Endophytic actinobacteria of medicinal plants: diversity and bioactivity

1Many types of microbial population such as bacteria and fungi have been found to be associated with the internal tissues of plant as endophytes. The term endophyte was coined by De Bary, which involves the existence of microorganisms inside the infested plant tissues without having negative effects on host plant (Schulz and Boyle ). Almost all the plants have been found to be infested with one or more endophytes (Petrini et al. ). The microbes are producers of growth promoting metabolites, insect and pest repellents, antimicrobials against plant pathogens, protectors in stress conditions and many more (Rya et al.; Staniek et al.; Rai et al., ). They also possess the potential to produce unique secondary metabolites, which can be exploited in pharmaceutical, agricultural and other industries. Thus, there is a growing interest of researchers in bioprospecting of endophytic microbial communities inhabiting the plants from various ecosystems.

2Actinobacteria are Gram-positive typically filamentous bacteria, and is a major phylum in the domain Bacteria (Ludwig and Klenk ). Actinobacteria are widely distributed in both terrestrial and aquatic ecosystems. They play important roles in decomposition of complex materials from dead plants, animals, algae and fungi and in recycling of the nutrients resulting in humus formation (Sharma ). Actinobacteria are an important and a large group of soil microbes with high potential of producing different bioactive metabolites including antimicrobial, anticancer and other pharmaceutical compounds (Fiedler et al.; Schulz et al. ). These microbes have been the largest producers of different antibiotics since the discovery of Penicillin in 1928 and provided the vast diversity of antibiotics against many deadly diseases. Total number of bioactive metabolites produced by microorganisms are around 23,000 out of which 10,000 (45 % of all bioactive metabolites) are produced by actinobacteria alone and among this group of bacteria, 7600 (76 %) compounds are reported from a single genus Streptomyces (Berdy ). This signifies their prime importance in the world of pharmaceuticals.

3It is well known that the medicinal plants are the rich sources of precious bioactive compounds. As a consequence of long term association of endophytes with such plants, the former may also participate in metabolic pathways and enhance its own natural bioactivity or may gain some genetic information to produce specific biologically active compound similar to the host plant (Stierle et al.; Eyberger et al.; Mitchell et al.; Kumar et al.; Chithra et al.; Rai et al., ). Therefore, the endophytes isolated from medicinal plants are of immense significance.

4The beneficial interactions of endophytic actinobacteria with plants are being considered as an important area of research. These endophytic actinobacteria are attractive source of novel bioactive compounds and therefore, many research groups are involved in the study of their bioactivities and industrial applications. The present review is focused on the advances in endophytic actinobacteria isolated from medicinal plants including their diversity and broad-spectrum bioactivities.

5Different methods have been used by researchers for isolation of endophytic actinobacteria. Takahashi and Omura emphasized that the diversity of actinobacteria depend mainly on the methods of isolation. The most frequently employed method for their detection and enumeration involves isolation from surface-sterilized host plant tissue. Isolation of endophytic actinobacteria depend on various factors, which include- host plant species, age and type of tissue, geographical and habitat distribution, sampling season, surface sterilants, selective media and culture conditions (Hallmann; Gaiero et al. ).

6In general, the isolation protocol involves the collection of plant parts such as leaves, stem, roots that should be processed freshly or stored at 4 °C until isolation within 24 h. These explants are washed in running tap water to remove adhered epiphytes, soil debris or dust particles on the surface, followed by surface sterilization using one or more different surface sterilizing agents. The most commonly used surface sterilants include ethanol and a strong oxidant or general disinfectant like household bleach (NaOCl) with 2–5 % (w/v), available chlorine (for 2–4 min). Qin et al. and Dochhil et al. applied combination of 5 % sodium chlorate (NaClO3), 2.5 % sodium thiosulfate (Na2S2O3), 75 % ethanol and 10 % sodium bicarbonate (NaHCO3) as sterilizing agents to inhibit the growth of fungal endophytes. The strength of sterilizing chemicals depends on permeability of the sample. Otherwise, the internal tissues will be sterilized (Hallmann et al. ). All the explants are finally rinsed with sterile distilled water, divided into small fragments (1 cm for steam or roots and 1 cm 2 for leaves) and inoculated on appropriate agar medium. In another method, the surface sterilized plant tissues are macerated and thoroughly homogenized with phosphate buffer or other suitable liquid medium. This suspension is serially diluted as 10−1 to 10−5 and spread on agar medium in order to obtain endophytic actinobacteria. The media are supplemented with antifungal antibiotics such as nystatin and cycloheximide (50 or 100 μg/ml) to suppress the fungal growth. After incubation at 26 ± 2 °C for 15–30 days, individual colonies with characteristic actinomycete morphology emerging out from the plant tissue are isolated. The pure cultures of the isolates are obtained by streaking on fresh media plates. The efficacy of the surface sterilization method, resulting from lack of microbial growth, can be authenticated by inoculating the last washing water into the same media plates.

