Antifungal activity of Artemisia annua endophyte cultures against phytopathogenic fungi

Abstract

Artemisia annua, well recognized for its production of antimalarial drug artemisinin, is seldom attacked by any of phytopathogenic fungi, which could be partially associated with the presence of endophytes. Present investigation is aiming at disclosing whether the endophytes inside A. annua produce antifungal substances. A total of 39 endophytes were isolated and fermented, and the ferment broth was evaluated in vitro for the antifungal activity against crop-threatening fungi Gaeumannomyces graminis var. tritici, Rhizoctonia cerealis, Helminthosporium sativum, Fusarium graminearum, Gerlachia nivalis and Phytophthora capsici. These plant pathogens are still causing wheat take-all, sharp eyespot, common rot, scab, snow mould, and pepper phytophthora blight, respectively. Out of 39 endophytes investigated, 21 can produce in vitro substances that are inhibitory to all or a few of the tested phytopathogens whereas the rest yielded nothing active. Moreover, the most active broth of endophyte IV403 was extracted with EtOAc and n-butanol, and comparisons of the antifungal activity of the extracts indicated that the major active metabolites were EtOAc-extractable.

Introduction

Scientists are trying to devise new means to increase food production with the world population expanding rapidly. Unfortunately, severe cereal loss is still inevitable owing to plant diseases, particularly those caused by phytopathogenic fungi. Application of the synthetic fungicides has been considered to be one of the cheapest and most common approaches for the control. However, these chemicals usually take long timelines to be degraded completely causing heavy toxicity to human being, domestic animals, etc. (Fawcett and Spencer, 1970, Bajaj and Ghosh, 1975, Lingk, 1991). Like human pathogenic micro-organisms, phytopathogens are also prone to developing ‘drug’ resistances to decrease substantially the effectiveness of these pesticides (Rosenberger and Meyer, 1981). Accordingly, there is an urgent need to work towards the invention of safer antifungal agents which are expected to be renewable, non-petrochemical, naturally eco-friendly and easily obtainable.

During long-time evolution, plants, on which insects, micro-organisms and mammals are feeding, usually acquire self-defending capabilities by producing a variety of secondary metabolites such as alkaloids, terpenoids, steroids and aromatic compounds which are presumably unpleasant or even toxic to the ‘enemy’. Inside the tissue of nearly all the healthy plant, there are a lot of micro-organisms called ‘endophytes’. Endophytes are mutualistic to their host, at least some of them are thought to be making returns for the nutrition from the plant by producing special substances such as secondary metabolites to prevent the host from successful attack of fungi, pests and mammals. As a matter of fact, metabolites of endophytes were reported to inhibit a number of micro-organisms (Fisher et al., 1984a, Gurney and Mantle, 1993). To the best of our knowledge, Artemisia annua (belonging to the family Composite), well known for its biosynthesis of artemisinin, was found to be resistant to the common pests and phytopathogenic fungi. Is the resistance associated with the presence of endophytes? Are the endophytes capable of producing antifungal compounds that could be used for controlling the aforementioned crop-attacking fungi? We therefore undertake the present study to get hopefully a part of the answer to the questions.