Introduction

Endophytic fungi are a diverse and polyphyletic group of fungi, capable to colonize plant tissues asymptomatically [1, 2]. These fungi are well known to produce antimicrobial substances and are being investigated as a potential source of secondary metabolites [3,4,5,6]. The secondary metabolites derived from the endophytic fungi constitute a major class of desirable bioactive molecules [7, 8]. Exploitation of novel bioactive metabolites including alkaloids, macrolides, terpenoids, peptide derivatives colletotric acid, griseofulvin, etc. from the endophytic fungi of various habitats is noticeable [9,10,11,12]. The fungal metabolites have exhibited the antimicrobial, antineoplastic, immunosuppressive and cytotoxic activities [13]. Freshwater fungi are a specific group of conidial fungi that are most abundant in almost all running freshwater bodies throughout the world [14,15,16]. These fungi produce peculiar types of conidia, ranges from simple rounded spores to branched, triradiate or tetraradiate [17]. Waid [18] first time isolated these fungi as endophytes from the living roots of some plants, and later, these fungi were recovered worldwide [15, 19,20,21]. Antimicrobial compound ‘Anguillosporal’ from Anguillospora longissima and A. crassa has resulted in the discovery of novel antimicrobial compound, from these fungi [22]. There may be a good scope for novel antimicrobial compounds within these fungi [23,24,25,26,27], but these fungi have not been surveyed extensively as antimicrobial producer, perhaps owing to their ecological peculiarities. Therefore, the present investigation was carried out to evaluate the antagonistic activity of an isolated root endophytic freshwater fungus A. longissima against some pathogenic fungi.

Living root samples were collected from the riparian plants, growing in the ravine area of Nainital, Kumaun Himalaya, India. Collected root samples were processed for the isolation of endophytic freshwater fungi following Singh and Sati [27]. The isolates were identified with the help of relevant monographs and papers [17]. Among the isolates, fungus A. longissima was evaluated for antagonistic activity against eight plant pathogenic fungi, viz. Alternaria alternata, Botrytis cinerea, Colletotrichum falcatum, Fusarium oxysporum, Pyricularia oryzae, Rhizoctonia solani, Sclerotium rolfsi and Tilletia indica. Malt extract agar 2% (MEA, HiMedia) medium was used to culture and maintain the isolated freshwater fungus. Potato dextrose agar (PDA, HiMedia) medium was used to maintain the pathogenic fungi and to assess the antagonistic activity.

Antagonistic activity was assessed through dual culture method, using 5-mm fungal discs, cut from the apical margin of used fungus (as antagonist) and from the pathogenic fungi (as test fungi), and placed in 90-mm Petri plate containing ≈ 25 ml medium [28]. In well diffusion method; one well was filled with 200 µl of fungal crude extract and second well was used for test pathogenic fungus and then incubated at 25 ± 2 °C for 5 days. Fungal crude extract was prepared following Bugni and Ireland [12]. The resulting crude extract was finally dissolved in 2.5 ml of methanol and used as crude extract for antagonistic activity [29]. Observations were recorded periodically following Pandey et al. [28], by measuring the radial growth of test fungi in two directions; R1 (radius in opposite direction) and R2 (radius in direction of the used fungus) and per cent inhibition were calculated following Sati and Arya [30].

$$ {\text{Inhibition }} \% \, = \,R_{1} - R_{2} /R_{1} \times 100 $$

On the basis of morphological analyses, the fungal isolate was identified as A. longissima (Sacc. and P. Syd.) Ingold. Results of the antagonistic activity are summarized in Table 1, showing that the fungus possessed a strong inhibitory potential against the test pathogenic fungi. In dual culture method, maximum inhibition was recorded against pathogenic fungus T. indica (79.76%), followed by P. oryzae (58.21%), while it was found minimum against another test fungus F. oxysporum (35.73%). The growth of test pathogenic fungi was also inhibited to a varied magnitude of inhibition with 37.30% (for S. rolfsi) to 55.55% (for B. cinerea) by the used root endophytic fungus A. longissima (Fig. 1; Table 1). In well diffusion method, the maximum inhibition was recorded against the pathogenic fungus C. falcatum (62.96%), followed by B. cinerea (50.54%), whereas minimum inhibitory fungus was observed, P. oryzae (17.85%). The pathogenic fungi R. solani, S. rolfsi, A. alternata, T. indica and F. oxysporum, were also inhibited (Fig. 2; Table 1).

Table 1 Antagonistic activity of isolated root endophytic freshwater fungus A. longissima
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Fig. 1
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Antifungal activity of isolated root endophytic freshwater fungus A. longissima against test pathogenic fungi (dual culture method). AA—Alternaria alternata, BC—Botrytis cinerea, CF—Colletotrichum falcatum, FO—Fusarium oxysporum, PO—Pyricularia oryzae, RS—Rhizoctonia solani, SR—Sclerotium rolfsi and TI—Tilletia indica

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Fig. 2
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Antifungal activity of isolated root endophytic freshwater fungus A. longissima against test pathogenic fungi (well diffusion method). AA—Alternaria alternata, BC—Botrytis cinerea, CF—Colletotrichum falcatum, FO—Fusarium oxysporum, PO—Pyricularia oryzae, RS—Rhizoctonia solani, SR—Sclerotium rolfsi and TI—Tilletia indica

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Endophytic fungi are a fertile ground of secondary metabolites [31]. The fungicidal activity of metabolites produced by endophytic fungi has been studied [32]. Shearer and Zare-Maivan [24] reported that freshwater fungi are potent inhibitor for their competitive species, and the production of antibiotic substances by these fungi has also been reported [26, 33, 34]. In the present investigation, the fungus A. longissima was found potent to produce antifungal substances that inhibited the growth of test fungi (Figs. 1, 2, 3; Table 1). It is also observed that the living mycelium (as 5-mm discs) and fungal metabolites (as crude extract) worked differently to inhibit the growth of test pathogenic fungi (Table 1). Freshwater fungi are generally slow growing fungi (compared to terrestrial fungi) on agar medium, and it was interesting to note that the used fungus was found capable to inhibit the growth of fast growing fungi (pathogenic fungi), during the dual culture assay that clearly indicates the antagonistic potency of isolated root endophytic fungus, and proved to be a good synthesizer of some antifungal substances those inhibited/suppressed the growth of all test pathogenic fungi. It was also noteworthy that the used fungus was less in biomass (visually) compared to the pathogenic fungi but was found more potent to inhibit the growth of competitive fungi (pathogenic fungi). Bugni and Ireland [12] suggested that endophytes are habitual to produce some antimicrobial substances. The isolated root endophytic fungus A. longissima was also found potent to synthesis some antifungal substances as a strong growth inhibition was also measured in diffusion assay (Fig. 1; Table 1). Zhang et al. [35] have reported the production of broad spectrum antifungal agents by the endophytic fungi.

Fig. 3
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Antagonistic activity of root endophytic aquatic fungus A. longissima against eight pathogenic fungi

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Although freshwater fungi have been studied extensively for their taxonomical studies, studies on antimicrobial potential of these fungi are reported only for few species. The present investigation clearly indicates that freshwater fungi synthesize antifungal substances to inhibit the growth of certain phytopathogens that may also be used for the exploitation of naturally occurring compounds, within these fungi, to compete the synthetic fungicides. Further, screening and characterization of the compounds responsible for the antagonistic activity are required.