Non-streptomycete Actinomycetes Research Articles

Cloning, expression and in silico studies of a serine protease from a marine actinomycete (Nocardiopsis sp. NCIM 5124)

A serine protease (N. protease), from Nocardiopsis sp., was cloned and expressed in Escherichia coli and investigated for its potential kinetic stability. Protein expression using two vectors, pET-22b (+) and pET-39b (+) was compared based on proper folding and soluble expression of the protein. pET-39b (+) was found to be a better vector for soluble expression of this protease containing disulfide bonds. In silico studies were also carried out for N. protease. Homology modeling suggested N. protease to be a member of PA clan of proteases. The phylogenetic analysis showed relatedness of N. protease to kinetically stable proteases. Molecular docking studies performed exhibited interaction of a peptide substrate with catalytic pocket of the enzyme. High temperature MD simulations were performed on N. protease to study its unfolding behavior and comparisons were made with αLP. A novel approach to study ‘cooperativity’ of protein unfolding was undertaken, wherein ‘P’ value analysis based on φ and ψ values of the protein was performed. Data showed sharper P value transition for αLP when compared to N. protease thus indicating relatively less kinetic stability of N. protease. Present study holds significance as the non-streptomycete actinomycetes group is least explored and ensures industrially important enzymes with exceptional stabilities.

Non-streptomycete actinomycetes nourish the current microbial antibiotic drug discovery.

OPINION article Front. Microbiol., 20 August 2013Sec. Antimicrobials, Resistance and Chemotherapy Volume 4 - 2013 | https://doi.org/10.3389/fmicb.2013.00240

A Novel Method Isolated Microorganisms in Soil Granule

A novel method isolated microorganisms in soil granule was built. The key steps included: repeated elutriation of soil by sterilized water, inoculation on the plates with the elutriated sediments, incubation of the plates and isolation of the actinomycetes by using selected culture medium. We formulated that most microflora included the dominant actinomycetes in the soil were carried away with the sterilized water in the elutriation procedure, some rare actinomycetes and few other microflora included bacteria were remained in the elutriated sediments, the other microflora were excluded to grew into colonies on the plates by using selective culture medium for actinomycetes in the elutriated sediments. Results showed the supposition. Non-streptomycete actinomycetes were isolated both from black soil samples from Chinese northeast area and compost samples from Chinese central area. Soil fungi in granule were isolated by using the selective conditions to favor fungi. The results showed that the method was effective.

Promotion of growth of bean ( Phaseolus vulgaris L.) in a calcareous soil by a phosphate-solubilizing, rhizosphere-competent isolate of Micromonospora endolithica

The aim of this study was to determine the potential of non-streptomycete actinomycetes (NSA) to solubilize insoluble phosphates in soil and to promote plant growth. Thirty-one NSA were isolated using Streptomyces phage and dry heat techniques, from a calcareous soil deficient in available phosphorus (P) in the United Arab Emirates. Nine of these NSA isolates solubilized powdered rock phosphate (PRP) in solid and liquid media. Five isolates were initially selected based on their rhizosphere competence. Among them, an isolate of Micromonospora endolithica caused a significant drop in pH in a liquid medium amended with PRP, solubilized considerable amounts of P, produced acid and alkaline phosphatases, as well as a variety of organic acids. In addition, this isolate of M. endolithica was chosen from among the five isolates because it also showed exceptional rhizosphere competence and a strong ability to colonize bean ( Phaseolus vulgaris L.) roots up to a depth of 14 cm. In the greenhouse, the application of M. endolithica to soil amended with either single super-phosphate (SP) or PRP significantly promoted the growth of roots and shoots of bean plants compared with those of plants grown in non-inoculated soil amended with SP or PRP. This was also evident in the significant increases in the concentration of available P in the soil and in the levels of N, P, K, S, Mg, Fe and Zn in the roots and those of N, P, K, S, Mg and Fe in the shoots of inoculated plants. The plant growth promotion by M. endolithica was most pronounced in the presence of SP as soil amendment compared to PRP. In comparison, a non-phosphate-solubilizing, non-rhizosphere-competent NSA isolate of M. olivasterospora failed to increase available soil P, nutrient levels in roots and shoots or to promote plant growth. Plant growth regulators tested do not appear to be involved in the plant growth stimulation observed. Neither M. endolithica nor M. olivasterospora produced detectable levels of indole-acetic acid, indole-pyruvic acid, gibberellic acid, isopentenyl adenine, isopentenyl adenoside or zeatin in vitro. This study is the first published report to demonstrate the potential of phosphate-solubilizing NSA to promote plant growth. In addition, it is also the first published report of rhizosphere-competent actinomycetes capable of solubilizing SP or PRP, in soils.

