Isolation and StructuralCharacterization of N-(naphthalene-1-yl) Propanamide, a HerbicidalCompound from Streptomyces sp. KA1-3

P. Priyadharsini, D. Dhanasekaran, and B. Kanimozhi

Abstract

Totally 15 actinobacterial isolates were screened for herbicidal activityagainst the weed. Among the 15 isolates, only five isolates KA1-3, KA1-4,KA1-7, KA2-6 and KA23A showed significant activity, particularly KA1-3had shown excellent herbicidal activity against Cyperus rotundus. Theherbicidal effect of Streptomyces sp. KA1-3 culture filtrates on germina-tion and seedling growth of C. rotundus was severely affected whencompared to control. Streptomyces sp. was extracted and purified.Chemical structure of the compound N-(naphthalene-1-yl) propanamidewas established on the basis of spectroscopic studies such as UV, FT-IR,H1NMR and MS. The present study concludes that Streptomyces sp.isolate will be used as bioherbicide against C. rotundus. Further studiesare required to confirm the activity of N-(naphthalene-1-yl) propanamideagainst C. rotundus under field conditions.

Keywords

Streptomyces sp. KA1-3 �Weeds � Herbicidal compound � Purification �Chemical structure

Introduction

Microorganisms being the pioneer colonizer ofthis earth planet, as come to stay, have cosmo-politan conglomerates of highly compatibleorganisms. Microorganisms with its 3.8 billion

year biosynthetic experience remain nature’s bestchemists and treasure house for a variety of novelbiologically active metabolites. The term micro-bial bioprospecting refers to the search of micro-organisms for biologic products or the utilizationof microbial cell as a whole for human benefit andenvironmental applications. In general, themicrobial bioprospecting starts from the collec-tion of environmental samples to the identifica-tion and application of specific bioproducts.

Of the scores of soilborne microorganisms,actinobacteria have been reported to be prolificproducers of a variety of biochemicals. Herbicidalactivity of the compounds excreted by the

P. Priyadharsini � D. Dhanasekaran (&) �B. KanimozhiDepartment of Microbiology, School of LifeSciences, Bharathidasan University, Tiruchirappalli,Tamil Nadu, Indiae-mail: dhansdd@gmail.com

R. K. Velu (ed.), Microbiological Research In Agroecosystem Management,DOI: 10.1007/978-81-322-1087-0_13, � Springer India 2013

187

streptomyces group came into the limelight withthe discovery of bialaphos from S. hygroscopicusand S. viridochromogenes (Ogawa et al. 1973).Anisomycin from Streptomyces sp. is the firstmicrobial product used for the development of thesynthetic herbicide methoxyphenone whichexhibits excellent activity against barnyard grassand crabgrass (Yamada et al. 1974; Ito et al.1974). Herboxidiene from Streptomyces sp. is apotent soil-based herbicide for many weedspecies (Issac et al. 1992). Other promisingphytotoxins from Streptomyces sp. are isoxazole-4-carboxylic acid, nigericin, vulgamycin,ansamitocin, herbimycin and pyridazocidin(Babczinski et al. 1991; Kobinata et al. 1991;Gerwick et al. 1997).

Since weeds are opportunistic species, rapidlymodifying their behavior with a high phenologi-cal plasticity to suit better any newly prevailingconditions, concern is growing among farmersand weed scientists regarding recent develop-ments in weed control technology. There is a greatfear about the future situation and what solutionswill be required, if some species of weeds becomewell adapted to this technology. Omura et al.(1979, 1984) have reported the herbimycin, anti-biotic Streptomyces isolates. We have alreadyreported that the bioherbicidal activity of Strep-tomyces isolates against Ehinochilora crus-galliL, Gynandropsis pentaphylla L, Amaranthusspinosus L, Cyperus rotundus L, Amaranthusviridis L, Cassia occidentalis L and Echinochloaoryzicola L (Dhanasekaran et al. 2012). Thepresent study describes the extraction, purifica-tion and the structural elucidation of the bioactivecompound from a liquid culture of Streptomycessp. KA1-3. The herbicidal activity of the com-pound is also addressed.

Materials and Methods

Collection of Samples

The whole plant seeds of C. occidentalis L andrhizomes of C. rotundus L were collected frompaddy fields in and around Tiruchirappalli.

