aerial parts are used in the treatment of kidney stones , hypertension , fever and cough ( http://www.forestry.gov.my/ ) .
the earlier studies have shown that endophytic microorganisms isolated from medicinal plants produce the same metabolites as their hosts .
therefore , there is a great potential in exploring endophytes as a source of therapeutic natural products . however , despite several traditional medicinal applications of nepenthes spp .
the objective of this study was to isolate and to identify the endophytic bacteria ( eb ) from nepenthes spp .
namely , nepenthes ampullaria , n. gracilis , n. macfarlanei , n. mirabilis , n. rafflesiana and n. sanguinea were collected from frim , selangor , malaysia .
however , leaves and twigs from one to three individual plants of nepenthes spp . ,
namely , n. alba , n. albomarginata , n. gracillima and n. sanguinea were collected from the gunung jerai ( gj ) , gurun , kedah , malaysia .
leaves with petioles were thoroughly washed under running tap - water and the surface - sterilization of plant material samples was carried out as reported elsewhere .
the stem pieces were soaked in 70% ethanol and flamed to make their surface sterile .
aseptically , the leaf and stem tissue pieces were inoculated in the petri plates containing luria - bertani ( lb ) agar medium .
the plates were incubated in an incubator at 37c ( 3c ) for 18 - 20 h in the dark .
the isolation , cultivation of endophytes , amplification of 16s rdna , sequencing of 16s rdna , identification of endophytes and rooted phylogenetic tree construction was carried out as reported by bhore et al .
incubation of the inoculated leaf discs and stems pieces on lb agar medium enabled cultivable eb to grow , and the colonies of grown eb were visible on the margins of the leaf and stem tissues .
all 96 isolates were identified based on 16s rdna sequence blast ( megablast ) hits analysis . the annotated 16s rrna gene fragment ( 16s rdna )
nucleotide sequences of all isolates have been submitted to the international dna database ( genbank / ddbj / embl ) under accession numbers : jf819686-jf819713 and jf938974-jf939041 .
were from the bacilli ( 59.4% ) class , followed by gammaproteobacteria ( 35.4% ) and betaproteobacteria ( 5.2% ) [ table 1 ] .
the genera of endophytic bacterial isolates ( ebis ) isolated from 9 nepenthes plant species the 16s rdna multiple sequence alignment output from clustalw was used in the construction of a rooted dendrogram .
rooted dendrogram showing clustering of 44 diverse types of endophytic bacteria ( eb ) isolated from nine nepenthes spp .
the accession number of 16s rdna ( rrna gene ) sequence of the respective isolate is given in front of the species name
in this short and snappy study , we isolated and identified 96 isolates from nine nepenthes spp . eb have been reported from several medicinal plants , for instance glycyrrhiza spp .
however , to the best of our knowledge , our study is the first to illustrate diversity and types of eb in nine stated nepenthes spp .
comparison between the annotated 16s rdna fragment sequences from isolates and the sequences from genbank / ddbj / embl database using the blastn program revealed the identity of the respective isolates .
the rooted dendrogram clearly showed the clustering of the species from the bacilli , betaproteobacteria and gammaproteobacteria groups .
endophytes are found abundantly in various plant species studied to date , and soil bacteria such as bacillus spp . , pseudomonas spp . and azospirillum spp .
however , we did not find any pseudomonas spp . and azospirillum spp . in our isolates .
during isolation of eb , the growth medium used might be directly affecting the number and type of endophytic microorganisms that can be isolated from the plant tissues .
the tissue samples used in the isolation of eb were from a single or few plants of each nepenthes spp .
the location and the conditions in which plant species are grown also determine the types of endophytes in it .
we have used plant samples from nepenthes spp . that were collected from their wild habitat in gj and diverse collection available at frim .
this could be the reason for the wide diversity of eb in the studied nepenthes spp .
it is important to note that from 96 isolates , 22 isolates ( representing 15 species ) belonged to the enterobacteriaceae family , which contains human enteric pathogens .
a number of species from the enterobacteriaceae family have been reported as endophytes , viz entrobacter cloacae and klebsiella pneumonia in maize , entrobacter asburiae in cotton , and klebsiella spp . and
entrobacter cloacae in banana . of the 44 species of eb isolated from nepenthes spp .
however , we have not found any published record that reported acinetobacter soli , bacillus cibi , b. horneckiae , b. indicus , b. koreensis , b. stratosphericus , citrobacter gillenii , c. youngae , kluyvera ascorbata , providencia alcalifaciens and serratia liquefaciens as endophytes . perhaps , this is the first study that reports these bacterial species as endophytes .
however , the benefits derived by nepenthes spp . from these bacterial endophytes and its quantum are not clearly understood yet . from this study
contains diverse types of cultivable eb , and that the majority of bacterial endophytes ( 59.4% ) were from the bacilli class .
nonetheless , these research findings could serve as a foundation in further research on the therapeutic properties of nepenthes spp . in correlation with their bacterial endophytes .
we hypothesize that in nepenthes spp . , these eb might be involved in producing bioactive compounds of pharmaceutical importance , and further research is required to ascertain the same .
 background : nepenthes species are used in traditional medicines to treat various health ailments . however , we do not know which types of endophytic bacteria ( eb ) are associated with nepenthes spp.objective:the objective of this study was to isolate and to identify eb associated with nepenthes spp.materials and methods : surface - sterilized leaf and stem tissues from nine nepenthes spp . 
 collected from peninsular malaysia were used to isolate eb . 
 isolates were identified using the polymerase chain reaction - amplified 16s ribosomal dna ( rdna ) sequence similarity based method.results:cultivable , 96 isolates were analyzed ; and the 16s rdna sequences analysis suggest that diverse bacterial species are associated with nepenthes spp . majority ( 55.2% ) of the isolates were from bacillus genus , and bacillus cereus was the most dominant ( 14.6% ) among isolates.conclusion:nepenthes spp . 
 do harbor a wide array of cultivable endophytic bacteria . 
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION