Symbiosis (2011) 55:39–46 DOI 10.1007/s13199-011-0142-2
Assessment of diversity, distribution and antibacterial activity of endophytic fungi isolated from a medicinal plant Adenocalymma alliaceum Miers Ravindra N. Kharwar & Satish K. Verma & Ashish Mishra & Surendra K. Gond & Vijay K. Sharma & Talat Afreen & Anuj Kumar
Received: 7 July 2011 / Accepted: 19 November 2011 / Published online: 6 December 2011 # Springer Science+Business Media B.V. 2011
Abstract A study was conducted for isolation, identification and antibacterial potential of fungal endophytes of Adenocalymma alliaceum Miers., (Bignoniaceae), a medicinal shrub vine plant which has long history for its usages in curing various disorders. A total of 149 isolates of endophytic fungi representing 17 fungal taxa were obtained from 270 segments (90 from each stem, leaf and petiole) of this plant. Hyphomycetes (77.85%) were the most prevalent, followed by Ascomycetes (8.05%) and Coelomycetes (4.03%) respectively. A considerable amount of fungal isolates was kept under (10.07%) Mycelia-Sterilia (MS). Leaf harboured maximum colonization of endophytic fungi (72.22%) which was greater than stem (67.78%) and petiole (25.54%). The Jc similarity index was maximum (0.619) between stem vs leaf followed by leaf vs petiole (0.571) and stem vs petiole (0.428). The dominant endophytic fungi were Alternaria alternata, Aspergillus niger, Stenella agalis, Fusarium oxysporum, Curvularia lunata and Fusarium roseum. Among twelve endophytic fungi tested for antibacterial activity, crude extracts of nine endophytic fungi (75%), showed antibacterial potential against one or more clinical human pathogens. Alternaria alternata, Curvularia lunata, Penicillium sp. and Chaetomium globosum exhibited significant antibacterial activity against 4 of 5 R. N. Kharwar (*) : S. K. Verma : A. Mishra : S. K. Gond : V. K. Sharma : T. Afreen Mycopathology and Microbial Technology Laboratory, Department of Botany, Banaras Hindu University, Varanasi 221005, India e-mail:
[email protected] A. Kumar Department of Botany, Buddha P. G. College, Kushinagar, UP, India
tested pathogens, showing broad spectrum activity. This investigation explains the value of sampling from different tissues of a host plant for the greater species diversity, and additionally, the antibacterial screening of some endophytic fungi from this specific medicinal plant may represent a unique source for many of the useful antibacterial compounds. Keywords Antibacterial . Adenocalymma alliaceum . Colonization frequency . Diversity . Endophytic fungi
1 Introduction Endophytes are all those microorganisms that colonise and cause asymptomatic infections in healthy plant tissues (Wilson 1995), and provide an effective protection to the concerned hosts against array of biotic and abiotic stresses (Omacini et al. 2001 and Redman et al. 2002). Endophytes in general, are very important and viable components of microbial biodiversity. Fungal endophytes are ubiquitous among terrestrial plants (Petrini et al. 1982 and Carroll 1988), having been reported in algae (Hawksworth 1987), lichen (Petrini et al. 1990 and Li et al. 2007), mosses (Schulz et al. 1993), conifers (Carroll and Carroll 1978), and angiosperm (Clay 1988; Rodrigues 1994; Hyde et al. 1997; Verma et al. 2007 and Gond et al. 2007). The fungi are considered hyperdiverse, as 1.5 million of species have been estimated (Hawksworth 2001) and only 6–7% of this has been described so far while, rests are still awaiting their chance to be introduced to existing microbial world. Temperate plants are maximally studied for endophytic fungi, thus the fungal endophytes of the tropical plants need to be explored to describe for the remaining 93% of mycoflora (Murali et al. 2007).
