Endophytic Actinomycetes Research Articles

Multiple, Novel Biologically Active Endophytic Actinomycetes Isolated from Upper Amazonian Rainforests

Microbial biodiversity provides an increasingly important source of medically and industrially useful compounds. We have isolated 14 actinomycete species from a collection of approximately 300 plant stem samples from the upper Amazonian rainforest in Peru. All of the cultured isolates produce substances with inhibitory activity directed at a range of potential fungal and bacterial pathogens. For some organisms, this activity is very broad in spectrum while other organisms show specific activity against a limited number of organisms. Two of these organisms preferentially inhibit bacterial test organisms over eukaryotic organisms. rDNA sequence analysis indicates that these organisms are not equivalent to any other cultured deposits in GenBank. Our results provide evidence of the untapped biodiversity in the form of biologically active microbes present within the tissues of higher plants.

Plant growth promotion and biological control ofPythium aphanidermatum, a pathogen of cucumber, by endophytic actinomycetes

To evaluate the potential of Actinoplanes campanulatus, Micromonospora chalcea and Streptomyces spiralis endophytic in cucumber roots, to promote plant growth and to protect seedlings and mature plants of cucumber from diseases caused by Pythium aphanidermatum, under greenhouse conditions. Three endophytic isolates, out of 29, were selected through tests aimed at understanding their mechanisms of action as biocontrol agents and plant growth promoters. When applied individually or in combination, they significantly promoted plant growth and reduced damping-off and crown and root rot of cucumber. The combination of the three isolates resulted in significantly better suppression of diseases and plant growth promotion, than where the plants were exposed to individual strains. The three selected actinomycete isolates colonized cucumber roots endophytically for 8 weeks, promoted plant growth and suppressed pathogenic activities of P. aphanidermatum on seedling and mature cucumber plants. The results clearly show that the endophytic, glucanase-producing actinomycetes used, especially as a combined treatment, could replace metalaxyl, which is the currently recommended fungicide for Pythium diseases in the United Arab Emirates. These endophytic isolates also have the potential to perform as plant growth promoters, which is a useful attribute for crop production in nutrient impoverished soils.

A promising strain of endophytic Streptomyces sp. for biological control of cucumber anthracnose

In a survey for effective biocontrol agents of Colletotrichum orbiculare, causal agent of cucumber anthracnose, 43 (MBCu series) and 135 (MBPu series) endophytic actinomycete strains were recovered from surface-sterilized organs of cucumber (Cucumis sativus) and pumpkin (Cucurbita moschata) plants, respectively. The strains were cultured with C. orbiculare on IMA-2 agar medium to determine their in vitro antagonistic ability. Eleven strains that strongly inhibited hyphal growth of the pathogen were selected as potent antagonists. Detached cotyledons of cucumber were soaked in a spore suspension of an antagonistic strain 1 day before challenge-inoculation with the pathogen, and six of these antagonists (MBCu-32, 36, 42, 45, and 56, and MBPu-75) significantly reduced the number and size of the lesions on the cotyledons compared to the untreated control. In the same way, these six strains inhibited lesion development on attached leaves of 3-week-old cucumber seedlings. Strain MBCu-56, the most suppressive of the strains, was selected for further tests. Its suppressiveness increased as concentrations increased; pretreatment of leaves with the strain at 107, 108 and 109 cfu/ml suppressed disease by 72, 79 and 93%, respectively. These results strongly indicated that MBCu-56 has strong potential for controlling cucumber anthracnose. Based on the taxonomic characteristics and 16S rDNA sequence, MBCu-56 was identified as Streptomyces sp. With scanning electron microscopy, the substrate mycelia of the strain were seen to colonize the surface of leaves above the cuticle. Some hyphae also penetrated and grew underneath the cuticle.

