INTRODUCTION:

Microorganisms which can live within plant tissues for all part of their life cycle without causing any visible symptoms are known as endophytes^1,2.Endophytes inhabits in various tissues, seeds, roots, stem and leaves³. Endophytic bacteria can actively colonize plants ,these bacteria exist in variety of tissues types within numerous plant species, suggesting their existence in all higher plants. Endophyte can live within plants without causing disease symptoms⁴. These endophytic bacteria can actively promote growth and also play role in disease control⁵. Purpose of the present study is to isolate and exemplify endophytic bacterial flora from selected Indian medicinal plants such as- Curcuma longa L, Eucalyptus globules Dehnh, Musa paradiasa L., and Pongamia glabra Vent., Vinca rosea (L.)G. DON, for the production of the extracellular enzymes, antimicrobial and antioxidant substances.

MATERIALS AND METHODS:

Mature and healthy plant materials were collected by sampling different parts of the selected medicinal plants, fifteen samples were taken from each tree five each from roots, inner bark and leaves, from each sample ten subsamples were separated further for isolation of endophytes. Samples were washed in running tap water for 10 min to remove, soil particles.

Surface sterilization was done using method described by Petrini et al.⁶, Samples were immersed in 70% ethanol for 1-3 min and 4% aqueous solution of sodium hypochlorite 1.5 min, again 1min with 70% ethanol and finally rinsed with sterile distilled water. Samples were selected by aseptic cutting using sterile knife and inner tissues were excised. Endophytic bacterial flora were isolated on Tryptic Soya agar and water agar media for incubation of 2-10 days at 25^oC to isolate endophytic bacteria.

Enzyme activity:

The amylolytic isolates were inoculated in nutrient agar⁷with 1% starch (g l^-1), pH 6.0.After incubation period culture plates were treated with iodine which allowed to observed clear zone around colonies.

Proteolytic activity:

The proteolytic activity was determined using milk agar⁸.Milk was separately autoclaved and added in 1% (v/v) pH 7-7.4. The microorganism was inoculated and incubated for 28- 48 hrs and plates were observed for zone of proteolysis.

Lipase activity:

Isolates were grown on to the medium having (gl^-1) peptone 10, NaCl 5, CaCl₂H₂O 0.1, agar 16, pH 6.0 with Tween 20 separately added after sterilization in 1 %( v/v).Clear zone around the colony indicates lipase activity⁷.

Cellulolytic activity:

Media was used having (gl^-1)Yeast extract 1, peptone 0.5,agar 16, 0.5 Na carboxy methyl cellulose according to Samanta et al⁹. After incubation plates were flooded with 0.2 aqueous Congo red and distained with 1M NaCl for 15 min. Clear zone surrounding the colony indicated cellulase activity.

Catalytic activity:

Loop full cultures of isolates were inoculated into the tube containing H₂O₂ solution and release of Oxygen bubbles indicates positive catalase activity¹⁰.

Antifungal activity:

Antifungal activity was screened using dual culture method described by Narayan¹¹ with some modification in which both endophyte isolate and test fungi were inoculated in same media plate. Five days old culture disks (5mm diameter)of test fungi were inoculated at the centre of potato dextrose agar plate and endophytic bacteria were spot inoculated at three corner of the plate and incubated for four to eight days at 27⁰ C. Antifungal activity was indicative as mycelia growth of test fungus prohibited in the direction of active endophyte, the level of inhibition was calculated by subtracting the distance (mm)of fungal growth in the direction of an antagonist colony from the endophyte growth radius. The width of inhibition zone between the pathogen and endophyte was evaluated as inhibition zone and ranked as. +, <2mm; ++ >, 2-10mm; +++, 10 > mm.¹⁴.

Antibacterial activity:

Antibacterial activity was screened using dual culture technique as describe above using some some variation. Suspension of 24 hrs old culture of pathogenic bacteria was spread on sterile nutrient agar plate on to which five day old disc (5mm diameter) of endophytic fungi was kept and incubated at 27⁰c for 24 -48 hrs. Antibacterial activity was calculated by measuring zone of inhibition produce by endophytic fungi against pathogenic bacteria.