7Various types of growth media have been described by the authors for the isolation of endophytic actinobacteria such as starch casein (Küster and Williams ), starch casein nitrate (SCNA), actinomycetes isolation, soybean (Williams and Davies ), chitin-vitamin B (Hayakawa and Nonomura ), tap water-yeast extract (TWYE; Crawford et al. ) agars and humic acid vitamin B (HV), yeast extract casamino acid (YECA), synthetic (Mincer et al. ), modified Gausse (Ivantiskaya et al. ) and glycine–glycerol (Küster ) media. Zhao et al. also underlined the need of using wider range of isolation methods to acquire more knowledge about species diversity of actinobacteria within medicinal plants. A modified method employed by Machavariani et al. describes the pre-treatment of leaves with solutions of heteroauxin and zircon, which helped to isolate and increase the numbers of rare actinobacteria from medicinal plants.

8Current identification and classification of actinobacteria are based a polyphasic approach, comprising morphological, physiological and molecular studies (Goodfellow et al. ) based on each taxon should be described and differentiated from related taxa. The sequencing of highly conserved macromolecules, notably 16S rRNA genes, has provided valuable data for constructing phylogenies at and above the genus level (Ludwig and Klenk ). The DNA: DNA relatedness, molecular fingerprinting and phenotypic techniques are methods of choice for delineating taxa at and below the rank of species (Rosselló-Mora and Amann ). Distinguishing phenotypic differences are required for the description of a new species (Wayne et al. ). Exploring the diversity of endophytic actinobacteria is indispensable for screening of beneficial strains and understanding their ecological niche.

9Endophytic actinobacteria are able to associate with their host at a very early stage of the plant development (Hasegawa et al. ). Minamiyama et al. noticed in SEM studies that mycelia of Streptomyces galbus, which was spread on the surface of the tissue-culture medium in which rhododendron seedlings were growing, grew on leaf surfaces and entered into the leaf tissues via stomata. Further, they also observed that the internal mycelia grew out of stomata after internal multiplication within host leaves. Moreover, the authors observed that within host leaves, hyphae of S. galbus were present individually or in colonies in intercellular spaces but not inside epidermal or mesophyll cells.

10The maximum endophytic actinobacteria have been recovered from roots followed by stems and least in leaves (Qin et al.; Gangwar et al. ). The woody plants conferred far greater diversity of actinobacteria in comparison to herbaceous plants. The high rate of occurrence of actinobacteria in roots as compared to other tissues is very common. This underlines the fact that the actinobacteria are natural dwellers of soil that easily come in contact with the roots of plants and may form the symbiotic association with them by entering the plant tissues. The results obtained by Nimnoi et al. suggested that different locations within the plant also differ in the diversity of actinomycete flora. Strobel and Daisy reported that the greater diversity of endophytes is probable to occur in the tropical and temperate regions. Du et al. analyzed the endophytic diversity of 37 medicinal plants and reported 600 actinobacteria belonging to 34 genera and 7 unknown taxa. The authors depicted that there was no direct relationship between host plants and their endophytic flora regarding the utilization of sole carbon source, fermentation of carbon sources for production of acids and enzymes, rather the physiological characteristics of endophytic isolates were related to the geographical distribution of their host plants.

