Pseudo alkaloids .2????? ?????Proper alkaloids .1

2.1.1. Proper alkaloid: they also known as true alkaloids we can describe them as the fallow: Basic Nitrogen part of a heterocyclic ring system, Chemically complex, Derived biosynthetically from amino acids, especially the cyclic ,Amino acids: Phe, Tyr, Try, Many physiologically active, and have limited distributions in the plant kingdom for example the strychnine alkaloids from the dried seeds of Strychnos vux vomica. Small tree found from India to

Northern Australia. Fruit is a large berry with a hard coat and pulpy interior containing 3-5 fleshy grey seeds ? contain ~1.2% strychnine, 60mg can kill an adult by the way we can say that the highly potential of it s activity is by the base of it molecular structure( Habermehi, G. 1983, Mann, J. 1992.).

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And as we can see strychnine, simply coupled with L-tryptophan one of the most important amino acid?? in cells vitality (Albert?s ET al.1993).

2.2.2. Pseudo alkaloid: Nitrogen containing (physiologically active) compounds not derived from amino acids, the purine ring is gradually elaborated by piecing together small components from primary metabolism for example caffeine the alkaloids which extracted from coffee

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2.2.3. Proto alkaloids:? they have?? physiologically active, Nitrogen atom is outside the ring system and this is the reason for nominated them as proto alkaloids well the alkaloids such as ephedrine or colchicines are one of the good examples of these kinds of alkaloids which play medicinally rolls in the industry of drugs. COLCHISINE the copesetic alkaloid of the Colchicum autumnale plant the family of Liliaceae which sustainability band through

Tubulin in a compeer of 1:1 (the Antimitotic potential) and thus inhibits the assembly of microtubules and as a consequence the meiotic spindle of dividing cells disappear suddenly after colchicines treatment and the chromatids no longer separated but whereas the animal cells die under this condition the plant cells become polyploidy? and? leads in breeding programs, using this alkaloid as an anticancer because of its Antimitotic potential widespread but after a while because of its damage to cells skeleton colchisin lay away and another alkaloid by the base of it by the name of colcemide use in this aim well this alkaloids because of their

Lipophilicity potential, simply absorb by cells and its clear why the family of colchicum plants are not attack by herbivores (Teuscher E et al.1995).

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2.3. The effect of Alkaloids on Neurotransmitter Receptors: the nerve single transduction in the central nervous system and in neuromuscular junction is mediated by receptor proteins residing in the membrane which directly or indirectly coupled with ion channels the neurotransmitters involve include (Alberts et al.1993). among others adrenalin(NA),serotonin, dopamine, histamine, glycine, GABA, and acetylcholine have been deduced to ion channels ,to many plant alkaloids have the similar molecular structure? with these neurotransmitters for? instance acetylcholine? and histamine in sting hairy roots of Urtica or serotonin and dopamine in several species, and the acts can be:

I. The receptor itself through inhibition or overstimulation

II.The enzymes that deactivate neurotransmitters after they have bound to a receptor

III.Transport process, which are important for storage of the neurotransmitters in synaptic

IV.Enzymes involved in the biosynthesis of a neurotransmitter (Mothes, K.et al.1985, Robinson, T. A. 1981).

Table I. some of the alkaloids as inhibitors of Neurotransmitters

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Enzyme????????????????????????????????? Natural Substrate?????????????????????????? ?????????Alkaloid???????????????? ?????occurrence (plant)

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?? Acetylcholine esterase ??????????????????????????Acetylcholine??????????????????????????? Physostigmine (eserine)??? Physostigma veneosum?????????????????????????

??????????????????????????????????????????????????????????????????????????????????????????????????????? Berberine????????????????????? ?????several papaveracea

?????????????????????????????????????????????????????????????????? ?????????????????????????????????????????????????Coptisine?????????????????????? ????several papaveracea

???????????????????????????????????????????????????????????????????????????????????????????????????? ???????????????Galanthamine??????????????? ???several Amaryllidaceae

????????????????????????????????????????????????????????????????????????????????????????????????????? ??????????????Chaconine?????????????????????? ?Solanum

?????????????????????????????????????????????????????????????????????????????? ?????????????????????????????????????Solanine???????????????????????? ??Solanum

???????????????????????????????????????????????????????????????????????????????????????????????????????? ???????????Solanidine??????? ????????????????Solanum

????????????????? ??????????????????????????????????????????????????????????????????????????????????????????????????Huperzine A???????????????? ????Huperzia srrata

?? Monoamine oxidase????????????????????????? ???NA. dopamine, Serotonin??????????? Harmaline???????????????? ???????Pegamum

????????????????????????????????????????????????????????????????????? histamine?????????????????????????????? Harmine?????????????????????????? Peganum

???????????????????????????????????????????????????????????????????????????????????????? ???????????????????????????Ephedrine??????????????????????? Ephedra

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The other location for alkaloids effects are the neurotransmitters? which closely coupled and band with ion channels (Na+ ?K? , Ca? ? Cl?) as a fact the receptors or ion channels both are incipit for nervous signals? and as a consequences affected? one of them? by inheritors or resonators? could occasion the vitality of animal cells (Rosenthal, et al.1992).

All animals need to transport nutrients, hormones, ions, signals compounds, O2 and CO2 between the different organs of the body well this achieved in higher animals through blood in the circulatory system.Inhibitorrs of its motor, the heart ,were discussed earlier . But the synthesize of red blood cells is also vulnerable and can inhibited by antimiotic alkaloids , such as vinblastin or colchisin and some allelochemicals have hemolytic properties, such as saponins and steroidal alkaloids , these compounds complex membrane sterls and make cells leaky these effected consequently effected the vitality of animal cells(Teuscher E. and Lindequist, U. 1994).

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Table II .The Alkaloids which known as inheritors or resonators for nervous signal transmission

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?????????????????? Receptor?s?? ?????????????????????????Ligand???? ???????????????????????????Alkaloids???????????? ???????????????Occurrence(plant)

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??? ???Acetylcholine receptor ???????????????Acetylcholine???????????????? ????????????Nicotine????????????????????? ??????Nicotinana, many other plants

???????? ??????????????????????????????????????????????????????????????????????????????????????????????????C-toxiferine??????????????? ????????strychnos

?????????? ????????????????????????????????????????????????????????????????????????????????????????????????Tubocurarine?????? ??????????????Chondodendron

? ?????????????????????????????????????????????????????????????????????????????????????????????????????????Cytosine and other QA??? ?Several legumes

???????? ??????????????????????????????????????????????????????????????????????????????????????????????????Lobeline???????????????????? ????????Lobelia

????????? ?????????????????????????????????????????????????????????????????????????????????????????????????Anabasine???????????????? ????????Anabasis , Nicotiana

????????? ????????????????????????????????????????????????????????????????????????????????????????????????Hyoscyamine (atropine)???? Atropa, Hysoscyamus, Datura.

????????? ????????????????????????????????????????????????????????????????????????????????????????????????Scopolamine???????????????????? ??Several Solanaceae

????????? ????????????????????????????????????????????????????????????????????????????????????????????????Arecoline????????????????????????????? Areca

????? Adrenergic receptors ????????Noradrenaline/adrenaline???????????? ????Norlaudanosoline?????????????? ??Papaveraceae

???????? ?????????????????????????????????????????????????????????????????????????????????????????????????Ephedrine, norephedrine? ???Ephedra

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Its amazing that the plant alkaloids even could show allergic sign in the animals cell? which its refers to the immunological responds to the? plants alkaloids? for instance alkaloids such as coumarins, furanocoumarine, hypercin , helenalin , Activation or repression of the immune response are certainly targets that were selected during evaluation as an antiherbivore strategy(Luckner et.al.1990).

2.4. Mechanism of Allelochimical Activities in Antiviral, Antimicrobial and Allelopathic Interactions of alkaloids:

There are circumstantial evidence that some alkaloids such as Quinolizidine alkaloids that protect the producing plants against viruses, bacteria , fungi, and other plants , relative to alkaloids animal interaction , these modes of action have been less well or hardly at all, a number of antimicrobial alkaloids such as sanguinarine, quinine , or berberine intercalate with viral and microbial DNA to bind it well these compounds may thus inhibit process such as DNA replication and RNA transcription which are vital for the microorganisms, protein biosynthesis in ribosome?s is another vulnerable target for plant alkaloids even the stability of biomembranes can be disrobed by steroidal alkaloids(Luckner et.al.1990).

? In another view even herbicidal properties or germination inhibition which can be observed in plant-plant interactions, can also proceed via the above mentioned mechanisms but interaction with growth hormones and their metabolites also must be considered (Wagner, H. 1993).

?

3. Counclusion:

Plants, as a renewable source with low energy consumption that can offer complex biochemical syntheses, will be even more compatible in the future. Well all in all alkaloids augment which is far from complete shows that many plant alkaloids inhibiter or over stimulate central process at the cellular or organ level. in this completion only a limited of structures have been discussed .In many instance , plants contain mixture of related alkaloids , which only differ for particular substitution patterns well this allelochemical properties are requisite for chemical defense compound in an ecological context? for surviving of plants but also constitute the base for their exploitation in medicine or agriculture, well after all of these dilemma situation we should use medicines by the source of plants in a secure and caution way ??but to many experiments demands through this complicated way.

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4. References:????????????????

Alberts, B. D., Lewis, J. Raff, M., Roberts, K. and Watson, J. D. 1993. .Molecular Biology of the Cell.3rd ed . Garland. New York. Habermehi, G. 1983. Gifttiere and ihre Waffen. Springer. Berlin. Harborne, J. B. 1993. introduction to Eclogical Biochemistry.4 th ed. Academy press. san Diego. Luckner, M. 1990 Secondery Metabolism in Microorganism.Plants and Animals .Springer, Berlin. Luckner, M. 1990. Secondary Metabolism in Microorganisms, Plants and Animal, Springer, Berlin mann,J..1992, Murder,Magic and Medicine.Oxford University Press. London. Mann, J. 1992. Murder Magic and medicine.Oxfordr University press .London. Mothes, K., Schutte, H. R. and Luckner, M. 1985. Biochemistery of Alkaloides .VCH. Weinheim. Robinson, T. A. 1981. The Biochemistry of alkaloids, Springer. Berlin. Rosenthal, G. A. and Berenbaum, M. R. 1992. Herbivores: Theair Interaction with Secondary Plant Metabolites vol.Academic Press, San Diego. Teuscher E. and Lindequist, U. 1994. Biogene Gifte. Fischer. Stuttgart. Urk H, Schipper D, Breedveld GJ, Paul RM, Scheffers WA, van Dijken JP (1989) Biochim Biophys Acta 992: 78 Scopes RK (1989) In: van Unden N (ed), Alcohol toxicity in yeast and bacteria, CRC Press Inc, p. 89 Wagner, H. 1993. Pharmazeutische Biologie . 2. Drogen and ihre Inhalisstoffe, Fisher, Stuttgart. Zhou, G.M. 1980. Studies on useful compounds of Bai-Zhi for healing Yin- Hsieh Ping. Chung-Chen-Yau Res. 4: 33. (In Chinese). Zhou, G.M., C.G. Yu, Y.C. Han, and C.T. Mun. 1988. Studies on Bai-Zhi. IV. The toxicity test of useful compounds. Med. J. China Hospital 8: 220-221. (In Chinese).

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