Annual Meeting, Québec, Quebec, 2006, The Canadian Phytopathological Society / Réunion annuelle, Québec, Québec, 2006, La Société canadienne de phytopathologie

Annual Meeting, Québec, Quebec, 2006, The Canadian Phytopathological Society / Réunion annuelle, Québec, Québec, 2006, La Société canadienne de phytopathologie

Non-streptomycete actinomycetes as biocontrol agents of soil-borne fungal plant pathogens and as plant growth promoters

Among soil microorganisms, bacteria and fungi and to a lesser extent actinomycetes, have received considerable attention as biocontrol agents of soil-borne fungal plant pathogens and as plant growth promoters. Within actinomycetes, Streptomyces spp. have been investigated predominantly, mainly because of their dominance on, and the ease of isolation from, dilution plates and because of the commercial interest shown on the antibiotics produced by certain Streptomyces spp. Many of non-streptomycete actinomycetes (NSA) taxa are therefore rarely reported in literature dealing with routine isolations of biocontrol agents and/or plant growth promoters from plant and soil. It is clear that special isolation methods need to be employed in routine isolations to selectively isolate NSA. Some interesting information exists, albeit in relatively few reports compared to that on other microorganisms, on the biological activities of NSA, especially in relation to their mechanisms of action in the biological control of soil-borne fungal plant pathogens and plant growth promotion. This review presents an overview of this information and seeks to encourage further investigations into what may be considered a relatively unexplored area of research. Certain soil environmental factors, especially in horticultural systems, could be manipulated to render the soil conducive for the biological activities of NSA. A variety of NSA isolated by selective methods have not only shown to be rhizosphere competent but also adapted for an endophytic life in root cortices. Some of the NSA, including endophytic strains that have shown potential to suppress soil-borne fungal plant pathogens, are able to employ one or more mechanisms of antagonism including antibiosis, hyperparasitism and the production of cell-wall degrading enzymes. Strains of NSA promote plant growth by producing plant growth regulators. Enhancement of plant growth by the antagonists are considered to help the host by producing compensatory roots that mask the impact of root diseases.

Rhizosphere-competent isolates of streptomycete and non-streptomycete actinomycetes capable of producing cell-wall-degrading enzymes to controlPythium aphanidermatumdamping-off disease of cucumber

Fifty-eight streptomycete and 35 non-streptomycete actinomycetes were isolated from cucumber rhizosphere soil. These isolates were screened for the production of cell-wall-degrading enzymes using mycelial ( Pythium aphanidermatum (Edson) Fitzp.) fragment agar. Eighteen promising isolates were screened for their competence as root colonizers. Eight isolates showing exceptional rhizosphere competence significantly inhibited, in vitro, P. aphanidermatum, the causal agent of postemergence damping-off of cucumber ( Cucumis sativus L.) seedlings. The four most inhibitory isolates ( Actinoplanes philippinensis Couch, Microbispora rosea Nonomura and Ohara, Micromonospora chalcea (Foulerton) Ørskov, and Streptomyces griseoloalbus (Kudrina) Pridham et al.) produced in vitro β-1,3-, β-1,4-, and β-1,6-glucanases and caused lysis of P. aphanidermatum hyphae. None of these produced volatile inhibitors or siderophores. Only S. griseoloalbus produced diffusible inhibitory metabolites, whilst A. philippinensis and Micromonospora chalcea parasitized the oospores of P. aphanidermatum. These four isolates were subsequently tested in the greenhouse, individually or as a mixture, for their ability to suppress damping-off of cucumber seedlings in soil with or without cellulose amendment. The treatment, which included all four isolates in soil amended with cellulose, was significantly superior to all other treatments in suppressing damping-off and was nearly as good as the metalaxyl treatment. Results show that there is a potential to use a mixture of antagonistic rhizosphere-competent actinomycetes along with cellulose amendment rather than fungicides for the field management of this disease. This is the first study that has involved the screening of rhizosphere-competent non-streptomycete actinomycetes capable of producing cell-wall-degrading enzymes, for the management of Pythium diseases.