11The measures of functional biodiversity may be more reliable and powerful than the taxonomic measures in order to recognize mechanistic basis of diversity and its effects on the plant-endophyte interactions (Parrent et al. ). Species distribution and biological diversity of endophytic actinobacteria of medicinal plants are extensively influenced by ecological environment (Hou et al. ). El-Shatoury et al. interpreted that the plant species can be separated into three clusters representing high, moderate and low endophytic diversity on the basis of generic diversity analysis of endophytes. The authors also reported that the endophytes represent high functional diversity, based on forty four different traits including catabolic and plant growth promotion traits and such traits may characterize a key criteria for successful habitation of endophytes within the endosphere. Furthermore, the stress-tolerance traits were more predictive measure of functional diversity of endophytic actinobacteria (El-Shatoury et al. ).

12Hasegawa et al. reported a new genus of actinobacteria namely Actinosynnema, from a grass blade, which was probably the first report of an actinomycete of plant origin. A comprehensive literature survey has revealed the huge diversity of endophytic actinobacteria isolated from interior tissues of stem, leaves and roots of medicinal plants (Table 1). Taechowisan et al. studied the diversity of actinobacteria residing in medicinal plants based on their morphology and the amino acid composition of the whole-cell extract and analysed the percentage of endophytic actinobacteria recovered from different explants: 64 % isolates from roots, 29 % from leaves, and 6 % from stems of 36 different plant species.

13Janso and Carter also assessed the diversity of endophytic actinobacteria, including those from medicinal plants, albeit by ribotyping with Pvu II restriction enzyme to digest the genomic DNA. Ribotypes were then compared to each other using appropriate software (Janso and Carter ). The authors have found that 85 % of 123 isolates studied were determined to be unique at the strain level. The isolates were classified to six families and 17 different genera. Streptomyces accounts for the dominant genus, which is most commonly isolated as endophytic actinomycete (Qin et al.; Zhao et al.; Shutsrirung et al.; Gangwar et al. ) while others include genera such as Micromonospora, Actinopolyspora, Saccharopolyspora, Nocardia, Oerskovia, Nonomuraea, Streptoverticillium, Microbispora, Streptosporangium, Promicromonospora and Rhodococcus (Verma et al.; Zhao et al. ). Some rare actinobacteria like Dietzia, Blastococcus, Dactylosporangium, Actinocorallia, Jiangella,Promicromonospora, Oerskovia, Microtetraspora and Intrasporangium were also reported as endophytes (Qin et al.; Zhao et al.; Qin et al.; El-Shatoury et al. ). A novel halotolerant actinomycete was isolated from a salt marsh plant Dendranthemaindicum collected from the coastal region of China (Zhang et al. ). New species of endophytic actinobacteria such as Rhodococcuscercidiphylli and Saccharopolysporaendophytica were isolated from leaf of Cercidiphyllum japonicum (Li et al. ) and root of Maytenus austroyunnanensis (Qin et al. ), respectively. Du et al. proposed a new genus and species, Allonocardiopsis opalescens gen. nov., sp. nov., based on the polyphasic taxonomic study, within the suborder Streptosporangineae. Wang et al. studied the diversity of uncultured microbes associated with medicinal plant Mallotus nudiflorus and concluded that actinobacteria were the most dominant microbes, covering about 37.7 % of whole endophytic isolates.

14In, Qin and co-workers studied the diversity of endophytic actinobacteria recovered from root, stem and leaf tissues of Maytenus austroyunnanens which was collected from tropical rainforest in Xishuangbanna, China. Later the authors concluded the diversity of isolates by combination of cultivation and culture-independent analysis and based on 16S rRNA gene sequencing. Further by using different selective isolation media and methods total of 312 actinobacteria were isolated from above plants which were affiliated with the order Actinomycetales (distributed into 21 genera). Based on a protocol for endophytes enrichment, three 16S rRNA gene clone libraries were constructed and 84 distinct operational taxonomic units were identified and they distributed among the orders Actinomycetales and Acidimicrobiales, including eight suborders and at least 38 genera with a number of rare actinobacteria genera. Moreover, six genera from the order Actinomycetales and uncultured clones from Acidimicrobiales were found to be unknown and reported as first time endophytes. This study confirms abundant endophytic actinobacterial consortium in tropical rainforest native plant and suggests that this special habitat still represents an underexplored reservoir of diverse and novel actinobacteria of potential interest for bioactive compounds discovery.

