Remember Me
Or use your Academic/Social account:


Or use your Academic/Social account:


You have just completed your registration at OpenAire.

Before you can login to the site, you will need to activate your account. An e-mail will be sent to you with the proper instructions.


Please note that this site is currently undergoing Beta testing.
Any new content you create is not guaranteed to be present to the final version of the site upon release.

Thank you for your patience,
OpenAire Dev Team.

Close This Message


Verify Password:
Verify E-mail:
*All Fields Are Required.
Please Verify You Are Human:
fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Cholerton, Linda Jane
Languages: English
Types: Unknown

Classified by OpenAIRE into

mesheuropmc: food and beverages
Brassica napus is a commercially important crop worldwide and its use is quickly increasing due to its beneficial oil products and biofuel demands. Yield can be lost through infection by a fungal pathogen, Leptosphaeria maculans, the causal agent of stem canker (blackleg). An early indication of the presence of stem canker is a lesion (leaf spot) on the cotyledons or early leaves. The leaf spot stage of the disease was used in this work to ascertain if biological control agents applied both individually and in combination decreased the lesion area and also to quantify the amount of L. maculans DNA present using quantitative polymerase chain reaction (qPCR). \ud The natural production of antibiotics by some bacteria is a commonly found form of antagonistic biological control. Bacillus amyloliquefaciens and Pseudomonas chlororaphis spp. aureofaciens 30.84 evaluated in this work both produce antibiotics and were assayed for their ability to provide control of Leptosphaeria maculans. \ud Known active strains and field isolates of Bacillus and Pseudomonas were tested as potential biocontrol agents in vitro and then used in in planta assays. The in planta assays using bacterial isolates applied individually indicated that all the bacteria gave statistically significant control of L. maculans at the leaf spot stage. Those isolates with highest activity were further evaluated in combination, to determine if improved control of leaf spot occurred. Firstly, however, it was important to confirm the two bacteria would be compatible and antibiotics would be produced. To this aim, an in vitro assay using mutant Chromobacterium violaceum confirmed Pseudomonas chlororaphis spp. aureofaciens upregulated antibiotic production using acyl-homoserine lactones, signalling molecules. Consequently, it was vital that the Bacillus applied with it did not produce lactonase which would denature these molecules. PCR was used to confirm the enzyme was not present. It was, however, shown using in planta assays that combinations of Bacillus and Pseudomonas did not halt the infection or growth of L. maculans, but appeared to lead to increased lesion size. \ud Colonisation of the cotyledons by the bacterial biological control agents applied onto the cotyledons was monitored by washing recovery, serial dilution, plating and colony counting along with qPCR of the DNA. All bacteria colonised successfully when applied individually. However, the populations decreased from the quantity at time zero when they were applied in combination, indicating they were unable to colonise the cotyledons successfully under those circumstances. \ud To quantify Leptosphaeria infection, the concentration of ergosterol, a fungal sterol, was quantified to measure the colonisation of cotyledons. Concentrations were assessed using high performance liquid chromatography (HPLC). This assay was not successful no free ergosterol could be detected. This was probably due to L. maculans either having small amounts of ergosterol in its cell membranes, or having most of the ergosterol esterfied and unsuitable for quantification using this method. \ud Polymerase chain reaction (PCR) was used to ascertain the presence of fungal hyphae within asymptomatic regions of cotyledons. It was found that the fungal DNA was detected within all areas of the cotyledon irrespective of whether the leaf spot could be seen. This result highlights the unreliability of the common method of visually assessing the presence and/or severity of L. maculans infection using leaf spot area. \ud To monitor the populations of bacteria and the fungus in real time, DNA was extracted from the cotyledons and quantified using quantitative PCR (qPCR). The amount of L. maculans DNA isolated decreased when the BCAs were applied individually, and increased when the BCAs were applied in combination (when compared with the amount isolated from the control cotyledons). These results confirmed earlier, non-molecular assessments. \ud To provide a benchmark for biocontrol activity, fungicides used in the control of leaf spot on oilseed rape were tested under the standard experimental conditions. Whilst control was obtained, it was not as effective as when used in the field, probably due to the formulations being optimised for field conditions. Fungicides targeted at wheat pathogens were also tested for control against L. maculans. Field application rates of these fungicides were not successful, as all damaged the epidermis of the cotyledon, resulting in death of the plant. Application of ¼ field rate still induced epidermal damage in all cotyledons except those sprayed with Q8Y78 (now called Refinzar®), where a necrotic lesion could be seen without pycnidia, at day 15 after inoculation.
  • The results below are discovered through our pilot algorithms. Let us know how we are doing!

