Evaluation of Alternaria alternata Isolates for Metabolite Production Isolated from Different Sites of Varanasi, India

Introduction

Phytotoxins and mycotoxins produced by fungal plant pathogens are generally low molecular weight secondary metabolites that exert toxic effects on host plants and animals, respectively. A widespread distribution of Alternaria fungi, belonging to the Dematiaceae of the Hyphomycetes in the Fungi Imperfecti, is found in nature. Alternaria exhibits all kind of life forms found in fungi; they act as plant pathogens, weak facultative parasites, saprophytes and endophytes [1]. Alternaria are known to produce several metabolites which are toxic to plants and animals, and are designated as phytotoxins and mycotoxins, respectively [2, 3, 4]. The metabolites produced by Alternaria exhibit a wide variety of biological activities ranging from phytotoxic, cytotoxic, and antimicrobial activities. Owing to possess such diverse properties, the metabolites of Alternaria have drawn the attention of many chemists, pharmacologists, and plant pathologists to consider them as tools in research programs as well as in application studies [5, 6].

Members of the genus Alternaria are known to produce a wide range of phytotoxic secondary metabolites which affect a large number of plants on which the fungus is found [7]. These phytotoxins include alternariol, alternariol monomethyl ether (AME), altenuene, altenuic acid, tenuazonic acid (TA), tentoxin, alternaric acid, AK- toxin and AAL-toxin and posses a broad range of biological and metabolic effects [8]. Sometimes these phytotoxic metabolites have been referred to as ‘host- specific’ because they are toxic only to the host that is susceptible to the pathogen which produces the toxic metabolite and if they induce nearly all symptoms of the disease are considered to be definitive chemical probes in the study of disease susceptibility and physiological stress at the molecular level. Alternaria alternata is a ubiquitous fungus and one of the most frequently occurring species of genus Alternaria which is of particular interest because it produces a number of harmful secondary metabolites. Some of the secondary metabolites produced by Alternaria alternata include alternariol, alternariol monomethyl ether, altenuene, tentoxin, tenuazonic acid and many more. The

metabolites alternariol and alternariol monomethyl ether have been found in sorghum, sunflower seeds, barley, wheat, oats, tomatoes, mandarin oranges, pepper and melons. The pathogen produces brown to black spots on leaves and stems that may coalesce into larger lesions. Affected leaves may turn yellow then drop, leaving the fruit exposed to sunburn. In severe infection of pathogen on tomato, their symptoms of disease appear on the stems and fruits as freckles, spots or lesions. Alternaria alternata has also been reported to be a major causal agent for post harvest loss of crops, fruits and vegetables [9]. The present study was taken with an objective to isolate several isolates of Alternaria alternata from different vegetables being cultivated in different field locations of Varanasi and screen them for production of metabolite(s).

Discussion

Members belonging to the genus Alternaria are amongst the common fungi found in soil and are agents for spoilage of commercially important crops [1]. Alternaria generally attacks the aerial parts of its host. In the leafy vegetables, symptoms of Alternaria infection typically start as a small, circular, dark spot. As the disease progresses, the circular spots may grow to ½ inch or more in diameter and are usually brown to black in colour. In the present work isolation was carried out on potato dextrose agar medium from infected tissues collected from the field. The fungus Alternaria alternata was consistently isolated and identified based on morphological characters [15]. Alternaria is a cosmopolitan fungal genus widely distributed in soil and organic matter. It includes saprophytic, endophytic and pathogenic species. At least 268 metabolites from Alternaria fungi have been reported in the past few decades. The metabolites from Alternaria fungi can be grouped into several categories which include nitrogen-containing compounds, steroids, terpenoids, pyranones (pyrones), quinones, phenolics, etc.

Several metabolites are unique to one Alternaria species, but most metabolites are produced by more than one species [9]. For the detection and quantification of the secondary metabolites produced by Alternaria alternata in broth culture TLC and HPLC studies have been very widely and efficiently used [14, 16, 17]. In the present study also the identification of the metabolites revealed that the isolates produced several metabolites in the culture filtrate as was evident from TLC analysis and the same was supported by HPLC chromatograms. Alternaria metabolites act as phytotoxins to plants or as mycotoxins to humans and animals. They have been examined to have a variety of biological activities and functions, which mainly include the effects on plants, cytotoxic and antimicrobial activities. Plant pathogenic Alternaria species can affect cereals, vegetables and fruit crops in the field and during storage. Alternaria fungi contamination is responsible for some of the world’s most devastating plant diseases, causing serious reduction of crop yields and considerable economic losses. The metabolites from plant pathogenic fungi are usually toxic to plants and are called phytotoxins. They are further divided into host-specific and host non-specific toxins. The host-specific toxins (HSTs) are toxic only to host plants of the fungus that produces the toxin [18, 6]. Another definition seems to be more acceptable that the host-specific toxins are toxic to plants that host the pathogen, but have lower phytotoxicity on non-host plants [19, 20]. Most HSTs are considered to be pathogenicity factors, which the fungi producing them require to invade tissue and induce disease [21]. All isolates of the pathogen that produce HST are pathogenic to the specific host. All isolates that fail to produce HSTs lose pathogenicity to the host plants. Plants that are susceptible to the pathogen are sensitive to the toxin. Host non-specific Alternaria phytotoxins can affect many plants regardless of whether they are a host or non- host of the pathogen [18, 6]. Host non-specific nitrogen- containing phytotoxins include tenuazonic acid, porritoxin and tentoxin. Tentoxin, a cyclic tetrapeptide from A. alternata, inhibited chloroplast development, which phenotypically manifests itself as chlorotic tissue [22]. Tentoxin was suggested to exert its effect on chlorophyll accumulation through over energization of thylakoids [23]. Tenuazonic acid was investigated in Chlamydomonas reinhardtii thylakoids which revealed that TA inhibited photophosphorylation with the action site at QB level [24]. Host non-specific pyranone phytotoxins include radicinin, deoxyradicinin, alternaric acid, alternuisol, altertenuol, dehydroaltenusin, alternariol, alternariol 9-methyl ether, and alternuene. They are very common non-specific phytotoxic metabolites of Alternaria species [9]. The potential applications of Alternaria metabolites as antitumor agents, herbicides, and antimicrobials as well as other promising bioactivities have led to considerable interest within the pharmaceutical community. Chemical syntheses have been achieved for a few bioactive metabolites such as AAL-toxin TA1, maculosin, AM-toxin I, alternariol, alternariol 9-methyl ether, altenuene, isoaltenuene, neoaltenuene, altertoxin III, zinniol, altenusin and alterlactone. In recent years, more and more Alternaria fungi have been isolated as plant endophytic fungi from which large amounts of bioactive compounds have been structurally characterized. Another approach is to discover novel bioactive compounds from the Alternaria fungi isolated from marine sources. These Alternaria fungi could be the rich sources of biologically active compounds that are indispensable for medicinal and agricultural applications [25].

Conclusions

In the present study 12 different isolates of A. alternata were obtained from different locations of Varanasi infecting different plant types. After preliminary screening through TLC and HPLC all the isolates were found to be producers of certain number of metabolites in the culture media. Now, these metabolites need further biophysical and biochemical characterization to warrant their applicability in different sectors.

Acknowledgement

The author MM is thankful to RGNF-(UGC) for providing the necessary facilities during the course of this study. The author is thankful to the Head Department of Botany and Programme Coordinator, Centre of Advanced Study in Botany, Banaras Hindu University for providing necessary research facilities for the experiments.