15The plant endosphere consists of a large variety of microbial endophytes, which constitute a complex micro-ecosystem (El-Shatoury et al. ). A vast diversity of secondary metabolites in actinobacteria may occur due to the natural adaptations to environment, as a part of competition for common resources such as plant matter in soil. It has been observed that the genes responsible for the production of individual secondary metabolites were found almost always located as a cluster in the genome and referred to as biosynthetic gene clusters (Doroghazi and Metcalf ). Although, there is no data available about full genome sequencing on actinobacteria from medicinal plants it has been known, that whole genomes of Streptomyces sp. and non-Streptomyces non-endophytic actinobacteria such as Streptomyces avermitilis MA-4680 (Ōmura et al.; Ikeda et al. ) and Streptomyces coelicolor A(3)2 (Bentley et al. ) as well as Saccharopolyspora erythraea NRRL 23338 (Oliynyk et al. ), Salinispora tropica CNB-440 (Udwary et al. ) contain around 20 or more natural product biosynthetic gene clusters for the production of known or predicted secondary metabolites (Goodfellow and Fiedler ). The potential of actinobacteria isolated from medicinal plants, especially of non-productive non-streptomycete ones to produce secondary metabolites can be estimated by detection of polyketide synthase (PKS) (both I and II type) and nonribosomal peptide synthetase (NRPS) genes (Janso and Carter ). The authors studied 29 strains and all of them produced bands of the expected size for NPRS and majority of them possessed PKS (66 % of PKSI and 79 % of PKSII type) genes. However, some of the pathways encoded by these genes may not be functional. The above study suggests that the non-productive actinobacteria possess the genetic capacity to produce secondary metabolites, if cultivated under proper growth conditions (Janso and Carter ).

16Amongst prokaryotes, members of Actinobacteria, notably the genus Streptomyces, remains the richest source of valuable natural products (Pandey et al.; Newman and Cragg; Lu and Shen; Olano et al.; Berdy ). The diverse arrays of bioactivities of endophytic actinobacteria are further classified into pharmaceutical and agricultural applications and are illustrated below in detail.

17In recent years, many of novel antibiotics synthesized by endophytic actinobacteria recovered from medicinal plants found to be active against bacteria, fungi and viruses. Moreover, these antibiotics demonstrated their activity at significantly lower concentrations (Table 2). This indicates the strong and broad spectrum microbiocidal potential of the antibiotics originating from endophytic actinobacteria, mainly of the genus Streptomyces.

18Day by day due to excessive use of antibiotics, the multi-drug resistance capacity of pathogens is becoming more and more severe. The scientists all over the world are endeavouring continuously to search new antibiotic compounds in order to tackle this problem. Here endophytic microbes, especially actinobacteria appear as a source of novel and active compounds to combat the increasing number of multidrug-resistant pathogens. Out of 65 strains of endophytic actinobacteria 12 strains were able to suppress penicillin-resistant Staphylococcus aureus, belonging to the genus Glycomyces and majority of them were Streptomyces isolated from plants Achyranthes bidentata, Paeonia lactiflora, Radix platycodi and Artemisia argyi (Zhang et al. ). Wang et al. displayed moderate antiviral activity against influenza virus type A subtype H1N1 of perlolyrine, 1-hydroxy-β-carboline, lumichrome, 1H-indole-3-carboxaldehyde from Jishengella endophytica with IC50 value of 38.3, 25.0, 39.7, and 45.9 μg ml−1, respectively. Further, they also suggested that 1-hydroxy-β-carboline could be a promising new hit for anti-H1N1 drugs.

19Larvicidal activity of Streptomyces sp. isolated from Artemisia herba-alba, Echinops spinosus, Balotta undulate and Mentha longifolia was observed by El-Shatoury et al.. The authors studied cytotoxic effect against larvae of Artemia salina was positive for 27 out of 41 endophytic actinobacteria and of these, nine isolates, mainly from Artemisia and Echinops exhibited high mortality rate reaching to 100 % death after 12 h. Similarly, Streptomyces albovinaceus and S. badius isolated from plants of family Asteraceae were also found to have significant larvicidal potential against first and fourth instar stages of Culexquinquefasciatus (mosquito larvae) (Tanvir et al. ). They illustrated strong larvicidal activity (80–100 % mortality) of six isolates while four isolates showed potent larvicidal activity (100 % mortality) at the fourth instar stage.

