Introduction

Fungal endophytes, which are a universal and indispensable constituent of a plant microbiome, have a long evolutionary history as plant symbionts (Krings et al. 2007). Foliar fungal endophytes (FFE) which reside inside leaf tissues are ubiquitous and have been isolated from leaves of plants of the Arctic (Zhang and Yao 2015), Antarctic (Rosa et al. 2009), deserts (Suryanarayanan et al. 2005; Massimo et al. 2015), tropical (Arnold et al. 2000; Suryanarayanan et al. 2011), temperate (Matsumura and Fukuda 2013), and mangrove forests (Suryanarayanan et al. 1998; Rajamani et al. 2018). Such a long-time symbiosis which is unrestricted by the geography and taxonomy of the host plants goes to show that endophytism is a successful life strategy among fungi. Recent investigations have revealed many functional roles of FFE in the establishment and performance of plants. They enhance their host plant’s ability to tolerate abiotic (Chitnis et al. 2020) and biotic (Estrada et al. 2013) stressors. Furthermore, it is envisaged that a plant’s response to climate change could be modulated by the endophytes it harbours (Suryanarayanan and Uma Shaanker 2021). Such features of FFE which modify their plant host’s traits have encouraged viewing them as alternatives to genetic modifications and dependence on chemicals to improve crop production (Vega 2018; Chitnis et al. 2020). A few FFE persist in fallen leaves to initiate litter degradation and thus help in nutrient recycling (Prakash et al. 2015; Guerreiro et al. 2017). Furthermore, fungal endophytes are known to produce many industrially important bioactive metabolites (Pimentel et al. 2011). Despite such appealing characteristics of fungal endophytes, few studies address their interactions with plants and co-occurring microbes and within tissue distribution (Suryanarayanan 2013). For instance, although it is well established that certain genera such as Colletotrichum, Guignardia (Phyllosticta), Pestalotiopsis, Diaporthe (Phomopsis), and Xylaria exist as dominant FFE irrespective of their plant hosts’ taxonomic or geographic connectivity (Suryanarayanan et al. 2018), there is no information on fungi present in low frequencies (minor species) inside leaves. The roles of minor species in a community could be disproportionate to their numbers. It is known that minor species of soil bacteria reduce invasion by alien species, protect plants from pathogen invasion, and determine plant’s responses during rhizosphere colonization (Dawson et al. 2017). Among plant communities, minor species influence the abundance of the dominant species (Boeken and Shachak 2006) and functional similarities between dominant and minor species aid in the resilience of ecosystem function under changing environmental conditions (Walker et al. 1999). Hence, we analysed the results obtained from our study on 75 tree species of 32 families growing in three different types of tropical forests to understand the pattern of distribution of minor FFE exhibiting very low frequency of occurrence — a colonization frequency of less than 5% in any tree leaf.

Materials and methods

Collection sites

Twenty-five woody tree species, each group from tropical dry thorn (DT), dry deciduous (DD), and montane evergreen (ME) forests situated in the Nilgiri Biosphere Reserve (Latitude 11°33′0″N, Longitude 76°37′30″E), were sampled for their FFE. These are not primary forests; they receive a mean annual rainfall of 700–1000 mm, 1000–1400 mm, and 1300–3000 mm, respectively (Kodandapani et al. 2009; Suryanarayanan et al. 2011). All collections were made from private lands or unprotected regions and the data accumulated in a 10-year study (from 2000 to 2009) are analysed here.

Sample collection

The most common tree species recorded in each forest type were screened (Table 1). Trees with more than 10-cm diameter at breast height (DBH) were chosen since trees of these forests do not have discernable annual rings for age determination. Three individual trees for each host species were chosen and 20 mature, healthy, symptomless leaves were collected (total 60 leaves/tree species) and screened for endophytes. Leaf sampling was done only during the months of November, December, and January, thus avoiding the dry and monsoon seasons.

Table 1 Tree species and the forest type studied for minor FFE
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Endophyte isolation

From each leaf, three segments (0.5 cm2) were cut from its apical, middle, and basal regions of the lamina and the 180 segments thus obtained from 60 leaves for each host species were surface disinfected by the method of Suryanarayanan et al. (1998). From these, 150 segments were randomly selected and plated on chloramphenicol-amended Potato Dextrose Agar (PDA) medium contained in 9-cm diam. petri dishes. Such petri dishes (10 segments/dish) were incubated in a light chamber (12-h light:12-h dark cycle) for 28 days at 26 ± 1 °C to induce sporulation in the growing endophytes (Suryanarayanan 1992). The surface imprinting method of Schulz et al. (1998) was followed to ensure the effectiveness of the surface disinfection in killing the epiphyllous microbes. The tissue segments were observed periodically and the fungi growing out of them were isolated and identified based on their spore morphology and spore development (Ellis 1976; Subramanian 1971; Sutton 1980; Onions et al. 1981; Ellis and Ellis 1988; Nag Raj 1993; Hyde et al. 2000). Sterile mycelia which differed in their colony structure (colour and growth characteristics in culture) were treated as morphospecies. Foliar fungal endophytes which had a colonization frequency (CF%) of less than 5% were considered minor species for the analysis. In very few cases, genera such as Alternaria, Colletotrichum, Fusarium, Pestalotiopsis, Phomopsis, Phyllosticta, Sporormiella, and Xylaria exhibited < 5 CF%; these were not considered for the analysis since they are universally dominant FFE (Suryanarayanan et al. 2011). Representative endophyte fungal cultures were deposited in the culture collection centres of India (The Microbial Type Culture Collection (MTCC) and National Fungal Culture Collection of India (NFCCI)); these include dry thorn forest, Corynespora sp. (MTCC 8466), Drechslera rostrata (NFCCI 2312, GB), and Lasiodiplodia theobromae (MTCC 10,347); dry deciduous forest, Corynespora sp. (MTCC 8467) and Nigrospora oryzae (MTCC 8465); and montane evergreen forest, Chaetomium sp. (MTCC 8385) and Sordaria fimicola (MTCC 10,342). In a parallel work addressing the technological potential of endophytes, Curvularia sp. and Drechslera rostrata isolated as minor endophytes were identified using their ITS sequences and their GenBank accession numbers are KF135619 and HQ909080, respectively.

Statistical analyses

The colonization frequency (CF%) of each endophyte was calculated by the method of Hata and Futai (1995).

$${\text{CF\% = }}\frac{{\text{Number of segments colonized by each endophyte}}}{{\text{Total number of segments observed}}} \times 100$$

Margalef’s richness index (R1) was used to measure the species richness and Fisher’s α, which is less affected by the abundance of common species (Magurran 2004), was used to estimate the species; R1 = S − 1/ln (n), where S is the total number of species in the community and “n” is the total number of individuals observed. Fisher’s α was calculated by S = a*ln (1 + n/a), where S is the number of taxa, n is the number of individuals, ln is the natural logarithm, and a is the Fisher’s alpha. Using the software BiodiversityPro, a principal component analysis (PCA) was done to detect any difference in the minor endophyte assemblage of trees from different forests.

Qualitative test for antifungal metabolites

Since Lasiodiplodia was the most common among the minor endophytes obtained in the present study, isolates of this fungus were screened for the production of antifungal metabolites by the autobiogram method (Schulz et al. 1995). Each isolate was grown in 200 mL Potato Dextrose broth at 26 °C for 3 weeks. The mycelium was filtered, and the culture filtrate was extracted with methanol and concentrated to 1.5 mL under vacuum. The methanol extract (50 μL) was spotted on a TLC (20 × 20 cm silica gel 60 F254-coated aluminium sheet, Merck, Germany) sheet and was developed in a dichloromethane:methanol (9.6:0.4) solvent system. After drying, the chromatogram was sprayed with a suspension of Cladosporium cucumerinum spores in 2% glucose solution and incubated for 5 days at 26 °C. The appearance of inhibition zone in the growth of the C. cucumerinum on the TLC plates confirmed the production of antifungal secondary metabolite(s). As a control, methanol (50 μL) was run and tested.

Qualitative test for production of biomass degrading enzymes

The production of biomass degrading enzymes such as cellulase, β-glucosidase, laccase, lipase, pectinase, pectate transeliminase, and protease was detected by agar plate assay after growing the fungus on a suitable medium for 5 days (Rohrmann and Molitoris 1992; Thirunavukkarasu et al. 2017). For detecting cellulase activity, the fungus was grown on yeast extract and peptone agar medium containing Na-carboxymethylcellulose (0.5%). Then, the colony was flooded with 0.2% Congo red and destained with 1 M NaCl. Yellow areas around the fungal colony indicated cellulase activity. Laccase activity was visualized by growing the fungus in glucose, yeast extract, and peptone agar medium with 0.05 g α-napthol/L. Appearance of blue colour in the growth medium due to the oxidation of α-naphtol indicated the presence of laccase. For detecting lipase activity, the fungus was grown in peptone agar medium with 1% Tween 20. Clearing or precipitation around the fungal colony indicated lipase activity. For detecting pectinase, the fungus was grown in pectin agar medium (5.0 g pectin, 1.0 g yeast extract) and then the colony was flooded with 1% aqueous solution of hexadecyl trimethyl ammonium bromide. A clear zone that formed around the fungal colony indicated pectinolytic activity. Pectate transeliminase production was seen at pH 7.0 and pectinase activity at pH 5.0 of the medium. For detecting protease activity, the fungus was cultured in glucose yeast peptone agar medium amended with 0.4% gelatin. The growth was then flooded with an aqueous saturated solution of ammonium sulphate and the presence of a clear zone around the fungal colony confirmed the production of the enzyme. β-Glucosidase activity was detected by growing the fungus on yeast extract peptone liquid medium with 0.5% of Na-carboxymethyl cellulose and the culture filtrate was centrifuged and the supernatant was used for assay. One hundred millilitres of 4% agar in 0.2 M sodium acetate buffer (pH 5.0) was autoclaved and maintained at 50 °C. One hundred millilitres of 0.2% esculin (Sigma) was mixed with 6 mL of 1% FeCl3 solution and heated up to 50 °C in a water bath; this was mixed with the agar solution and poured in petri dishes (20 mL per plate). After solidification, four wells of 0.5-cm dia. were cut and 75 µL of a supernatant was poured into each well and incubated at 37 °C for 5 h. The appearance of brown colour around the well indicated enzyme activity (Saqib and Whitney 2006).

Results

We screened 11,250 leaf tissue segments (3750 for each forest type) of the 75 tree species for the presence of endophytes. We recorded 86 species belonging to 51 fungal genera (including sterile morphospecies) as minor endophytes (CF% < 5) from the 3 forests (Fig. 1, Table 2). From the 25 tree species screened from each forest type, we obtained 47, 35, and 51 minor species in DT, DD, and ME forests respectively (Table 2). Twelve fungi including Aspergillus, Cladosporium, Corynespora, Drechslera, Eurotium, Gliocladium, Lasiodiplodia, Nigrospora, Nodulisporium, Paecilomyces, Penicillium, and Phoma were present in the leaves of trees growing in all the forests studied.

Fig. 1
figure 1

Conidia of some of the minor species of endophytes. a Curvularia sp. b Lasiodiplodia theobromae c Nigrospora sp. d Pithomyces sp. Scale bars: a, b = 30 μm, c, d = 15 μm

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Table 2 Minor FFE (< 5% CF) from trees of DT, DD, and ME forests. No. of tree species harbouring an endophyte and its total CF%
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Lasiodiplodia spp. were not host restricted and could be isolated from 32 of the 75 tree species from the 3 different forests; the total CF% of this genus were 22, 22, and 10 in DT, DD, and ME forests, respectively. Aspergillus niger, Cladosporium cladosporioides, Corynespora cassiicola, Drechslera halodes, Eurotium sp., Gliocladium roseum, Lasiodiplodia theobromae, Nigrospora oryzae, Nodulisporium gregarium, Paecilomyces sp. 1, Penicillium sp. 1, and Phoma sp. 1 were present as minor species in all the forests (Table 2). Of these, C. cladosporioides, Lasiodiplodia theobromae, N. oryzae, Paecilomyces sp. 1, and Phoma sp. 1 showed a wider host range and were present in more than 10 different tree species. Some endophytes including Aspergillus sp. 3, sp. 4, and sp. 5, Beltrania rhombica, Chaetomium globosum, C. verrucichaeta, Curvularia eragrostidis, C. pallescens, Diplococcium sp. 1, Ellisiopsis sp. 1, Fusicoccum sp. 1, Gliocladium sp. 1, Graphium penicillioides, Humicola sp. 2, H. grisea, Monodictys levis, Paecilomyces sp. 2, Penicillium sp. 8, Phaeotrichoconis sp. 1, Phoma sp. 2, Pithomyces graminicola, Polynema sp. 1, Rhizopus sp. 1, Sordaria sp. 1, and sp. 2, and Ulocladium botrytis could be isolated from only one of the 75 trees sampled (Table 2).

Margalef’s richness R1 of FFE reduced from 8.7 to 3.6, 7.9 to 3.9, and 9.4 to 3.3 and Fisher’s α reduced from 12.6 to 4.5, 11.2 to 4.9, and 14.5 to 7.2 in DT, DD, and ME, respectively, when the minor FFE were not considered (Table 3).

Table 3 Minor FFE status of dry thorn, dry deciduous, and montane evergreen forests
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Since Lasiodiplodia exhibited a wider host range infecting 32 tree species and also occurred in all three forests, it was taken up for further study. Three isolates (VIG 454, 671, and 113) from three different tree hosts of DT and one isolate (VIG 307) from a tree host of DD forest were screened for enzyme production and antifungal activity (Table 4). Qualitative agar plate assay showed that out of the 4 isolates of this minor endophyte screened, 3 produced cellulase and β-glucosidase enzyme (Table 4); 2 were positive for protease; and 1 produced pectinase, pectate transeliminase, and elaborated laccase. An autobiogram test showed that 2 isolates produce antifungal metabolites (Table 4).

Table 4 Production of biomass degrading enzymes and antifungal compounds by isolates of foliar endophytic Lasiodiplodia spp
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Discussion

Unlike the mycorrhizae, endophytes have not been studied in detail for their roles in the ecosystem (Naranjo-Ortiz and Gabaldón 2019). There are limited studies on the diversity and ecological significance of FFE at the plant community level (Arnold and Lutzoni 2007; Sanchez-Azofeifa et al. 2012). Again, such studies focus on the dominant FFE species (Suryanarayanan et al. 2002, 2011, 2018; Rajamani et al. 2018), and thus information on the diversity and ecological importance of the minor FFE is lacking. Our current preliminary study underscores this gap in knowledge by looking at the diversity of minor FFE of three different types of tropical forests.

It is important for studies quantifying diversity to ensure that the sample size used effectively reflects the diversity. Using species accumulation and unique species curves (Longino 2000; Henderson 2003), we had determined earlier that a sample size of 25 tree species/forest type is sufficient to report the overall species diversity of FFE of these three forests (Suryanarayanan et al. 2011).

Generally, most communities are represented by a few dominant species and many species with low abundance (Walker et al. 1999) — a situation which is also exhibited by the FFE community. A leaf is densely colonized by 15–20 species of FFE such that their infection frequency is high; however, their CF% is skewed due to the dominance of one or two core species (which shows high CF%) (Fig. 2) (Vaz et al. 2018). Our earlier screening of single leaves of eight tree species of DD and 12 of ME confirmed that a similar trend exists in these forests (Suryanarayanan et al. 2011). Invariably, these core species are those of Colletotrichum, Pestalotiopsis, Phomopsis, Phyllosticta, and Xylaria; they also exhibit a wide host range and infect plants irrespective of the geographic and taxonomic distance of the host plants (Suryanarayanan et al. 2018). Probably owing to their dominance, these core endophytic fungi have been the subject of intense screening for their technological potential (Lu et al. 2000; Zou et al. 2000; Okane et al. 2003; Liu et al. 2010; Chapla et al. 2014; Ratnaweera et al. 2014; Yang et al. 2015) and interaction with their plant hosts (Chen et al. 2013; Mejía et al. 2014). In general, such core fungal species directly report any ecological shifts and their increased occurrence reflects increased stability of the system (Unterseher et al. 2011). Foliar fungal endophytes showing very low frequency of colonization are merely recorded in studies and no information is available regarding their host preference and distribution in the leaf tissue, and role in their plant host microbiome or the ecosystem. The present study showed that although their colonization frequency is very low, the occurrence of many different fungi as minor species influenced the FFE diversity. Both species richness and species diversity indices were lower for the endophyte community of the forests when the minor endophyte species were not considered (Table 3).

Fig. 2
figure 2

Representative diagram showing the pattern of occurrence of FFE in a tree species

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We also show that some minor endophytes including Lasiodiplodia have a wide host range, though there are some fungi such as Diplococcium with restricted host range (Slippers and Wingfield 2007). A PCA showed a clustering pattern of the 86 species of 51 genera of minor fungi despite the variations in the elevation, rainfall, and tree host taxonomy among the three forests (Fig. 3). This was suggestive of a broad ecological amplitude of the minor endophytes of these forests.

Fig. 3
figure 3

Principal component analysis for minor FFE

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The present study reveals for the first time that a broad host range may be characteristic of some minor FFE species such as Lasiodiplodia. Currently, twenty-six species of Lasiodiplodia are recognized (Coutinho et al. 2017); many of these cause different diseases including dieback, gummosis, leaf spot, and fruit rot. In the present study, this endophyte was isolated from 19 of the 32 families (32 of the 75 trees) as a minor fungus. It is intriguing that this fungus is not a core species in any plant species studied despite its ability to infect a broad range of plant hosts including trees of the Eastern Amazon (Vaz et al. 2018), Magnolia forests of China (de Silva et al. 2019), mangrove plants of Andaman Islands (Rajamani et al. 2018), and orchids of Northeastern India (Govinda Rajulu et al. 2016). The colonization frequency of L. theobromae as a minor endophyte is not static but increases in Piper betle as the leaf ages (Thirumalai et al. 2021). Thus, it needs to be determined if its limited colonization is due to its low competitive ability or dispersal limitation. The age of the leaf could also affect the CF% of this minor endophyte.

Several bacterial and fungal endophytes including species of Lasiodiplodia (Sakai et al. 2021) produce different types of antibiotics, siderophores, and volatile compounds which could determine the plant’s stable endobiome composition (Schulz et al. 2015; Hassani et al. 2018; González-Teuber et al. 2020; Christian et al. 2020). We observed that endophytic Lasiodiplodia spp. produce antifungal compounds and plant biomass degrading enzymes (Table 4). This fungus is known to occur as a litter degrading saprotroph in shed leaves in the DD forest (Prakash et al. 2015). These characteristics are exemplified by many core endophyte species of these forests (Govinda Rajulu et al. 2013; Prakash et al. 2015). Such functional redundancies with the dominant species of FFE along with their low infection density could render minor FFE unimportant. However, considering the importance of minor species in other communities such as plants, their diversity and influence on host performance need to be studied to appreciate their contribution to functional diversity. Although Unterseher et al. (2011) opine that minor species of ecological groups of fungi including phyllosphere, ectomycorrhiza, and arbuscular mycorrhiza are transient inhabitants and contribute negligibly to ecosystem functions, the situation could be different with FFE as they are known to influence several host traits (Harrison et al. 2021). Lasiodiplodia spp. are known to metabolize plant host defense compounds (Paolinelli-Alfonso et al. 2016) and produce phytohormones suggesting that they could affect their plant host’s physiology (Eng et al. 2016). Furthermore, latent pathogenic forms of this genus surviving as endophytes in plants are triggered to become pathogenic under environmental stress conditions (Paolinelli-Alfonso et al. 2016). Considering such traits, it is likely that in some plants or under certain ecological conditions, a minor FFE could function as a keystone species leading to a cascade of interactions which may ultimately determine the homeostasis of the plant endobiome (Vandenkoornhuyse et al. 2015; Banerjee et al. 2018). Furthermore, the endophyte community status in a leaf is not static but changes with the age of the leaf (Suryanarayanan and Thennarasan 2004) and factors such as dispersal limitation and local extinctions determine the microbial assembly (Peay et al. 2010). Hence, periodic sampling of leaves is essential to fully understand the status of minor FFE.

To sum up, our study using a large sample size suggests indirectly that minor FFE could have a disproportionate influence in selecting, constituting, and functioning of the plant endomicrobiome (Jones et al. 2019). We used the conventional methods for identifying the fungi because of the huge number of isolates obtained. With molecular methods, the actual number of minor foliar endophyte species in these forests could be less or more than that reported here. However, considering the projected roles of fungal endophytes in enhancing their host plants’ tolerance to abiotic and biotic stress (Chitnis et al. 2020) and resilience of plants to climate change (Suryanarayanan and Uma Shaanker 2021), it is essential to know the role played by minor endophyte species in individual plant microbiome and in the ecosystem.