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Title:   Author  item
                                            Bock, Cliveitem
                                            Shapiro Ilan, Daviditem
                                            Wedge, Daviditem
                                            Cantrell, Charles
  Submitted to: Journal of Pest Science
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 7/20/2013
                                    Publication  Date: 3/1/2014
                                    Citation: Bock, C.H., Shapiro Ilan, D.I., Wedge, D.E., Cantrell, C.L. 2014. Identification of the antifungal compound, trans-cinnamic acid, produced by Photorhabdus luminescens, a potential biopesticide against pecan scab. Journal of Pest Science. 87(1):155-162.Interpretive Summary: Pecan scab is the major disease limiting productivity and quality of pecan in the southeastern US. Biorational alternatives to conventional fungicides are desirable to minimise environmental risk and the risk of fungicide resistance. Metabolites produced by bacteria from the guts of entomopathogenic nematodes can suppress pecan scab, but the molecules responsible have not been identified. Extracts of the bacteria investigated to identify the major constituent responsible for the activity.  
Antifungal bioassays confirmed toxicity of the fractions to Colletotrichum species. The most fungitoxic fraction was purified and identified as trans-cinnamic acid (TCA) and further tested in-vitro against the pecan scab fungus, F. effusum. Zone of inhibition tests and tests with TCA incorporated into the growth media showed that TCA was toxic to F. effusum at concentrations of 148-200+ and 64+ μg ml-1 in solid and liquid culture, respectivvely. 
Naturally occurring anti-microbial products might offer an alternative to disease control in crops, helping minimize the risk of fungicide resistance, while minimizing any negative impact on the environment. Additional research is warranted to determine the potential to use TCA as a suppressive agent for pecan scab and other diseases.
                                Technical Abstract: Pecan scab (caused by Fusicladium effusum) is the major disease that limits the productivity and quality of pecan in the southeastern US. Alternatives to conventional fungicides are desirable and should be biorational, of low environmental risk with a reduced risk for fungicide resistance developing in the pathogen. Prior research showed that metabolites from the nematode symbiont P. luminescens can suppress pecan scab, but the bioactive molecules had not been identified. Extracts from P. luminescens were chemically investigated using a bioactivity-directed fractionation approach in an effort to identify the constituent(s) responsible for the activity.  
RESULTS: High throughput antifungal bioautography assays against Colletotrichum gloeosporioides, C. fragariae and C. actuatum were used to guide the fractionation.  One of the metabolites was purified and identified as trans-cinnamic acid (TCA) using silica gel chromatography followed by semi-preparative high performance liquid chromatography. In-vitro tests confirmed toxicity of TCA to C. gloeosporioides, C. fragariae and C. actuatum at 10 and 100 μg ml-1using fungal bioautography inhibition screening plates.  The antimycotic activity of TCA was further tested in-vitro against F. effusum. Zone of inhibition tests and tests with TCA incorporated into agar showed that TCA was toxic to F. effusum at concentration 148-200+ μg ml-1. Further tests incorporating TCA into liquid media demonstrated that TCA arrested all growth of F. effusum at a concentration as low as 64 μg ml-1, measured over a period of 21 days. 
CONCLUSION: Naturally occurring anti-microbial products might offer an alternative to disease control in crops, helping minimize the risk of fungicide resistance, while minimizing any negative impact on the environment. Additional research is warranted to determine the potential to use TCA as a suppressive agent for pecan scab and other diseases.


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