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Until recently, more than 8600 biologically active compounds have been reported from fungi with various usages (Bérdy 2005). In some cases, plant-associated fungi are able to make the same bioactive compounds as the host plant itself and the discovery of gibberellins in Fusarium fujikuroi and taxol from endophytic fungi, associated with Taxus brevifolia prove this assumption (Strobel and Daisy 2003). Taxol itself is the world’s first billion dollar anticancer drug and its main source is Taxus spp. Potentially, a fungal source of taxol would reduce its price and save the plant from extinction in some areas. Additionally, endophytes have been identified as an outstanding source of novel bioactive natural products like antibacterial, antifungal, anticancer, antimalarial, antioxidant, antiviral and immunosuppressive compounds (Verma et al. 2009), because many of them occupying millions of unique biological niches (higher plants) growing in so many unusual environments (Strobel et al. 2004). Since medicinal value of plants, their unusual longevity or the plant survives under insulted conditions, often harbour potential fungal endophytes that produce bioactive metabolites (Strobel et al. 2004). The present study was carried out on Adenocalymma alliaceum (Bignoniaceae), a highly medicinal, evergreen tropical shrubby vine plant that is native to the Amazon rainforest. This plant is known as ‘garlic creeper or lahsun lata’ as its leaves give strong garlic smell and flavour when they are crushed. In case of unavailability of garlic, leaf of this plant could be used as a substitute. Almost parts like root, leaf, bark and flower of the plant are well used by the indigenous people of the Amazon as medicine for curing various disorders. Despite having several compounds, it is considered analgesic, anti-inflammatory, depurative, purgative and widely used against arthritis, rheumatism, body aches, muscles pain, cholesterol and injuries (Dugasani et al. 2009). Leaves of A. alliaceum are also a common remedy for colds, flu, pneumonia, coughs, fever and headaches. Aaqueous extract of A. alliaceum had shown a good range of fungitoxic activity against fungi associated with deterioration of food commodities and herbal drugs with antiaflatoxigenic activity (Shukla et al. 2007). Flower of A. alliaceum possesses an effective antioxidant property (Scogin 1980). Only a few reports are available on isolation and diversity of endophytic mycoflora from Indian medicinal plants such as Azadirachta indica, Aegle mormelos, Eucalyptus citriodora, Terminalia arjuna, Adhatoda zeylanica, Bauhinia phoenicea, Callicarpa tomentosa, Clerodendron serratum, Labelia nicotinifolia and Crataeva magna (Nalini et al. 2005; Raviraja 2005; Tejesvi et al. 2005; Verma et al. 2007; Gond et al. 2007; Kharwar et al. 2010). Moreover, a considerable number of antimicrobial compounds have been isolated from endophytic fungi
R.N. Kharwar et al.
(Gunatilaka 2006 and Verma et al. 2009). As A. alliaceum is well documented for its various medicinal attributes, it was selected as a source plant to examine the population of endophytic fungi with their antibacterial potential. Thus, the aims of this study were to isolate and characterize the maximum number of isolates in selected plants to observe the diversity of fungal endophytes and their inhibitory activity to bacterial pathogens. Since, this is the maiden report of endophytic mycoflora of A. alliaceum with their antibacterial activity, thus we hope these initial steps of product discovery point out the way for future approaches to be taken by examining medicinal plants for their endophytes and then systematically studying product isolation and characterization of the bioactive compounds.
2 Materials and methods 2.1 Samples collection Healthy and asymptomatic stems, leaves and petioles of A. alliaceum were collected randomly from the campus of Banaras Hindu University, Varanasi during June–December 2009. Sampling was done on three plants of A. alliaceum in triplicate for each plant parts (stem, leaf and petiole) in this experiment. Nine samples were collected from each plant, three each from stems, leaves and petioles. All samples were immediately brought to the laboratory in an icebox, and stored at 4°C in refrigerator. Each sample tissues were used within 48 h from collection. Finally, 90 segments from each tissue part i.e. stem, leaf, and petiole were plotted for the isolation of endophytic fungi. 2.2 Surface sterilisation, isolation and identification All the samples were washed properly in running tap water followed by double distilled water before processing. To eliminate epiphytic microorganisms, all the samples were initially surface sterilised by using methodology adopted by Petrini et al. 1992. The samples were immersed in 70% ethanol for 1–2 min and then sterilized with aqueous sodium hypochlorite (4% available chlorine) for 2–5 min and then rinsed in 70% ethanol for 30 s. The tissues were then rinsed 3 times in sterile double distilled water and allowed to surface drying in sterile conditions. The samples were cut into small pieces using a sterile pinch cutter. Stems were cut into 0.25 cm thick section, leaf samples were cut into 0.5×0.5 cm square and petiole into 0.5 cm in length segments. Four to six segments of plant tissues were placed on each potato dextrose agar (PDA) Petri plate amended with streptomycin (150 μg/ml). A total of 270 segments (90