    • Glare, T., Caradus, J., Gelernter, W., Jackson, T., Keyhani, N., Kohl, J., Marrone, P., Morin, L., and Stewart, A. (2012). Have biopesticides come of age? Trends in Biotechnology 30, 250-258.
    • Griffiths, K.M., Bacic, A., Howlett, B.J. (2003). Sterol composition of mycelia of the plant pathogenic ascomycete Leptospheria maculans. Phytochemistry 62, 147-153.
    • Gudmestad, N.C., Arabiat, S., and Miller, J.S. (2013). Prevalence and impact of SDHI fungicide resistance in Alternaria solani. Plant Disease 97, 952-960.
    • Guetsky, R., Elad, Y., Shtienberg, D., and Dinoor, A. (2002). Establishment, survival and activity of the biocontrol agents Pichia guilermondii and Bacillus mycoides applied as a mixture on strawberry plants. Biocontrol Science and Technology 12, 705-714.
    • Guetsky, R., Shtienberg, D., Elad, Y., Fischer, E., and Dinoor, A. (2002). Improving biological control by combining biocontrol agents each with several mechanisms of disease suppression. Phytopathology 92, 996- 985.
    • Haegi, A., Catalano, V., Luongo, L., Vitale, S., Scotton, M., Ficcadenti, N., and Belisario, A. (2013). A newly developed real-time PCR assay for detection and quantification of Fusarium oxysporum and its use in compatible and incompativble interactions with grafted melon genotypes. Phytopathology 103, 802-810 Hartung, J.S., Pruvost, O.P., Villemot, I., and Alvarez, A. (1996). Rapid and sensitive colorimetric detection of Xanthomonas axonopodis pv. Citri by immunocapture and a nested polymerase chain reaction assay. Phytopathology 86, 95-101.
    • Hammond, K.E., Lewis, B.G., and Musa, T.M. (1985). A systemic pathway in the infection of oilseed rape plants by Leptosphaeria maculans. Plant Pathology 34, 557-565.
    • Hammoudi, O., Salman, M., Abuamsha, R., and Ehlers, R., (2012). Effectiveness of bacterial and fungal isolates to control Phoma lingam on oilseed rape, Brassica napus. American Journal of Plant Sciences 3, 773-779.
    • Handelsman, J., and Stabb, E.V. (1996). Biocontrol of soilborne plant pathogens. Plant Cell 8, 1855-1869.
    • Harman, G.E. (2000). Myths and dogmas of biocontol: Changes in perceptions derived from research on Trichoderma harzianum T-22. Plant Disease 377-393 Loh, J., Pierson, E.A., Pierson, L.S., Stacey, G., and Chatterjee, A. (2002). Quorum sensing in plant-associated bacteria. Current Opinion in Plant Biology 5, 285-290.
    • Lopez-Fernandez, L., Ruiz-Roldan, C., Pareja-Jaime, C., Prieto, Y., Kharaiwesh, A., and Roncero, M.I.G. (2013). The Fusarium oxysporum gnt2 encoding a putative N-acetylglucosamine transferase, is involved in cell wall architecture and virulence. PLoS ONE 8, DOI 10.1371 e84690.
    • López, M.M., Llop, P., Olmos, A., Marco-Noales, E., Cambra, M., and Bertolini, E. (2009). Are molecular tools solving the challenges posed by detection of plant pathogenic bacteria and viruses? Current Issues in Molecular Biology 11, 13-46.
    • Luti, K.J.K., and Yonis, R.W. (2013). Elicitation of Pseudomonas aeruginosa with live and dead microbial cells enhances phenazine production. Romanian Biotechnological Letters 18, 8769-8778.
    • Madacsy, L., Bokor, M., and Kozocsa, G. (1975). Carbenicillin half-life in children with early diabetes mellitus. Pediatric Research 9 (11), 868-868 Maddula, V., Pierson, E.A., and Pierson, L.S. (2004). Phenazines play a role in adhesion/biofilm formation in Pseudomonas aureofaciens strain 30- 84. Phytopathology 94, S64-S65.
    • Maddula, V., Pierson, E.A., and Pierson, L.S. (2008). Altering the ratio of phenazines in Pseudomonas chlororaphis (aureofaciens) strain 30.84: Effects on biofilm formation and pathogen inhibition. Journal of Bacteriology 190, 2759-2766.
    • Maddula, V., Zhang, Z., Pierson, E.A., and Pierson, L.S. (2006). Quorum sensing and phenazines are involved in biofilm formation by Pseudomonas chlororaphis (aureofaciens) strain 30-84. Microbial Ecology 52, 289-301.
    • Mahuku, G.S., Hall, R., and Goodwin, P.H. (1995). Co-infection and induction of systemic acquired resistance by weakly and highly virulent isolates of Leptosphaeria maculans in oilseed rape. Physiological and Molecular Plant Pathology 49, 61-72.
    • Mahuku, G.S., Hall, R., and Goodwin, P.H. (1995). A competitive polymerase chain reaction to quantify DNA of Leptosphaeria maculans during blackleg development in oilseed rape. Molecular Plant-Microbe Interactions 8, 761-767.