20Castillo et al. have found that one of the tested munumbicins type D was considerably active against the parasite Plasmodium falciparum, the most pathogenic plasmodium causing malaria, with IC50 of 4.5 ng ml−1. They also described that outstanding activity of each of the munumbicins against P. falciparum were within the range to be pharmacologically interesting with IC50 of 175,130, 6.5 and 4.5 ng ml−1in munumbicin A–D, respectively. Authors emphasized special interest of the munumbicins C and D because of their extremely low IC50 values. Furthermore, they also reported that munumbicins B, C and D did not cause any detectable lysis of human red blood cells up to a concentration of 80 µg ml−1. Therefore, they suggested that the ultimate development of these compounds as antimalarial or anti-infectious drugs may have to depend upon the synthesis of munumbicin derivatives that have reduced toxicity (Castillo et al., ).

21Among the range of bioactive compounds from endophytic actinobacteria of medicinal plants those with anticancer activity were also found. Castillo et al. extracted kakadumicin A, which inhibited the human breast cancer cell line BT20 with IC50 of 4.5 ng ml−1. Similarly, Igarashi et al. reported that human cancer cell lines NCI-H522, OVCAR-3, SF539, and LOX-IMVI were inhibited with IC50 in the presence of 2.9, 3.9, 5.0 and 7.1 mM of pterocidin extracted from Streptomyces hygroscopicus TP-A0451 isolated from Pteridium aquilinum. Lu and Shen reported cytotoxic activity of naphtomycin A from Streptomyces sp. CS isolated from Maytenus hookeri against P388 and A549 human tumor cells with IC50 0.07 and 3.17 mM, respectively. The cytotoxicity against A549 human tumor cells was also studied by Zhang et al.. The cell line was inhibited with value of 3.3 and 5.1 mg ml−1 in presence of 3-acetonylidene-7-prenylindolin-2-one and 7-isoprenylindole-3-carboxylic acid, respectively. Cytotoxic activity of 6-alkalysalicilic acids, salaceyins A and B from Streptomyces laceyi MS53 against human breast cancer cell line, SKBR3 with IC50 values of 3.0 and 5.5 mg ml−1 was noticed by Kim et al.. Anthraquinones named lupinacidins from Micromonospora lupine sp. were reported to inhibit growth of colon 26-L5 carcinoma cells in mice (Igarashi et al. ). Furthermore, anti-invasive effects of lupinacidins were also examined at non-cytotoxic concentrations. The authors reported lupinacidin A as more potent both in cytotoxic and anti-invasive activities than lupinacidin B, suggesting that the alkyl substituent present in lupinacidin A was involved in these activities (Igarashi et al. ).

22Caruso et al. reported an anticancerous drug paclitaxel from endophytic actinomycete Kitasatospora sp. isolated from inner cortical tissues of Taxus baccata. Another novel anticancer compound named brartemicin, a trehalose-derived metabolite, was extracted from the actinomycete Nonomuraea sp. isolated from Artemisiavulgaris. This new compound was capable of inhibiting the invasion of murine colon carcinoma 26-L5 cells with an IC50 value of 0.39 μM without any cytotoxicity (Igarashi et al. ). Taechowisan et al. evaluated 4-phenylcoumarins on human lung cancer cell lines, which was extracted from Streptomyces aureofaciens and found that 5,7-dimethoxy-4-phenylcoumarin can inhibit cell proliferations more actively when compared with 5,7-dimethoxy-4-p-methoxylphenylcoumarin. Moreover, the screening of 4-arylocoumarins for inhibitory effect on transplanted Lewis lung carcinoma (LLC) by intraperitoned administration has showed antitumor activity with T/C values of 80.08 and 50.0 % at doses of 1 and 10 mg kg−1 of 5,7-dimethoxy-4-p-methoxylphenylcoumarin and 81.5 and 44.9 % at doses of 1 and 10 mg kg−1 of 5,7-dimethoxy-4-phenylcoumarin. Authors have concluded that 5,7-dimethoxy-4-phenylcoumarin might be preventing or delaying formation of metastases and both 4-arylocoumarins by their low cytotoxicity to normal cells and effect in malignant cells could be recommended as chemopreventatives and in combined antitumor treatment (Taechowisan et al. ).