Assessment of diversity, distribution and antibacterial activity
segments of each plant parts stem, leaf and petiole) were plotted to observe the emergence of endophytic fungi. The plotted Petri plates were sealed with Parafilm™ to avoid contamination and incubated in a biochemical oxygen demand (BOD) incubator with humidity (Model Calton super deluxe, Narang Scientific Works, New Delhi) for 21 days at 12-h light/dark cycle at 26±2°C. The plates were observed regularly at two days interval, and hyphal tips of actively growing fungi were then transferred to fresh PDA plate. The endophytic fungi were identified according to their macroscopic and microscopic characteristics such as colony colour/and morphology, fruiting structures and spores morphology. Standard taxonomic manuals were used to identify the fungal genera and species (Ainsworth et al. 1973; Ellis 1976; von Arx 1978; and Barnett and Hunter 1998). All isolated and identified endophytic fungi were maintained in cryovials layered with mineral oil at room temperature and also in lyophilized form at −20°C in deep freezer (Blue Star). All the samples were deposited in the Department of Botany, Banaras Hindu University, India.
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of Medical Sciences (IMS) B.H.U. Varanasi. Endophytic isolates were cultivated in 500 ml Erlenmeyer flasks containing 200 ml Potato Dextrose Broth (PDB) and incubated for 25 days at 26±2°C under static condition in B.O.D. incubator. The broth of each flask was filtered through Whatman # 1 filter paper, extracted with 200 ml ethyl acetate thrice (100 ml each time). The ethyl acetate extract of each fungal isolate was then evaporated to dryness in vacuum rotary evaporator (IKA RV 10, Germany) and weighed. The crude extract was dissolved in methanol. The plain filter paper discs (5 mm dia) were impregnated with 10 μl of methanolic extract containing 5.0 mg crude extract.The paper discs impregnated with crude extract and dried in laminar hood were placed on the surface of the Mueller-Hinton medium already seeded with test bacterium in Petri Plate. One control disc impregnated with only 10 μl of methanol was also placed for each test bacterium. The plates were incubated at 37±2°C for 24 h and measured inhibition zones. Each test was done in three replicates and interpretation was based on average value of results.
2.3 Statistical analysis of data 3 Results and discussion The colonization frequency (%CF) of endophytic fungi was calculated using the formula given by Hata and Futai (1995).%CF 0 Ncol/Nt ×100 where, Ncol 0 Number of segments colonized by each fungus, Nt 0 Total number of segments studied. Simpson’s and Shannon–Wiener Diversity indices were calculated as per formula given below; Simpson’s Diversity ¼ 1
X
ðpiÞ2 ;
Shannon–Wiener diversity (H) 0 −∑ s (pi log pi), where pi 0 proportion of frequency of colonization of the ith species in a sample. Species evenness was calculated as follows EvennessðEÞ ¼ H=logðSÞ. Where S 0 species richness i.e. total number of species. Jaccard’s coefficient (Jc) of similarity was calculated using formula, Jc ¼ a=ða þ b þ cÞ , where a 0 No. of species occurring in both samples, b 0 No. of species restricted to sample 1, and c 0 No. of species restricted to sample 2.
3.1 Fungal diversity A total of 149 isolates belonging to 14 well identified taxa and 3 Mycelia-Sterilia (MS) of endophytic fungi were obtained from 270 segments (90 from each stem, leaf and petiole). All three tissues showed the variation in colonization frequency of endophytic mycoflora. Among the tissues sampled, maximum colonization frequency was found in leaf (72.22%) followed by stem (67.78%) and least in petiole with 25.54% (Fig. 1). Among identified taxa, Alternaria alternata, Aspergillus
25.54%
67.78%
Stem Leaf Petiole
2.4 Antimicrobial assay 72.22%
Twelve endophytic fungi were tested for antimicrobial activity against potentially pathogenic bacteria Shigella flexnii (IMS/GN1), Salmonella enteritidis (IMS/GN3), S. paratyphi (IMS/GN4), Pseudomonas aeruginosa (ATCC278553) and Morganella morganii (IMS/GN6) taken from Institutes
Fig. 1 Colonization percentage of endophytic fungi to different parts of Adenocalymma alliaceum
42 Table 1 Endophytic fungi isolated from different tissues of Adenocalymma alliaceum
R.N. Kharwar et al.