    • Maksymiak, M.S., and Hall, A.M., (2000). Biological control of Leptosphaeria maculans (anamorph Phoma lingam) causal agent of blackleg canker on oilseed rape by Cyathus striatus, a birds nest fungus. In: Proceedings of the British Crop Protection Council Conference: Pest and Diseases, Brighton, UK: 13-6 November.
    • Manjukarunambika, K., Ponmurugan, P., and Marimuthu, S. (2013). Efficacy of various fungicides and indigenous biocontrol agents against red root rot disease of tea plants. European Journal of Plant Pathology 137, 67- 78.
    • Marahiel, M.A., Nakano, M.M., and Zuber, P. (1993). Regulation of peptide antibiotic production in Bacillus. Molecular Microbiology 7, 631-636.
    • Marcroft, S.J., Sprague, S.J., Pymer, S.J., Salisbury, P.A., and Howlett, B.J. (2003). Factors affecting production of inoculum of the blackleg fungus (Leptosphaeria maculans) in south-eastern Australia. Australian Journal of Experimental Agriculture 43, 1231-1236.
    • Marra, R., Li, H., Barbetti, M.J., Sivasithamparam, K., Vinale, F., Cavallo, P., and Lorito, M. (2010). Proteomic analysis of the interaction between Brassica napus cv surpass 400 and virulent or avirulent isolates of Leptosphaeria maculans. Journal of Plant Pathology 92, 89-101.
    • Matarese, F., Sarrocco, S., Vannacci, G., Gruber, S., and SeidlSeiboth, V. (2012). Biocontrols of Fusarium head blight interactions between Trichoderma and mycotoxigenic Fusarium. Microbiology 158, 98- 106.
    • Matsuda, K., Tsuji, H., Asahara, T., Kado, Y., and Nomoto, K. (2007). Sensitive quantitative detection of commensal bacteria by rRNA-targeted reverse transcription-PCR (2007). Applied and Environmental Microbiology 73, 6695-6695.
    • Padgett, D.E., and Posey, M.H. (1993). An evaluation of the efficiencies of several ergosterol extraction techniques. Mycological Research 97, 1476- 1480.
    • Padgett, D.E., Mallin, M.A., and Cahoon, B. (2000). Evaluating the use of ergosterol as a bioindicator for assessing fungal response to water quality. Environmental Monitoring and Assessment 65, 547-558.
    • Pal, K.K., and McSpadden Gardener, B. (2011). Biological control of plant pathogens. The Plant Health Instructor 1-25.
    • Pana, J., Huang, T., Yao, F., Huang, Z.P., Powell, C.A., Qiu, S.X., and Guan, X. (2008). Expression and characterization of aiiA gene from Bacillus subtilis BS-1. Microbiological Research 163, 711-716.
    • Panjehkeh, N.. Saberyan, A., Azad, H.A., and Salari, M. (2011). Biological control of Phoma lingam, the causal agent of rapeseed blackleg by Trichoderma and Bacillus subtilis isolates. Iranian Journal of Plant Pathology 47, 3-5.
    • Park, E.Y., Lee, C.H., Bisesi, M., and Lee, J.Y. (2014). Efficiency of peracetic acid in inactivating bacteria, viruses and spores in water determined with ATP bioluminescence, quantitative PCR, and culture based methods. Journal of Water and Health 12, 13-28.
    • Parlange, F., Daverdin, G., Fudal, I., Kuhn, M.L., Balesdent, M.H., Blaise, F., Grezes-Besset, B., and Rouxel, T. (2009). Leptosphaeria maculans avirulence gene AvrLm4-7 confers a dual recognition specificity by the Rlm4 and Rlm7 resistance genes of oilseed rape, and circumvents Rlm4- mediated recognition through a single amino acid change. Molecular Microbiology 71, 851-863.
    • Parsek, M.R., Val, D.L., Hanzelka, B.L., Cronan, J.E., and Greenberg, E.P. (1999). Acyl homoserine-lactone quorum-sensing signal generation. Proceedings of the National Academy of Sciences of the United States of America 96, 4360-4365.
    • Paulitz, T.C., and Belanger, R.R. (2001). Biological control in greenhouse systems. Annual Review of Phytopathology 39, 103-133.
    • Pavlova, V., Cilev, G., Pacinovshi, N., Ristanovic, B., and Petreski, A. (2014). Examination of quality and hygienic correctness of the by-products obtained in manufacturing vegetables and fruits. Journal of Hygienic Engineering and Design 7, 100-106.
    • Pekkonen, M., Ketola, T., and Laakso, J.T. (2013). Resource availability and competition shape the evolution of survival and growth ability in a bacterial community. PLoS ONE 8, DOI: 10.1371.
    • Peng, D.L., Shandong, C., Chang, J., and Zhou, M., (2014). Combined application of Bacillus subtilis NJ-18 with fungicides for control of sharp eyespot of wheat. Biological control 70, 28-34.
  • No related research data.
  • No similar publications.

Share - Bookmark

Cite this article