Biocontrol of Amaranthus retroflexus and Rumes crispus by NLP phytotoxine, a selective bioherbicide

Document Type : Original Article


1 Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran

2 Student of Department of Agronomy, Faculty of Crop Sciences - University of Agricultural Sciences and Natural Resources, Sary, Iran


Non-beneficial and harmful weeds are plants that are unwanted, outside their home farms are growing and have the potential to exceed. This study was done in order to screening fungai and isolating NLP phytotoxine from them for selective biocontrol of Amaranthus retroflexus and Rumes crispus as a dicot, common and chemical herbicide resistance weeds. NLPs are effective just on dicot plants. Contaminated soil and dicotyledons plants were Collected from different regions of Iran. after collecting and culturing them, The effect of Supernatant from fungal cultures, was asseyd by spraying of 5 µl /cm3 of it mid 20 µl tween-20 on leaves of Amaranthus retroflexus,Rumes crispus and wheat as negative control that were cultured in MS media and pots in 3 replications with completely randomized design in laboratory and research green house of baqiatallah university. The effects were assessed according to numbering method. Finally, the QAT5and G7-1strains was selected from 9 top strains, because was more destructive than others on Amaranthus retroflexus and Rumes crispus respectively from necrosis to cell death with number 4 according to numbring method and has non-harmful effect on the wheat (Triticum aestivum). SDS-page results showed phytotoxine that was produced by QAT5 strain was a protein and this from G7-1 was non-protein. For better result on SDS-page protein was concentrated using by ammonium sulfat method, but about G7-1 again this outcomewas repeated. The protein purification of QAT5strain using FPLC showed the presence of a protein with about 24 kDa like other family members of this protein. Considering this fact that these phytotoxines according to the result had similarity features to what founded befor about NLPs, they are recommended as biocontrol factor of these weeds insteade of chemical herbicides.