Sr. No.
Endophytic fungi
% CF of endophytic fungi Stem
Leaf
Petiole
Total
1 2 3 4 5 6 7 8 9 10
Alternaria alternata Alternaria sp. Aspergillus niger Aspergillus fumigatus Curvularia lunata Fusarium oxysporum Fusarium roseum Penicillium sp. Trichoderma sp. Stenella agalis
7.78 3.33 4.44 6.67 3.33 0 6.67 5.56 0 6.67
7.78 2.22 7.78 2.22 8.89 13.33 5.56 0 5.56 8.89
3.33 0 5.56 0 2.22 2.22 0 0 3.33 0
6.3 1.85 5.93 2.96 4.81 5.18 4.07 1.85 2.96 5.2
11 12 13 14 15 16 17
Phomopsis sp. Chaetomium globosum Chaetomium sp. Rhizoctonia sp. Mycelia-Sterilia 1 Mycelia-Sterilia 2 Mycelia-Sterilia 3 Total
0 6.67 4.44 5.56 0 2.22 4.44 67.78
2.22 0 2.22 0 3.33 2.22 0 72.22
4.44 0 0 0 1.11 2.22 1.11 25.54
2.22 2.22 2.22 1.85 1.48 2.22 1.85 55.17
niger, Stenella agalis, Fusarium oxysporum, Curvularia lunata and Fusarium roseum were the most frequently isolated species with colonization frequencies 6.3%, 5.93%, 5.2%, 5.18%, 4.81% and 4.07% respectively (Table 1). The frequent occurrence of Alternaria alternata is in accordance with the study made on other medicinal plants in Varanasi, India like Aegle marmelos, Azadirachta indica, Eucalyptus citriodora and Nyctanthes arbor-tristis (Gond et al. 2007; Verma et al. 2007; Kharwar et al. 2010 and Gond et al. 2011). However, Penicillium sp. and Rhizoctonia sp. were the least frequent with colonization frequency 1.85% only whereas MS 1, 2, 3 had shared 1.48%, 2.22%, and 1.85% colonization frequency respectively.This finding suggests that mycelia sterilia can dominate over the rest of the fungal taxa in endophytic colonization. The taxa like Penicillium sp., Chaetomium globosum and Rhizoctonia sp. were specific to stem tissue only and were not found in leaf and petiole and probably this may be because of displacement of their spores from root and substrate specificity supported by stem (Cannon and Simons 2002). Maximum endophytes were recovered from leaf 43.62%, followed by stem 40.93% and petiole 15.43% respectively (Fig. 2). The greater recovery of endophytic fungal isolates from leaf samples compared to stems and petioles from this plant is supported by previous report of endophyte diversity in leaves, twigs, and roots of Gynoxis oleifolia (Fisher et al. 1995). The higher recovery of fungal endophytes from leaf compared to stem and petiole suggests that properties of leaf
tissues and wide surface area exposed to environment, certainly favours the spores adherence and deposition that may lead to be endophytes after due course of time and conditions (Kharwar et al. 2010 and Fisher et al. 1995). Collectively, among the total isolates recovered in this study, there were 74.47% Hyphomycetes, 4.03% Coelomycetes, 8.05% Ascomycetes and 10.07% Mycelia-Sterilia species (Fig. 3). However, not even a single coelomycetes from stem and ascomycetes from petiole was recovered, while hyphomycetes were omnipresent in all three tissues sampled.