23Another important group of compounds, which were found in endophytic actinobacteria from medicinal plants were alpha-glucosidase inhibitors (Pujiyanto et al. ). Twelve out of 65 isolates obtained from Tinospora crispa, Caesalpinia sappans and Curcuma aeruginosa were able to produce it. This inhibitor showed antidiabetic property by which it can retard the release of glucose from dietary complex carbohydrates and also delay absorption of glucose. Interestingly, it was observed that endophytic actinomycete BWA65 produced these inhibitors which showed doubled activity than its host plant (Tinospora crispa). Furthermore, the tissue cultured plants that were devoid of any endophyte had very low capability to produce inhibitor compounds (Pujiyanto et al. ). This indicates that the production of alpha-glucosidase inhibitors by this plant is largely due to the contribution of its endophytic actinobacteria. It also strengthens the hypothesis that there may be a phenomenon of inter-kingdom genetic transfer of some specific traits between the host plant and its endophytic counterpart.

24Similarly, Akshatha et al. isolated alpha-amylase inhibitor secreting endophytic actinobacteria S. longisporoflavus and Streptomyces sp. from well-known antidiabetic medicinal plants Leucas ciliata and Rauwolfia densiflora. Alpha-amylase inhibitors demonstrated antidiabetic activity similar to alpha-glucosidase inhibitors. The extracts obtained from these actinobacteria did not show insulin-releasing ability, instead it improved the ability of available insulin to pass glucose into muscles.

25Phenolic compounds are known as natural antioxidants, which provide protection by scavenging harmful free radicals. Endophytic Streptomyces sp. isolated from Alpiniaoxyphylla produced two active compounds 2,6-dimethoxy terephthalic acid and yangjinhualine A, which demonstrated considerable antioxidant activity (Zhou et al.; ). Out of the total endophytic actinobacteria isolated from medicinal plants, 66.6 % isolates demonstrated potent antioxidant activity (Tanvir et al. ). Antiinflammatory drugs are used to reduce the inflammations and this property was also shown by one of the endophytic actinomycete. Taechowisan et al. demonstrated the successful application of 5,7-dimethyloxy-4-p-methoxylphenylcoumarin and 5,7-dimethoxy-4-phenylcoumarin produced by Streptomyces aureofaciens as an antiinflammatory agents.

26The endophytic actinomyctetes can also be a source of metabolites, which promote or improve host plant growth as well as reduce disease symptoms caused by plant pathogens or various environmental stresses (Shimizu ). Several scientific investigations evidenced the plant growth promotion activity and secretion of plant growth hormones from endophytic actinobacteria. Dochhil et al. demonstrated the plant growth enhancement and higher seed germination percentage by the application of two Streptomyces sp. isolated from Centella asiatica. These strains were also evaluated for production of a plant growth promoter, indole acetic acid (IAA) which was found in much higher concentration as 71 g/ml and 197 g/ml. The isolates of the genus Nocardiopsis presented highest IAA production ability among all other actinomycete genera (Shutsrirung et al. ). In the field trials conducted by El-Tarabily et al., Actinoplanes campanulatus, Micromonospora chalcea and Streptomyces spiralis were applied individually and in combination to cucumber seedlings, which enhanced plant growth and yield.

27Igarashi and Igarashi et al. isolated pteridic acids A and B from Streptomyces hygroscopicus isolated from a stem of bracken (Pteridium aquilinum) as plant growth promoters with auxin-like activity. They found that pteridic acids induced the formation of adventitious roots in hypocotyl of kidney beans at 1 mM as effectively as auxin (indole acetic acid; IAA), a natural plant growth hormone. Additionally, authors noticed that pteridic acid A promotes the root elongation at 20 ppm. However, the rice germination was inhibited at 100 ppm of IAA. Gangwar et al. also found actinobacteria, mostly Streptomyces sp, capable of producing IAA. Plant growth promoters were produced within the range of 9.0–38.8 μg ml−1.