Isolation and Characterization of Endophytic Actinomycetes from Chinese Cabbage Roots as Antagonists to Plasmodiophora brassicae

This study was conducted to select endophytic actinomycetes as biocontrol agents against Chinese cabbage clubroot caused by Plasmodiophora brassicae. A total of 81 endophytic actinomycetes were isolated from surface-sterilized roots of Chinese cabbage that was grown on paddy field and upland soils collected from various locations in Korea. By using 16S ribosomal DNA (rDNA) gene sequencing, they were classified to 8 actinobacterial genera. The genus Microbispora (67%) was most frequently isolated, followed by Streptomyces (12%) and Micromonospora (11%). Three of the 81 isolates, when inoculated in germinated Chinese cabbage seeds and then transplanted to pots, effectively suppressed the occurrence of a post-inoculated strain of P. brassicae in the pots. They showed control values of 58% for strain A004, 33% for strain A004, and 42% for strain A018. Based on cell wall components, morphological characteristics, and phylogenetic analyses, the three antagonistic isolates were identified as Microbispora rosea subsp. rosea (A004 and A011) and Streptomyces olivochromogenes (A018). Further researches on the field efficacy and action modes of the three actinomycetes are in progress.

Chitinolytic activity of endophyticStreptomycesand potential for biocontrol

Biological sources for the control of plant pathogenic fungi remain an important objective for sustainable agricultural practices. Actinomycetes are used extensively in the pharmaceutical industry and agriculture owing to their great diversity in enzyme production. In the present study, therefore, we evaluated chitinase production by endophytic actinomycetes and the potential of this for control of phytopathogenic fungi. Endophytic Streptomyces were grown on minimum medium supplemented with chitin, and chitinase production was quantified. The strains were screened for any activity towards phytopathogenic fungi and oomycetes by a dual-culture in vitro assay. The correlation between chitinase production and pathogen inhibition was calculated and further confirmed on Colletotrichum sublineolum cell walls by scanning electron microscopy. This paper reports a genetic correlation between chitinase production and the biocontrol potential of endophytic actinomycetes in an antagonistic interaction with different phytopathogens, suggesting that this control could occur inside the host plant. A genetic correlation between chitinase production and pathogen inhibition was demonstrated. Our results provide an enhanced understanding of endophytic Streptomyces and its potential as a biocontrol agent. The implications and applications of these data for biocontrol are discussed.

Selective Isolation and Ansamycin-Targeted Screenings of Commensal Actinomycetes from the “Maytansinoids-Producing” Arboreal Trewia nudiflora

To verify the hypothesis on the involvement of commensal actinomycetes in the biosynthesis of plant maytansinoids that belong to the ansamycin family, selective isolation and targeted screenings were conducted. In total, 164 endophytic actinomycetes isolates were obtained from the roots, twigs, seeds, callus, and germ-free seedlings of "maytansinoids-producing" Trewia nudiflora Linn. by selective isolation methods. Crude extracts of the isolates were screened by antifungal bioassay against Penicillium avellaneum UC-4376, thin-layer chromatography, and liquid chromatography-mass spectrometry detection according to the bioactivity as well as structural characteristics of maytansinoids. Respectively, 25.0%, 64.8%, and 26.7% of the extracts were positive in corresponding screenings. Ten isolates showed positive results in all three different screenings. To confirm on a genetic level, 28 representative isolates from 20 morphological groups were screened by polymerase chain reaction with 3-amino-5-hydroxybenzoic acid synthase gene and carbamoyltransferase gene primers, both of which were involved in the biosynthesis of ansamycins. According to the results of the targeted screenings, two isolates Streptomyces sp. 5B and Streptomyces sp. M27m3 might have the potential of producing ansamycins, which further enhanced the hypothesis that endophyte(s) might be involved in the biosynthesis of plant maytansinoids.

Antitumour and antimicrobial activities of endophytic streptomycetes from pharmaceutical plants in rainforest

The aim of this study was to screen antitumour and antimicrobial activities of endophytic actinomycetes isolated from pharmaceutical plants in rainforest in Yunnan province, China. Antitumour activity was studied by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and antimicrobial activity was determined by agar well diffusion method. The high bioactive endophytic isolates were identified and further investigated for the presence of polyketide synthases (PKS-I, PKS-II) and nonribosomal peptide synthetases (NRPS) sequences by specific amplification. The molecular identification confirmed that the 41 isolates showed significant activities were members of the genus Streptomyces. Among them, 31.7% of endophytic streptomycete cultures were cytotoxic against A549 cells, 29.3% against HL-60 cells, 85.4% against BEL-7404 cells, 90.2% against P388D1 cells, 65.9% were active against Escherichia coli, 24.4% against Staphylococcus aureus, 31.7% against Staphylococcus epidermidis, 12.2% against Candida albicans and no strain displayed antagonistic activity against Klebsiella pneumoniae. High frequencies of positive PCR amplification were obtained for PKS-I (34.1%), PKS-II (63.4%) and NRPS (61.0%) biosynthetic systems. Many endophytic streptomycetes isolated from pharmaceutical plants in rainforest possess remarkable and diverse antitumour and antimicrobial bioactivities. These endophytic streptomycetes are precious resources obtained from rainforests, and they could be a promising source for bioactive agents.

Endophytic Actinomycetes from Azadirachta indica A. Juss.: Isolation, Diversity, and Anti-microbial Activity

Endophytic actinomycetes from Azadirachta indica A. Juss. were screened and evaluated for their anti-microbial activity against an array of pathogenic fungi and bacteria. A total of 55 separate isolates were obtained from 20 plants, and 60% of these showed inhibitory activity against one or more pathogenic fungi and bacteria. Actinomycetes were most commonly recovered from roots (54.5% of all isolates), followed by stems (23.6%), and leaves (21.8%). The dominant genus was Streptomyces (49.09% of all isolates), while Streptosporangium (14.5%), Microbispora (10.9%), Streptoverticillium (5.5%), Sacchromonospora sp. (5.5%), and Nocardia (3.6%) were also recovered. Streptomyces isolates AzR 006, 011, and 031 (all from roots) had acute activity against Pseudomonas fluorescens, while AzR027, 032, and 051 (also all from roots) showed activity against Escherichia coli. Meanwhile, an isolate of Nocardia sp. from leaves (AzL025) showed antagonism against Bacillus subtilis. Overall, 32 of the 55 were found to have broad spectrum significant antimicrobial activity, while about 4% of them showed strong and acute inhibition to pathogenic fungi and bacteria. Isolates of Streptomyces AzR031, 008, and 047, Nocardia sp. AzL025, and Streptosporangium sp. AzR 021 and 048 are of particular interest because they showed significant antagonistic activity against root pathogens, including Pythium and Phytophthora sp. Thus, many of the isolates recovered from A. indica in this study may be used in developing potential bio-control agents against a range of pathogenic fungi and bacteria and in the production of novel natural antimicrobial compounds. These results not only further our understanding of plant-microbe interactions but also indicate that there is an untapped resource of endophytic microorganisms that could be exploited in the biotechnological, medicinal, and agricultural industries.

Antitumor Anthraquinones from an Endophytic Actinomycete Micromonospora lupini sp. nov.

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Cultivation of disease-resistant, tissue-cultured seedlings using endophytic actinomycetes

Cultivation of disease-resistant, tissue-cultured seedlings using endophytic actinomycetes

Interspecies variation of Kitasatospora recifensis endophytic from yam bean producing thermostable amylases in alternative media.

An endophytic actinomycete isolated from tubers of yam beam (Pachyrhizus erosus L. Urban) was classified as a novel species nominated Kitasatospora recifensis based in phenotypic and genotypic analysis (16S rDNA gene sequence). Monosporic culture using specific ISP2 media revealed three interspecies, which were identified by DNA southern hybridization (Wild strain 13817 W, Aerial Mycelium strain 13817 AM and Vegetative Mycelium strain 13817 VM). The strains were tested for the production of amylolitic enzymes in alternative media. Maximum yields for both enzymes were observed in starch-casein. Higher α-amylase was obtained with strain 13817 W in starch-urea, and amyloglucosidase with strain 13817 AM in starch-ammonium that are economic sources and may be important for industrial purposes. Type strain (DAUFPE 13817(T)=KCTC 9972(T)=DSM 44943(T)).

Antitumor anthraquinones from an endophytic actinomycete Micromonospora lupini sp. nov.

Two novel anthraquinones, lupinacidins A ( 1) and B ( 2), have been isolated from the culture broth of a new endophytic actinomycete belonging to the genus Micromonospora. Lupinacidins were found to show significant inhibitory effects on the invasion of murine colon 26-L5 carcinoma cells without inhibiting cell growth.

Disease resistance induced by nonantagonistic endophytic Streptomyces spp. on tissue-cultured seedlings of rhododendron

Of 82 strains of endophytic actinomycetes isolated from rhododendron plants, 12 were not antagonistic against Pestalotiopsis sydowiana, which is the causal agent of Pestalotia disease. Of these 12, MBR-37 and MBR-38 (identified as Streptomyces spp.) grew on IMA-2 medium. Tissue-cultured seedlings of rhododendron treated with these nonantagonistic strains showed less wilting and/or smaller lesions to P. sydowiana, although the degree of resistance was a little lower than that conferred by antagonistic Streptomyces galbus strain R-5. These seedlings accumulated the anthocyanin(s), suggesting that resistance induced by these strains could depend on activated defense responses associated with the phenylpropanoid pathway rather than with antibiosis.

Bioactivities of Endophytic Actinomycetes from Selected Medicinal Plants in the World Heritage Site of Saint Katherine, Egypt

Bioactivities of Endophytic Actinomycetes from Selected Medicinal Plants in the World Heritage Site of Saint Katherine, Egypt

Identification ofStreptomyces sp. Tc022, an endophyte inAlpinia galanga, and the isolation of actinomycin D

Some endophytic actinomycetes (120) were isolated from the roots ofAlpinia galanga. Identification of these endophytes was based on their morphology and amino acid composition of the whole-cell extract. Most isolates were classified aStreptomyces sp. (82), with the remainder belonging toNocardia sp. (11),Microbispora sp. (3) andMicromonospora sp. (2). Eight isolates were unclassified and 14 were lost during subculture. The strain identified as endophyticStreptomyces sp. Tc022 strongly inhibitedColletotrichum musae andCandida albicans. This endophyte was cultured, the agar was extracted with organic solvent and the extract was purified on a column of silica gel to give a major component, which was identified to be actinomycin D on the basis of spectroscopic dat Actinomycin D showed antifungal activity againstColletotrichum musae andCandida albicans with the MIC of 10 and 20 mg ml−1, respectively.

Isolation of Endophytic Actinomycetes from Different Cultivars of Tomato and their Activities Against Ralstonia solanacearum in Vitro

The populations of endophytic actinomycetes from healthy and wilting tomato plants (tomato cultivars resistant and susceptible to Ralstonia solanacearum) grown in three different sites from Guangzhou, Guangdong Province, South China were investigated by cultivation methods. Most of the isolates belonged to streptomycetes. The Aureus group of Streptomyces was the most frequently isolated group. The population composition of Streptomyces varied according to tomato cultivars, physiological status and soil types. The proportions of antagonistic Streptomyces strains from healthy plants were higher than that from wilting plants (P 0.05), but the difference was found from the different sampling sites (P < 0.05). The percentage of bacterial cell wall-degrading streptomycetes from wilting tomato was higher than that from healthy plants (P < 0.05). These results indicated that the cultivar of the host plant, physiological status and sampling sites would influence the proportion of endophytic streptomycetes with different physiological traits. Diversity of endophytic Streptomyces and their physiological diversity should be involved in developing potential biocontrol agents.

Endophytic Actinomycetes and Their Interactions with Host Plants

Endophytic Actinomycetes and Their Interactions with Host Plants

An Endophytic Actinomycete, Streptomyces sp. MBR-52, That Accelerates Emergence and Elongation of Plant Adventitious Roots

More than 100 isolates of endophytic actinomycetes were obtained from field-grown rhododendron. By root-growth tests using cucumber germlings, one isolate, MBR-52, was selected as a final candidate to accelerate rooting of tissue-cultured rhododendron. Re-isolation test proved that when tissue-cultured seedlings of rhododendron were treated with MBR-52 in flasks, this isolate colonized in the seedlings and survived there even after their transplanting in soil. Another transplanting test revealed that emergence of adventitious roots was prominently accelerated in the MBR-52-treated tissue-cultured seedlings, suggesting that MBR-52 might release some type of rooting-promoting plant hormone(s). In addition to nucleotide sequence analysis of 16S rDNA, cultural, physiological, chemical and morphological characterizations showed that MBR-52 belongs to a strain of Streptomyces sp. MBR-52 certainly gives a great practical advantage to shorten the acclimatization period of tissue-cultured seedlings in warm and humid environment which very frequently increases the disease-infection risk of the seedlings.

Scanning electron microscopy of some endophytic streptomycetes in snakevine-Kennedia nigricans

Soils of all types and locations have generally served as the major sources of streptomycetous bacteria. These organisms are the source of nearly 80% of the world's antibiotics. Now, it is realized that Streptomyces spp. (within the group of prokaryotic filamentous bacteria known as actinomycetes) can exist as endophytes within the interstices of some higher plants. While it is sometimes possible to isolate one or two different streptomycetes from certain plants, most plants are free of these organisms. However, the snakevine (Kennedia nigricans) of the Northern Territory of Australia has yielded at least 39 different endophytic actinomycetes (95% of them being Streptomyces spp.) Most of these isolates possessed no detectable antibiotic properties, while at least seven had antibacterial and antifungal activities. Examination of eight selected cultures by scanning electron microscopy (SEM) as well as environmental scanning electron microcopy (FEI ESEM FEG) (FEI Company, Hillsobro, Ore., USA) revealed unusual patterns, structures, and features of the spores and hyphae of these microorganisms. For instance, as revealed by ESEM FEG for the first time, it has become obvious that extremely fine hair-like structures (average 25-49 nm with gold-coated specimens) exist on the spores and hyphae of some endophytic streptomycetes. The biological purpose of these hair-like protrusions is unknown. Both SEM and ESEM FEG can be effectively used as tools in identification and elucidation of the biology of these organisms. In addition, unusual colony morphology, observed with the unaided eye can very easily be used to distinguish some of these isolates since characteristic donut and pseudo-horn shaped colonies appeared in culture.

Isolation and characterization of endophytic streptomycete antagonists of fusarium wilt pathogen from surface-sterilized banana roots

A total of 131 endophytic actinomycete strains were successfully isolated from surface-sterilized banana roots. These isolates belonged to Streptomyces ( n = 99), Streptoverticillium ( n = 28), and Streptosporangium ( n = 2) spp. The remaining 2 isolates were not identified. About 18.3% of the isolates inhibited the growth of pathogenic Fusarium oxysporum f. sp. cubense on banana tissue extract medium. The most frequently isolated Streptomyces sp. strain S96 was similar to Streptomyces griseorubiginosus. About 37.5% of the S. griseorubiginosus strains were antagonistic to F. oxysporum f. sp. cubense. The antagonism of strain S96 was lost when FeCl 3 was introduced into the inhibition zone. In vivo biocontrol assays showed that the disease severity index (DSI) was significantly ( P = 0.05) reduced and mean fresh weight increased ( P = 0.001) in plantlets treated with strain S96 compared to those grown in the absence of the biocontrol strain. These findings indicate the potential of developing siderophore-producing Streptomyces endophytes for the biological control of fusarium wilt disease of banana.