  1. Bridges, D.C., Crop losses due to weeds in Canada and United States, Weed Science Society of America, Weed Loss Committee, Champaign, IL, 1992, pp. 403.
  2. Thomas, A.G., Wise, R.F., Dew’s Alberta weed survey (1973–1977). Weed Survey Series Publ. 85-3, Agriculture Canada, Regina, SK. 134, 1985.
  3. Thomas, A.G., Wise, R.F., Clayton, G., Port Vermillon area of Alberta weed survey in cereal and oilseed fields. Weed Survey Series Publ. 86-4, Agriculture Canada, Regina, SK. 98, 1986.
  4. Doyon, D., Bouchard, C.J., Néron, R., Répartition géographique et importance dans les cultures de quatre adventices du Québec: Abutilon theophrasti, Amaranthus powellii, Acalypha rhomboideaet Panicum dichotomiflorum, Nat. Can, 1986, vol. 113, pp. 115–123.
  5. Gronwald, J. W., Resistance to photosystem II inhibiting herbicides. Pages 27–60 inS. B. Powles and J. A. M. Holtum, eds. Herbicide resistance in plants: Biology and biochemistry. CRC Press, FL, 1994.
  6. Heap, I., International survey of herbicide resistant weeds, [Online] Available: [July 2003], 2003.
  7. Heap, I. M., The world’s worst herbicide-resistant weeds. Proc, Weed Sci, Soc. Am, 2002, vol.  42, pp. 227 (Abstr.).
  8. Warwick, S.I., Weaver, S.E., Atrazine resistance in Amaranthus retroflexus (redroot pigweed) and A. powellii (green pigweed) from southern Ontario. Can J Plant Sci, 1980, vol. 60, pp. 1485–1488.
  9. Allard, R.W., Genetic systems associated with colonizing ability in predominantly self-pollinating species. In: The Genetics of Colonizing Species (eds HG Baker & GL Stebbins). 49–75. Academic Press, New York, USA, 1965.
  10. Hulten, E., Atlas over vaxternas utbredning i norden. Generalstabens Litografiska Anstalts Fo¨ rlag, Stockholm, Sweden, 1950.
  11. Meusel, H., Jager, E., Weinert, E., Vergleichende Chorologie der Zentraleuropa ischen Flora. Karten Gustav Fischer Verlag, Jena, Germany, 1965
  12. Tonev, Т., Manual of integrated weed control and farming culture. HAI – Plovdiv, 2000.
  13. Pino, J., Biologia i Dinamica de Poblacions de Rumex obtusifolus L. en conreus d,alfals (Medicago sativa L.) a la Plana d,Urgelle. PhD thesis. Universitat de Barcelona, Spain,1995.
  14. Humpreys, J., Jansen, T., Culleton, N., Macnaeidhe, F. and Storey, T., Soil potassium supply and Rumex obtusifolius and Rumex crispus L. abundance in silage and grazed grassland swards. Weed Res, 1999, vol. 39, pp. 1-13.
  15. Benvenuti, S., Macchia, M., Miele, S., Light, temperature and burial depth effect on Rumex obtusifolius seed germination and emergence. Weed Res, 2001, vol. 41, pp. 177-186.
  16. Van Eekeren, N., Jansonius, P., Ridderzuring beheersen; Stand van zaken in onderzoek en praktijk. Louis Bolk Instituut, Driebergen, pp. 45, 2005.
  17. Van Eekeren, N., Fecher, L., Smeding, L., Prins, U., Jansonius, P., Controlling broad-leaved dock (Rumex obtusifolius) in grass clover mixtures. Grassland Sci Eur, 2006, vol. 11, pp. 396-398.
  18. Guzzella, L., Pozzoni, F., Giuliano, G., Herbicides contamination of surficial groundwater in northern Italy. Environ Pollut, 2006, vol. 142, pp. 344-353.
  19. El-Shahawy, T.A., Abd-El Salam, I.S., Investigations into Pesticidal Properties of Certain Fungal Extracts for the Potential Uses in Controlling Weeds and Plant Pathogens. Res J Agr Biol Sci, 2011, vol. 7, pp. 174-181.
  20. Hoagland, R.E., Microbial allelochemicals and pathogens as bioherbicidal agents. Weed Technol, 2001, vol. 15, pp. 835-857.
  21. Najafi, H., Nonchemical weed management methods, Kankash E-Danesh Press. Mashhad, 2007, pp. 198. 
  22. Singh, j., Quereshi, S., Banerjee, N., Pandey, A.K., Production and Extraction of Phytotoxins from Colletotrichum dematium FGCC# 20 Effective against Parthenium hysterophorus L. Brazilian archives of biol and technol, 2010, vol. 53, pp. 669-678.
  23. Li, Y.Q., Sun, Z.L., Zhuang, X.F., Xu, L., Chen, S.F., Research progress of microbial herbicides. Crop Protect, 2003, vol. 22, pp. 247-252.
  24. Staats, M., Baarlen, P.V., Schouten, A., Jan, A.L., Kan, V., Bakker, F.T., Positive selection in phytotoxic protein-encoding genes of Botrytis species. Fungal Genet Biol, 2007, vol. 44, pp. 52–63.