28Endophytic actinobacteria are able to employ additional means of fungal antagonism such as chitin enzymes and siderophores. Chitin is the most characteristic polysaccharide of the fungal cell wall. Endophytic actinobacteria are able to produce fungal cell wall degrading enzymes especially by the production of chitinase (El-Tarabily and Sivasithamparam ). The role of siderophores produced by endophytic microorganisms has been paid more attention because these metabolites are suggested to be involved in promoting the growth of host plants as well as antagonism to phytopathogen (Cao et al.; Tan et al.; Rungin et al. ). El-Shatoury et al. reported actinobacteria from Achilleafragrantissimawhich were either capable of producing chitinases or siderophores and also showed remarkable inhibitory activity against phytopathogenic fungi. Chitinases produced by the endophytic actinomycete Actinoplanes missouriensis (El-Tarabily; El-Tarabily and Sivasithamparam ) were reported to cause hyphal lysis and reduction in conidial germination. The studies by El-Shatoury et al. were supported by Gangwar et al. where authors recorded production of hydroxamate-type of siderophore ranging between 5.9 and 64.9 μg ml−1 and catechol-type of siderophore in the range of 11.2–23.1 μg ml−1 by actinobacteria from Aloe vera, Mentha arvensis and Ocimum sanctum. In another investigation, El-Tarabily et al. applied endophytic Actinoplanes campanulatus, Micromonospora chalcea and Streptomyces spiralis to cucumber seedlings. As it reduced seedling damping-off as well as root- and crown- rot of mature cucumber plants caused by Pythium aphanidermatum successfully, authors suggested that these strains of endophytic actinobacteria can be employed as biological control agents.

29The 6-prenylindole, a new bioactive compound from Streptomyces sp. was studied by Igarashi. This simple molecule showed significant antifungal activity against plant pathogens, Alternariabrassicicola and Fusarium oxysporum. 6-prenylindole was first reported as a component of the liverwort (Hepaticae). This is an interesting example of the isolation of the same compound from plant and microorganism (Igarashi ). Similarly, Zhang et al. showed antifungal activity of one new prenylated indole derivative and tree known hybrid isoprenoids with IC50 values in range of 30.55–89.62 against phytopathogenic fungi Colletotrichumorbiculare, Phytophthora capsici, Corynespora cassiicola and Fusarium oxysporum. Lu and Shen reported antifungal activity of naphthomycins A and K extracted from Streptomyces sp. CS against Penicillium avellaneum UC-4376. Igarashi reported the compound fistupyrone from Streptomyces sp. isolated from a leaf of spring onion (Allium fistulosum) and determined as an inhibitor of spore germination of Alternaria brassicicola. The latter is the cause of black leaf- spot, a major disease of cultivated Brassica plant. Although fistupyrone did not show in vitro antifungal activity against A. brassicicola, it completely inhibited the infection of A. brassicicola by pretreating the seedlings with 100 ppm of the compound. Studies by Igarashi et al. revealed that fistupyrone did not give any effect on the growing hyphae but specifically suppresses the spore germination at 0.1 ppm.

30Thus, the metabolites obtained from these actinobacteria inhibit the phytopathogenic fungi and can be better and safer alternatives to the chemical fungicides, which pose potential environmental threat and mammalian toxicities. In terms of the availability, the endophytic actinobacteria are the rich and cost-effective source of numerous agro-based biological agents. So, it is desirable to evaluate more such compounds that might have different modes of action to protect the crops than the existing chemical fungicides and will also avoid the problems of cross-resistance.

31There is a pressing need to search for new therapeutic drugs, particularly anti-infective compounds due to the rapid increase of resistance in major known pathogens to front line antibiotics. Therefore, screening and isolation of promising strains of endophytic actinobacteria with antimicrobial properties which are relatively poorly investigated has increased the interest of researchers in both basic and applied fields. Clearly, more research on the formulation, development of novel technologies and methodologies is needed for employing them in the agricultural, medical and pharmaceutical fields.

32An extensive characterization and identification of the diverse population of endophytic actinobacteria associated with medicinal plants may also provide greater insight into the plant-endophyte interaction and evolution of mutualism. It is also important to understand the mechanism that enables these microbes to interact with their host plants may be of biotechnological potential. Several questions are yet to be answered. Is there any combination between the metabolic pathways of plants and endophytes, which together constitutes for particular bioactivity? What genetic control exists for synthesis of secondary metabolites similar to the host plants? In order to address this research area in depth, it is necessary to understand the physiology and biochemistry of endophytic actinobacteria as well as their defensive role and secondary metabolite producing ability inside the plants.