% Isolates Petiole 15% Stem 41%
Leaf 44%
Fig. 2 Recovery of endophytic fungal isolates from different tissues of A. alliaceum
% Distribution of endophytic fungi
Assessment of diversity, distribution and antibacterial activity
80 70 60 50 40 30 20 10 0
43
Stem Leaf Petiole Total
Fig. 3 Distribution of endophytic fungi to different fungal groups sampled from different tissues of A. alliaceum
Universal distribution of Hyphomycetes in all tissue samples with a high isolation frequency was in accordance with the findings reported in other angiospermic plants Terminalia arjuna, Aegle marmelos, Eucalyptus citriodora and Azadirachta indica (Tejesvi et al. 2005; Gond et al. 2007; Verma et al. 2007; Kharwar et al. 2010). Hyphomycetes are common fungal endophytes among plants that inhabit temperate, tropical and rainforest vegetations (Bacon and White 1994). Only two representatives of ascomycetes were recovered as Chaetomium globosum from stem and Chaetomium sp. from leaf (Table 1). The earlier reports describe that Chaetomium is a potent endophytic fungus producing several important bioactive products such as chaetopyranin C and D, cytoglobosin, radicicol, cochliodinol (Strobel et al. 2004; Tejesvi et al. 2005; and Kharwar et al. 2011). The endophytic fungal diversity was maximum in stem with Shannon-Weiner index 2.515 and Simpson index 0.9158 followed by leaf 2.379 and 0.8967 respectively, while petiole had the least endophytic diversity with Shannon-Weiner index 2.016 and Simpson index 0.862 (Table 2). Shannon-Weiner and Simpson indices of endophytic fungi isolated from stem and leaf sample tissues are significantly higher than earlier report in Terminalia arjuna, A. indica and Sesbania bispinosa (Tejesvi et al. 2005; Verma et al. 2007 and Anita et al. 2009). Although, leaf and stem have almost similar species richness but uneven isolates distribution exists in leaf due to presence of midrib and veins and thus shows less diversity than stem. The minimum endophytic diversity in petiole may be due to less surface
Table 2 Diversity indices of endophytic fungi in stem, leaf and petiole
Tissue type
Stem Leaf Petiole
area compared to leaf and stem for the infection of endophytic fungi (Fisher et al. 1995). Evenness was found maximum to stems 0.9806 followed by petiole 0.9453 and least to leaves 0.9277 (Table 2). Comparatively high evenness of endophytes in petiole than leaf tissues signifies the even distribution of isolates in petioles. Jaccard similarity index (Jc.) was found maximum between leaf and stem (0.619) followed by leaf and petiole (0.571). The minimum similarity was observed between stem-petioles (0.428) (Table 3). This is because of leaf and petiole is adjacent to each other and thus they show similar result. Comparatively, larger surface area to both organs stem and leaf provide also almost equal chance of infection to fungal flora. In present study diversity of fungal flora was intensively evaluated, while other microbes like bacteria and actinomycetes are also reported as endophytes by other studies (Tian et al. 2003 and Verma et al. 2009) which have been precluded due to media composition. These endophytic bacteria and actinomycetes may also interact in colonization of endophytic fungi in the same tissue. 3.2 Antimicrobial activity Development of drug resistance in human pathogenic bacteria, emergence of new diseases and adverse effects of chemically synthesised drugs need urgent demands of new natural antimicrobial agents which have no or low impact to environment as well as human health. The exhaustive exploitation of soil microflora as a source for bioactive compounds since longer time has reduced the opportunity of getting novel and potential antimicrobial product. Therefore, relatively less explored microbial endophytes are considered as promising source for producing variety of potential antimicrobial compounds (Strobel and Daisy 2003). In continuation of searching biologically active isolates, twelve endophytic fungi were selected to test their antimicrobial activity against 5 potent human bacterial pathogens (AShigella flexnii, B- Salmonella enteritidis, C- S. paratyphi, D- Pseudomonas aeruginosa, E- Morganella morganii) by disc diffusion assay (Fig. 4). Of 12, nine endophytic fungi (75%) inhibited the growth of at least one or more bacterial pathogens (Table 4) which is significantly higher than that of previous studies in which 8.3% isolates of endophytic fungi from Dracaena cambodiana and Aquilaria sinensis
Abundance
Species Richness
Shannon-Weiner index
Simpson Index
Species Evenness
61 65 23
13 13 8
2.515 2.379 2.016
0.916 0.897 0.862
0.981 0.928 0.945
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R.N. Kharwar et al.