  25. Bottiglieri, A., Zonno, M.C., Vurro, M., I bioerbicidi controle piante infestanti, L’informatore agrario, 2000, vol. 13, pp. 69-73.
  26. Amusa, N.A., Microbially produced phytotoxins and plant disease Management. Afr J Biotechnol, 2006, vol. 5, pp. 405-414.
  27. Bailey, B.A., Jennings, J.C., Anderson, J.D., The 24-kDaa protein from Fusarium oxysporum f. sp. Erythroxyli: occurrence in related fungi and the effect of growth medium on its production. Can J Microbiol, 1997, vol. 43, pp. 45–55.
  28. Jennings, J.C., Apel-Birkhold, P.C., Mock, N.M., Baker, C.J., Anderson, J.D., Bailey, B.A. Induction of defense responses in tobacco by the protein Nep1 from Fusarium oxysporum. Plant Sci, 2001, vol. 16, pp. 891–899.
  29. Feng, B.Z., Li, P.Q., Fu, L., Sun, B.B., Zhang, X.G., Identification of 18 genes   encoding necrosis-inducing proteins from the plant pathogen Phytophthora capsici (Pythiaceae: Oomycetes), Genet Mol Res, 2011, vol. 2, pp. 910-922.
  30. Scheres, B., Benfey, P., Dolan, L., Root Development, CR Somerville, Meyerowitz, The Arabidopsis Book, American Society of Plant Biologists, Rockville, 2001.
  31. Bailey, B.A., Collins, R., Anderson, J.D., Factors influencing the herbi- cidal activity of Nep1, a fungal protein that induces the hypersensitive response in Centaurea maculosa. Weed Sci, 2000, vol. 48, pp. 776–785.
  32. Akbar, M. Javaid, A., Evaluation of herbicidal potential of fungal metabolites against Phalaris minor. Afr J Microbiol Res, 2012, vol. 18, pp. 4053-4057.
  33. Obongoya, B.O., Wagai, S.O., Odhiambo, G., Phytotxic effect of selected crude plant extracts on soil-borne fungi of common bean. Afr Crop Sci J, 2010, vol. 1, pp. 15 – 22.
  34. Bailey, B.A., Purification of a Protein from Culture Filtrates of Fusarium oxysporum that Induces Ethylene and Necrosis in Leaves of Erythroxylum coca, Phytopathology, 1995, vol. 85, pp. 1250-1255.
  35. Jennings, J.C, Bailey, B.A., Anderson, J.D., Induction of ethylene biosynthesis and necrosis in weed leaves by a Fusarium oxysporum protein. Weed Sci, 2000, vol. 48, pp. 7–14.
  36. Towbin, H., Staehelin, T., Gordon, J., Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets, procedure and some applications. Proc Natl Acad Sci U S A, 1979, vol. 76, pp. 4350-4354.
  37. Kang, S.C., Park, S., Lee, D.G., Purification and Characterization of a Novel Chitinase from the Entomopathogenic Fungus Metarhizium anisopliae. J Invertebr Pathol, 1999, vol. 73, pp. 276–281.
  38. Namasivayam, K.S., Aruna, A., Herbicidal Activity of Mycelial and Cell Free Extracts of Fusarium oxysporum F.sp. Ciceris Against Rice Weed Cyperus Iria l. Res J Agric Biol Sci, 2010, vol. 6, pp. 728-731.
  39. Schouten, A., Baarlen,  P.V., Jan, A.L., Blackwell Publishing Ltd Phytotoxic Nep1-like proteins from the necrotrophic fungus Botrytis cinerea associate with membranes and the nucleus of plant cells, Laboratory of Phytopathology, Wageningen University, 2007.
  40. Pernollet, J., Sallantin, C., Salle-Tourne, M.M., Huet, J.C., Elicitin Isoforms from Seven Phytophtora Species: Comparison of their Physic- Chemichal Propertie and Toxicity to Tobacco and other Specie. Plant Pathol J, 1993, vol. 42, pp. 53-67.
  41. Dean, J.F.D., Anderson, J.D., The Ethylene Biosynthesis Inducing Xylanases II. Purification and Physical Characterization of the Enzyme Produced by Trichoderma viride, Plant Phsiol, 1991, vol. 95, pp. 316-323.
  42. Hufran, R.A., Wild, G.E., Sloderbeck, P.E., Description of three isozynte polyntorphisms associated with insecticide resistance in green bug (Homoptera: Aphididae) populations. J Econ Entomol, 1996, vol. 89, pp. 46-50.
  43. Shannag, H.K., Obeidat, W.M.,. Interaction between plant resistance and predation of Aphis fabae (Homoptera: Aphididae) by Coccinella septempunctata (Coleoptera: Coccinellidae), Ann Appl Biol, 2008, vol. 152, pp. 331-337.
  44. Charudattan, R., Biological control of weeds by means plant pathogens, Significance for integrated weed management in modern agro-ecology. Biocontrol, 2001, vol. 46, pp. 229-260.
  45. Gijzen, M., Nurnberger, T., Nep1-like Proteins from Plant Pathogens, Recruitment and      Diversification of the NPP1 Domain Across Taxa. Phytochem, 2006,vol.67, pp. 1800- 1807.