Seeds of Sesamum indicum L, Eleusine coracanaL and Zea mays L were collected from Tamiln-adu Agricultural College and Research Institute,Tiruchirappalli, Tamilnadu. The collected weedsand crops were identified based on morphologi-cal and taxonomical characteristics of stem, leaf,flower and seeds of the plant (Tadulingam et al.1932).

Actinobacterial Collection

About 15 isolates of Streptomyces sp. KA1-3were collected from Germplasm, Department ofMicrobiology, Bharathidasan University; col-lected isolates were subcultured on starch caseinagar slants and incubated at 28 ± 2 �C for10 days and is used for further analysis.

Production of Herbicidal Compoundsby Submerged Fermentation

All the Streptomyces isolates were inoculated onstarch casein agar plates and incubated at 28 �Cfor 7 days for mass preparation of mycelium.After the growth, Streptomyces sp. myceliumwas scraped and aseptically transferred into50 ml of starch casein broth (starch 1 %; casein0.03 %; calcium carbonate 0.002 %; potassiumnitrate 0.2 %; sodium chloride 0.2 %; magne-sium sulfate 0.005 %; pH 7.0 ± 0.2) prepared in250-ml Erlenmeyer flasks and incubated in arotary shaker at 120 rev/min for 48 h at 28 �C.The broth cultures were transferred to sterilecentrifuge tubes and centrifuged at 10,000 rpmfor 10 min. The supernatants were collected andused as a test sample to screen the herbicidalactivity.

Extraction of Herbicidal Compoundfrom Streptomyces sp.

After fermentation, the production medium wascollected and centrifuged at 10,000 rev/min for30 min at 4 �C to separate the supernatant and

188 P. Priyadharsini et al.

mycelium. Extracellular metabolites present infermentation broth were extracted by liquid–liquid extraction using equal volume of ethylacetate in a separating funnel for 24 h. Then, theethyl acetate portion was kept under reducedpressure for evaporation to obtain the extracel-lular metabolite (Radhakrishnan et al. 2007).

Screening for Herbicidal Activityof Streptomyces sp. KA1-3 by MoistChamber Technique

It was performed by placing a sterile filter papermoistened with 3 ml crude extract in a Petri dish.Fine seeds of Cassia occidentalis L and C. rotun-dus were placed on the each plate. The moistchamber was incubated at 28 �C for 4 days indark and observed for the germination. Similarprocedure was followed for control plate withoutculture filtrate. The experiment was repeated 3times for the isolates that expressed the herbicidalactivity to confirm the observation (Mallik 1997).

Screening of Herbicidal Activityof Streptomyces sp. by Rolled TowelPaper Assay

The rolled paper towel assay was used to evaluatethe efficacy of coating crop and weed seeds withspores of Streptomyces sp. strain to test the growthinhibition of seeds. Streptomyces sp. spore sus-pensions were obtained by flooding 10-day-oldstarch casein agar plate culture with sterile

distilled water. Surface sterilized seeds of cropsand weeds were soaked for 30 min in Strepto-myces sp. spore suspensions and air-dried in alaminar flow hood. The seeds were spread on amoistened sterile 26- to 30-cm paper towel. Thetowel was rolled, placed in a plastic bag andincubated for 5 days at room temperature. Threetowels were used for each treatment, and theexperiment was carried out three times.

Growth and Vigor Index of Seedlings

From each replication, seedlings were selectedfor at random and shoot height and root lengthwere measured. The shoot height was measuredfrom collar to the tip of the long primary leafand root length from the collar to tip of theprimary root, and respective mean valves arecalculated. The vigor index of the seedlings wascalculated by using the formula suggested byAbdualbaki and Anderson (1973).

VI ¼ Root lengthþ Shoot heightð Þ�% of seed germination

Table 1 Screening for herbicidal activity of Strepto-myces sp. KA1-3 by moist chamber technique

Sl.No

Isolates % of seed germination

Cassiaoccidentalis L

Cyperusrotundus L

1 KA1-3 30 25

2 KA1-7 60 33

3 KA2-6 60 33

4 KA2-3A

45 66

5 Control 92 87

Control KA1-3

Control KA1-3

Fig. 1 Screening for herbicidal activity of Streptomycessp. KA1-3 by moist chamber technique

Isolation and Structural Characterization of N-(naphthalene-1-yl) Propanamide 189

Purification of the Compoundby Thin-Layer Chromatography

The herbicidal compound was purified by usingthin-layer chromatography. The compound wasdissolved in 200 ll of ethyl acetate. With the helpof capillary tube, the sample was spotted at thebottom of the silica gel, and then, it was placed inthe developing beaker containing mobile phase.The solvent was allowed to run till it reaches abouthalf a centimeter below the top of the plate. Then,the sheet was kept in a closed chamber to visualizethe separated compounds as clear spots. Rf value

of the spot separated on the TLC plate wasdetermined. Rf value is equal to the movement ofsolute from the origin/movement of the solventfrom the origin.