Table 3 Jaccard’s similarity index (Jc) of endophytic fungi in stem, leaf and petiole
Stem Leaf Petiole
Stem
Leaf
Petioles
1
0.619 1
0.428 0.571 1
showed antimicrobial activity (Gong and Guo 2009), whereas 27.6% of endophytic strains of Camptotheca acuminata displayed antimicrobial activity against some pathogens (Lin et al. 2007). It is interesting that four endophytic fungi, Alternaria alternata, Curvularia lunata, Penicillium sp. and Chaetomium globosum inhibited the growth of 4 out of 5 tested human pathogens (80%), showing their broad spectrum antibacterial activity (Table 4, Fig. 4). Antimicrobial activity of endophytic Alternaria sp. and Penicillium sp. was also reported by earlier workers (Phongpaichit et al. 2006 and Li et al. 2007). Several antimicrobial and potent cytotoxic compounds have been identified and characterized from Chaetomium globosum, Aspergillus fumigatus, A. niger, Curvularia sp., Fusarium oxysporum, Penicillium sp. and Phomopsis sp. isolated from different hosts (Kharwar et al. 2011) and recovery of these endophytes from this host, make us enthusiastic to characterize them further. Javanicin an antibacterial napthaquinone is reported from neem endophyte Chloridium sp. (Kharwar et al. 2009). Another interesting aspect of fungal endophytes is to produce antimicrobial volatile compounds (VOCs) as they are reported from mitosporic xylariales fungi Muscodor albus and M. vitigenus isolated from Cinamomum zeylanicum (Strobel et al. 2001). Recently, some hydrocarbon derivatives as major constituents of diesel fuel are reported for the first time from a fungal endophyte Gliocladium roseum, NRRL 50072 (Strobel et al. 2008) which further our insight towards interesting aspects of fungal endophytes potential. Fig. 4 Disc diffusion assay for antibacterial activity of endophytic fungi* against Shigella flexnii (a) and Salmonella enteritidis (b). *CG- Chaetomium globosum, RS- Rhizoctonia sp., PS- Phomopsis sp., SA- Stenella agalis, TS- Trichoderma sp., FO- Fusarium oxysporum, CLCurvularia lunata, AN- Aspergillus niger, C- Control
a
Table 4 Antibacterial activity of crude extracts (5.0 mg per disc) of endophytic fungi Adenocalymma alliaceum Fungal Endophytes
Diameter of inhibition zone Aa
B
C
D
E
Alternaria alternata Aspergillus niger Aspergillus fumigatus Curvularia lunata Fusarium oxysporum Fusarium roseum Penicillium sp. Trichoderma sp. Stenella agalis Phomopsis sp.
++b − ++ − − ++ ++ − − −
+ − + +++ + − + ++ − −
++ − − + ++ ++ + − − −
++ − − ++ − − +++ ++ − −
− + − + ++ + − + − −
Chaetomium globosum Rhizoctonia sp.
+++ −
+ −
++ −
++ −
− −
a
A- Shigella flexnii (IMS/GN1), B- Salmonella enteritidis (IMS/GN3), C- S. paratyphi (IMS/GN4), D- Pseudomonas aeruginosa (ATCC27853), E- Morganella morganii (IMS/GN6)
Diameter of inhibition zone (+) 0 6 mm (++) 0 6–1.0 mm (+++) 0 More than 10 mm, (−) 0 No activity b
The fascinating dimension of fungal endophyte is the biofabrication of metal nanoparticles and their usages in biomedicine, agriculture and industries (Torney et al. 2007 and Verma et al. 2010). The overall findings of this study denote that Adenocalymma alliaceum was sampled for the first time to isolate endophytic fungi and interestingly harbour good diversity with greater antimicrobial activity. The fungi isolated from host A. alliaceum and their promising antibacterial activity against human pathogens encourage us to exploit these endophytes to isolate bioactive compound up to structural identity and their minimum inhibitory concentration (MIC) against array of pathogens. Work in this line is already under progress.
b
Assessment of diversity, distribution and antibacterial activity Acknowledgement The authors are thankful to the head (Prof BR Chaudhary), Dept. of Botany, BHU Varanasi for extending necessary facilities. Authors express thank to Prof. Gopal Nath (Institute of Medical Sciences, BHU) for providing human pathogens. SKV, AM, SKG and VKS are thankful to CSIR/UGC, New Delhi for providing fellowship. The financial support to RNK, from DST, New Delhi (FNo. SR/SO/PS-78/09 dated. 10-5-2010) is highly appreciated.
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