Inhibition zone diameter index :+++> 10mm, ++5-10mm+<5mm –no zone

Assay of Peroxidase activity:

Endophytic bacterial isolates were inoculated in nutrient broth and incubated for 24-48 hrs on rotary shaker at 37^oC ,broth culture were centrifuged at 5000 rpm for 15 min. Supernatant were used as crude enzyme source .The assay was carried out by using protocol described by Addy and Goodman¹² with some modification. The reaction mixture made was consisting 3ml of buffered pyrogallol [0.05M pyrogallol in 0.1M Phosphate buffer (pH 7.0) and 0.5 ml of 1%H₂O₂.1ml of enzyme extract was added to this mixture and change in O.D was measured at 430 nm for every 30 seconds for 2 minutes. The Peroxidase activity was calculated using an extinction coefficient of oxidized pyrogallol (4.5litres/mol)

Figure 1.Endophytic bacterial isolates from roots of M.paradiasica (a) and P.glabra (b)

Figure 2.Cellulase activity by isolates KB2 from stem of P. glabra (c) and proteolytic activity by SB1 from leaves of V. rosea (d)

Table: 1 Screening of Endophytic Bacteria for their possible Antimicrobial and Anioxidation activity

Isolates	Grams reaction	Tissue	Plants	Extracellular enzyme					Antimicrobial activity		Antioxidation activity
Isolates	Grams reaction	Tissue	Plants	A P C CS L					AF AB		Antioxidation activity
SBLV1	Gram positive bacillus	L	V.rosea	++	+++	+++	-	-	-	-	++++
HBRC1	Gram positive cocco bacillus	RZ	C.longa	+	+	+++	-	+	-	--	++
HBRC2	Gram positive bacillus	RZ	C.longa	+++	+	+++	-	-	++	-	-
HBRC3	Gram negative bacillus	RZ	C.longa	-	-	+++	-	-	+++	++++	-
HBRC4	Gram negative bacillus	RZ	C.longa	-	-	-	-	-	++++	-	-
HBRC5	Gram negative cocco bacillus	RZ	C.longa	-	-	-	-	-	+++	-	-
NBEL1	Gram positive rods	R	E.globules	-	+++	+++	-	-	++++	+	-
NBEL2	Gram negative rods	L	E.globules	-	-	+++	-	-	-	-	-
NBEL3	Gram negative rod	L	E.globules	-	-	+++	-	-	-	-	-
NBEL4	Gram Negative rods	L	E.globules	-	-	+++	-	-	-	-	-
NBEL5	Gram positive rods	L	E.globules	-	+++	+++	-	-	-	-	-
NBEL6	Gram positive cocco bacillus	R	E.globules	-	-	+++	-	-	++++	++++	+++
KBRP1	Gram negative rods	R	P.glabra	-		++	-	-	++	-	+++
KBRP2	Gram negative rods	S	P.glabra	-	++	-	+	-	-	-	+
KBRP3	Gram positive rods	L	P.glabra	-	++	-	-	-	-	-	-
KBRP4	Gram positive rods	S	P.glabra	-		-	+	-	++++	-	+++
KBRP5	Gram positive rods	R	P.glabra	-	++	++	-	-	+++	-	-
BBR1	Gram positive rods	R	M.Paradiasica	-	-	++	-	-	++	-	-
BBR2	Gram positive rods	R	M.Paradiasica	-	-	++	-	-	-	-	-
BBR3	Gram positive rods	R	M.Paradiasica	-	-	++	-	-	+++	-	-
BBR4	Gram positive rods	R	M.Paradiasica	-	-	++	-	-	-	-	-
BBR5	Gram positive rods	R	M.Paradiasica	-	-	++	-	-	-	-	-
BB6	Gram positive rods	R	M.Paradiasica	+++	+++	++	+	+	++++	-	-

L: Leaves; S: Stem; R: Roots; RZ: Rhizomes, AB: Antibacterial activity; AF: Antifungal activity

Enzyme activity: +, 1mm; ++, <3mm, +++, < 5mm , Antifungal activty: +, < 2mm; ++, 2-10mm;

Antibacterial activity :+++, >10mm, Inhibition zone diameter index :+++> 10mm, ++5-10mm+<5mm,

Antioxidation activity: +, Positive; ++, Good activity, +++Excellent activity

Figure 3 : Tissue specificity of isolated endophytic bacteria from selected medicinal plants

Medicinal plants

CC: C.longa, EG: E.globules, MP: M.paradiasica, PG; P.glabra,

VR: V.rosea

%CF = Number of segment colonized by endophyte X 100

Total segments analyzed

RESULTS AND DISCUSSION:

This investigation is to describe indigenous bacterial endophytes isolated from Indian medicinal plants. The diversity of collection of twenty three endophytic bacteria isolated from different tissues of the hosts was assessed for their possible enzyme, antimicrobial and antioxidation activities. Isolates were and morphologically distinct and

greater parts were Gram positive. However, in2002 Zinniel¹³ reported an equal presence of Gram negative and Gram positive bacteria from some plants. Endophytic bacteria SBLVl from leaves of V.rosea showed best enzymatic activity such as amylolytic, proteolytic and catalolytic activities along with strong antioxidation activity. Isolate BBR6 from leaves M .paradiasica was having good quality amylolytic, proteolytic, catalase activities and less positive activity for enzyme cellulose and laccase, endophyte BBR6 also exhibits superior antifungal activity .Rest of isolate like BBR2, BBR4, and BBR5 from leaves of M.paradiasica were positive only for enzyme catalase.NBEL6 endophyte from roots of E.globules was having strong antimicrobial activity against pathogenic bacteria and fungi Protease vulgaris and Aspergillus niger respectively along with antioxidation activity .Bacterial endophytes HBRC3 and HBRC4 from rhizomes of C.longa were tough candidates from which HBRC3 showed colossal antimicrobial activity against Protease vulgaris and Aspergillus niger. Bacteria HBRC4 have only burly antifungal activity. Endophytic bacteria KBRP4 from P.glabra exhibits large antifungal and antioxidation activity. Other isolates from selected medicinal plants were less active as per enzyme, antimicrobial and antioxidation activities were concerned. Endophytic bacteria isolated from selected medicinal plants were tissue specific, bacterial flora isolated from preferred medicinal plants were in following range leaves>stems>roots (Figure 3)

It was found that numbers of endophytic isolates from roots of plants were more active as per selected activities were concerned.

By meaning an endophyte cannot be considered to source disease in plants. The distinction between a plant pathogen and endophyte is not always understandable .It seems that the bacteria best adapted for living inside plants are naturally selected. Endophytic bacteria are found in legume nodules as well. Total bacterial population sizes on inoculated leaves varied by about 30 folds¹⁴. It is well recognized that bacterial endophytes are capable of suppressing nematodes proliferation also. Change in plant physiology can lead to the development of a distinct endophytic population¹⁵. Endophytic bacteria are found in roots, stem, leaves, seeds, fruits, flowers, tubers, and ovules ^{16, 15}. Enzymes are used in manufacturing food, beverages, confectioneries, textile and leather processing and help simplifying the processing of raw materials. Wood inhabiting fungi serve as a potential source of exoenzymes ^{17, 18,19,20,21}. Antioxidants are compounds that inhabit or delay the oxidation of other molecules by inhabiting the initiation or propagation of oxidizing chain reactions. Synthetic antioxidants are compounds with phenolic structures of various degrees of alkyl substitution, whereas natural antioxidants can be phenol compounds (Tocopherols, Flavonoids and Phenolic acids), nitrogen compounds(alkaloids ,chlorophyll derivatives, amino acids and amines).or carotenoids as well as ascorbic acid. Synthetic antioxidants, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been used from last some years but now are out of the use of because of their carcinogenicity. So need of natural antioxidants are more increased. Antioxidant metabolites are often produce by endophytic microbes Isopestacin, (Figure.4) Pestacin (Figure.5) were isolated from Pestalotiopsis microspora from plant Terminalia morobensis, native of the Papua New Guinea²²

Figure.4 Isopestacin

Figure.5 Pestacin

Recently biological controls or the uses of microorganisms or their secretions to prevent diseases offer an attractive alternative or supplement to disease management without the negative impact of chemical control²³ .Plants have long provided mankind with a source of medicinal agents, with natural products once serving as source of all drugs ²⁴. So endophytic microorganisms are excellent sources of bioactive natural products that can be use to satisfy demand of Pharmaceutical and Medical Industries .Study demonstrated the occurrence and diversity of culturable endophytes in Curcuma longa L Eucalyptus globules Dehnh, Musa paradiasa L., Pongamia glabra Vent., Vinca rosea (L.)G. DON.

CONCLUSION:

Medicinal plants selected for isolation of endophytes showed presence on diverse group of endophytic bacteria with variety of antimicrobial activities. Future research will give more attention to explore all these actions.

ACKNOWLEDGMENT:

We are thankful to principal Moolji Jaitha College, Jalgaon for providing us facilities to complete the research.

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Received on 18.10.2010 Modified on 02.11.2010

Asian J. Research Chem. 4(2): February 2011; Page 296-300