Herbicidal Effect of N-(naphthalene-1-yl) Propanamide

The compound was reconstituted 0.1 gm in 10 mlwater, and the sample was loaded on Whatman’sNo. 4 filter papers inside 9-cm glass Petri plates.Filter paper was air-dried. Seeds of C. occidentalisand C. rotundus were placed over the moistened

Table 2 Screening of herbicidal activity of Streptomyces sp. KA1-3 by rolled paper towel assay

Sl. No Plants Growth parameters Isolate

KA1-3 Control

1 Cassia occidentalis L % of seed germination 25 100

Root length (cm) 5.7 ± 1.06 9.4 ± 1.20

Shoot length (cm) 7.4 ± 0.84 11.7 ± 0.8

Dry weight (gm) 0.04 ± 0.1 0.05 ± 0.1

Vigor index 327.5 2110

2 Cyperus rotundus L % of seed germination 0 67

Root length(cm) 0 ± 0 5.5 ± 4.9

Shoot length (cm) 0 ± 0 7.7 ± 5.3

Dry weight (gm) 0 ± 0 0.74 ± 4.5

Vigor index 0 884.4

3 Sesamum indicum L % of seed germination 100 75

Root length (cm) 6.5 ± 1.0 5.3 ± 3.7

Shoot length (cm) 4.7 ± 1.4 3.6 ± 2.8

Dry weight (gm) 0.06 ± 0.2 0.03 ± 2.3

Vigor index 1120 667.5

4 Zea mays L % of seed germination 100 100

Root length (cm) 16.3 ± 1.0 14.7 ± 1.3

Shoot length (cm) 15.5 ± 1.2 14.3 ± 1.6

Dry weight gm 0.09 ± 0.6 0.04 ± 0.4

Vigor index 3180 2870

5 Eleusine coracana L % of seed germination 100 100

Root length (cm) 4.5 ± 0.4 3.6 ± 0.3

Shoot length (cm) 4.1 ± 0.5 4.4 ± 0.5

Dry weight (gm) 0.09 ± 0.1 0.05 ± 0.2

Vigor index 800.6 800

190 P. Priyadharsini et al.

filter paper. Similar procedure was followed forcontrol plate with distilled water. The moistchamber was incubated at 28 �C for 7 days in darkand observed for the seed germination.

Structure Elucidation of the Compound

The melting point of the compound was deter-mined on a Buchi-540 melting point apparatus.The UV spectra of the compound were measuredat 200–400 nm using Ultraviolet–visible spec-trophotometer (Shimadzu UV 1601). Infraredspectra of the compound were obtained using aFourier transform infrared spectrometer (BrukerFT-IR instrument equipped with AT-XT Goldengate accessories). The spectra were collectedwithin a scanning range of 400–4,000 cm-1. Thespectra were analyzed for various functionalgroups.

The proton NMR (JEOL GSX-400 MHZ-GSX 500) spectra of the compound wereobtained by using a dimethyl sulfoxide as asolvent and further confirmed by mass spec-troscopy (Finnigan MAT 8230). The structure ofthe compound was established with the help ofspectral data obtained from various spectro-scopic techniques. The 3D structure of thecompound was obtained using ChemDraw soft-ware (Ultra 8.0)

Results

All weed and crop seeds were collected frompaddy fields in Tiruchirappalli district, and fifteenStreptomyces isolates among that five isolatesshowed significant activity and one isolateStreptomyces sp. showed excellent activity whichwere obtained from Germplasm, Department ofMicrobiology, Bharathidasan University. Theherbicidal effect of extracellular metabolites ofStreptomyces sp. was screened for crop seeds andweeds (Table 1 and Fig. 1). One isolate waschosen for further herbicidal activity. TheStreptomyces sp. biomass–treated crop seedssuch as S. indicum, E. coracana, Z. mays andweed seeds of C. occidentalis L and rhizomes of

C. rotundus L were tested for plant growth ger-mination and herbicidal assay. The roll towelmethod was implemented to discover the growthparameter variation in the above-mentioned cropplants and weeds (Table 2 and Fig. 2). The resultof the growth-promoting assay has shown thatthere is no growth variation in normal plants

Sesamum indicum L

Cyperus rotundus L

Cassia occidentalis L

Elusine corocona L

Zea mays L

KA1-3Control

Fig. 2 Screening of herbicidal activity for Streptomycessp. KA1-3 by rolled paper towel assay

Isolation and Structural Characterization of N-(naphthalene-1-yl) Propanamide 191

when compared to the control. But distinctgrowth variation was observed in weeds. Theshoot and root growth of C. rotundus wasseverely affected when compared to control. Thepresent result has clearly indicated that theextracts of Streptomyces sp. culture significantly

inhibit the root and shoot growth in C. rotundusunder in vitro condition.

The solvent system chloroform:methanol(30:70) was found to have good separation withsingle spot. The Rf value of the spot was foundto be 0.678. The compound was readily soluble

Fig. 3 UV spectrum of N-(naphthalene-1-yl) propanamide

Fig. 4 FT-IR spectra of N-(naphthalene-1-yl) propanamide

192 P. Priyadharsini et al.

in water, methyl sulfoxide but insoluble inmethanol, ethanol, chloroform and acetone. Thespectral data UV, FT-IR, H1NMR and MSobtained for the compound were used to estab-lish the structure of the compound UV-VisFigs. 3 and 4 (kmax 382; FT-IR cm-1 3406.67(NH), 2914, 1645, 1406 (C = O)); H1NMRFig. 5 (DMSO 500 MHz); 8.5 (1H, S, NH),7.6–7.7 (3H, m, Arh), 7.6–7.5 (1H, Arh), 7.5–7.4(2H, Arh), 7.4–7.2 (1H, Arh) and 2.6–2.7 (2H, q,CH2), 1.3–1.4 (3H, t, CH3). Based on thespectral data, the structure of the compoundextracted from Streptomyces sp. was identifiedas N (naphthalene-1-yl) propanamide, and themolecular formula was determined as C13H13NOand molecular weight obtained by MS as 199(Figs. 6, 7 and Table 3).

Discussion

The biochemical analysis and allelopathicactivity of the Streptomyces sp. revealed that thecompound was potential to reduce 80 % inhi-bition of C. rotundus. These findings are similar

to the findings of Bae et al. (1993) and parallel tothat of Dhanasekaran et al. (2012). It has beenanalyzed and reported that the potential actino-bacterial isolates inhibit the germination, androot and shoot growth of C. rotundus. Batainehand Hameed (2008) had reported that the culturefiltrate of Streptomyces caused significantphytotoxic effect against Amaranthus retroflexusseeds indicated by complete inhibition for seedgermination, when applied in 1:1 dilution withsterilized distilled water. Kadir and Charudattan(2000) determined that Dactylaria higginsii hadpotential as a bioherbicide agent for purplenutsedge. D. higginsii resulted in significantreductions in shoot numbers (72 %), shoot dryweight (73 %) and tuber dry weight (67 %).

Finally, the present study identifies that thepotential herbicidal compound extracted fromStreptomyces sp. can pave way for furtheranalysis of the compounds to discover themechanism and mode of actions. The discoveryof novel bioherbicides with new chemicalstructures and new site of actions suggests thepossibility of a genetically manipulating ofmicroorganism to yield more utilizing. It is clear

Fig. 5 H1NMR (DMSO, 500 MHZ) of N-(naphthalene-1-yl) propanamide

Isolation and Structural Characterization of N-(naphthalene-1-yl) Propanamide 193

Fig. 6 Mass spectra for N-(naphthalene-1-yl) propanamide

NH

O

CH3

Fig. 7 3D and 2D structure of N-(naphthalene-1-yl)propanamide

Table 3 Herbicidal effect of the N-(naphthalene-1-yl)propanamide

Sl.No

Isolate % of seed germination inhibition

Cassiaoccidentalis L(%)

Cyperusrotundus L (%)

1 KA1-3 80 80

2 Control 100 100

194 P. Priyadharsini et al.

that the mechanisms of plant growth promotionand biocontrol are not mutually exclusive butinterdependent processes. Many of the proper-ties exhibited by actinobacteria are also thoseexhibited by effective biocontrol agents, whichmake their use in agriculture as an attractivealternative to agrochemicals.

Conclusion

From this work, it is finally concluded with theinhibitory effect of Streptomyces sp. KA1-3against the weeds by its compound. This studywill lead and support to produce bioherbicidesfor weed inhibition, growth and improvement,yield of economically important crops.

Acknowledgments The author wishes to acknowledgethe support of UGC-Rajiv Gandhi National Fellowship.

References

Abdualbaki AA, Anderson JD (1973) Vigour indexdetermination of soy bean seed by multiple criteria.Crop Sci 13:630–633

Babczinski P, Dorgerloh M, Lobberding A, Santel HJ,Schmidt RR, Schmitt P, Wunsche C (1991) Herbi-cidal activity and mode of action of vulgamycin.Pestic Sci 33:439

Bae YS, Ehoi YC, Lee JK, Zee JO, Park YS (1993) Aplant growth-inhibitory substance produced by Strep-tomyces sp. J Agri Sci 349–352

Bataineh MB, Hameed M (2008) Screening for soilStreptomyces from north Jordan that can produceherbicidal compounds. Pol J mic 57:297–305

Dhanasekaran D, Ambika K, Thajuddin N, PannerselvamA (2012) Allelopathic effect of actinobacterial iso-lates against selected weeds. Arch phytopath andplant pro 45(5):505–521

Gerwick BC, Fields SS, Chapin EL, Cleveland JA, HeimDR (1997) Pyridazocidin, a new microbial phytotoxinwith activity in Mehler’s reaction. Weed Sci45:654–657

Issac MB, Ayer SW, Elliot RC, Stonard RJ (1992)Herboxidine: a potent phytotoxic polyketide fromStreptomyces sp. A7847. J Org Chem 57:7220–7226

Ito K, Futatsuya F, Hibi K, Ishida S, Yamada O,Munakata K (1974) Herbicidal activity of 3,30-dimethyl-4-methoxybenzophenone(NK-049) in paddyfields. I. Herbicidal characteristics of NK-049 onweeds. Weed Sci 18:10

Kadir J, Charudattan R (2000) Dactylaria higginsii aFungal Bioherbicide Agent for Purple Nutsedge(Cyperus rotundus). Biol Cont 17:113–124

Kobinata K, Seikido S, Uramoto M, Ubukata M, OsadaH, Yamaguchi I, Isono K (1991) Isoxazole-4-carbox-ylic acid as a metabolite of Streptomyces sp. and itsherbicidal activity. Agric Biol Chem 55:1415

Mallik MB (1997) Isolates of soil actinomycetes withpotential for phytotoxin production. J Chem Ecol23:2683–2693

Ogawa Y, Yoshida H, Inouye S, Niida T (1973) Studieson a new antibiotic SF-1293. III Synthesis of a newphosphorus containing amino acid, a component ofantibiotic SF-1293. Meiji Seika Kenkyu Nempo13:49

Omura S, Iwai Y, Takahashi Y, Sadakane N, NakagawaA, Oiwa H, Hasegawa Y, Ikai T (1979) Herbimycin, aantibiotic produced by a strain of Streptomyces.J Antibiot 32:255–261

Omura S, Murata M, Hanaki H, Hinotozawa K, Oiwa R,Tanaka H (1984) Phosalacine, a new herbicidalantibiotic containing phosphinothricin: fermentation,isolation biological activity and mechanism of action.J Antibiot 32:829–835

Radhakrishnan M, Balaji S, Balagurunathan R (2007)Thermotolerant actinomycetes from the Himalayanmountain-antagonistic potential, characterization andidentification of selected strains. Malays Appl Biol36(1):59–65

Tadulingam C, Venkatanarayanan G, Anstead RD (1932)A handbook of some South Indian weeds. MadrasGovernment Press, Chennai

Yamada O, Kaise Y, Futatsunga F, Ishida S, Ito K,Yamamato H, Munakata K (1974) Studies on theplant growth-regulating activities of anisomycin andtoyocamycin. Agric Biol Chem 36:2013–2015

Isolation and Structural Characterization of N-(naphthalene-1-yl) Propanamide 195