An endophytic chitinase-producing isolate of Actinoplanes missouriensis, with potential for biological control of root rot of lupin caused by Plectosporium tabacinum

Twenty-one streptomycete and 15 non-streptomycete actinomycetes were isolated from surface-disinfested lupin roots and evaluated for their potential to produce chitinase and to inhibit the growth of Plectosporium tabacinum, the causal agent of lupin root rot in Egypt. The most inhibitory isolate was identified as Actinoplanes missouriensis which produced relatively high levels of chitinase and degraded the hyphae of P.�tabacinum in vitro, causing extensive plasmolysis and cell-wall lysis. A crude culture filtrate of A. missouriensis exhibited antifungal activity and significantly (P < 0.05) reduced spore germination and germ-tube growth of the pathogen. The antagonist was recovered from inside the root at all samplings up to 8 weeks after inoculation, indicating that the roots of healthy lupin may be a habitat for the endophyte. A. missouriensis significantly (P < 0.05) reduced the severity of root rot under glasshouse conditions. An endophytic isolate of Actinoplanes italicus incapable of producing chitinase and a mutant strain of A. missouriensis that did not produce detectable levels of chitinase, did not lyse hyphae of P. tabacinum or reduce root rot in the glasshouse experiments, although colonisation of the lupin root by both these isolates was similar to that of the chitinase-producing wild-type isolate of A. missouriensis. This study is the first record of control of a soil-borne plant pathogen by a chitinolytic actinomycete, endophytic in plant roots.

Total microbial activity and microbial composition of a mangrove sediment are reduced by oil pollution at a site in the Arabian Gulf.

In a study carried out to determine the effect of oil pollution on the microbiota of sediment associated with mangroves in the United Arab Emirates, sediment samples were collected from oil-polluted and nonpolluted mangrove sites. The levels of the total recoverable hydrocarbons and the polycyclic aromatic hydrocarbons assayed were noticeably higher in the polluted sediment. Microbial activity as measured by the hydrolysis of fluorescein diacetate and by the total populations of the culturable aerobic and anaerobic bacteria, streptomycete and non-streptomycete actinomycetes, and filamentous fungi and yeasts was significantly (P < 0.05) lower in the polluted than in the nonpolluted sediment. The estimated total aerobic and anaerobic hydrocarbon-utilizing bacteria were significantly (P < 0.05) higher in the polluted than in the nonpolluted sediments. Four days after the addition of the water-soluble fractions of the light Arabian crude oil to the nonpolluted sediment, at 10 different concentrations, there was a significant (P < 0.05) reduction (65%) in the microbial activity of the sediment compared with that of nonamended sediment. Concentrations of water-soluble fractions at 0.1% and above significantly and progressively reduced microbial activity, with total cessation of activity recorded at levels >50%. This study is the first to evaluate the effect of oil pollution on aerobic and anaerobic microbial flora of sediment of mangrove communities.

Biological control of Sclerotinia minor using a chitinolytic bacterium and actinomycetes

Isolates of 85 bacteria and 94 streptomycete and 35 nonstreptomycete actinomycetes were obtained from a lettuce‐growing field in Al‐Ain, United Arab Emirates, on colloidal chitin agar, and screened for their ability to produce chitinase. Twenty‐three bacteria and 38 streptomycete and 15 nonstreptomycete actinomycete isolates produced high levels of chitinase and were examined in vitro for their ability to suppress the growth of Sclerotinia minor, a pathogen causing basal drop disease of lettuce. The three most suppressive isolates were examined further for their production of β‐1,3‐glucanase and antifungal activity as well as their ability to colonize the roots and rhizosphere of lettuce in vitro and in planta. The three isolates, Serratia marcescens, Streptomyces viridodiasticus and Micromonospora carbonacea, significantly reduced the growth of S. minor in vitro, and produced high levels of chitinase and β‐1,3‐glucanase. Streptomyces viridodiasticus also produced antifungal metabolite(s) that significantly reduced the growth of the pathogen in vitro. When the pathogen was presented as the sole carbon source, all three isolates caused extensive hyphal plasmolysis and cell wall lysis. Serratia marcescens and St. viridodiasticus were competent to varying degrees in colonizing the roots of lettuce seedlings after 8 days on agar plates and the rhizosphere within 14 days in pots, with their competency being superior to that of M. carbonacea. All three isolates, individually or in combination, were antagonistic to S. minor and significantly reduced incidence of disease under controlled glasshouse conditions.

Use of randomly amplified polymorphic DNA as a means of developing genus- and strain-specific Streptomyces DNA probes.

We have analyzed 20 randomly amplified polymorphic DNA (RAPD) primers against 36 Streptomyces strains, including 17 taxonomically undefined strains, 25 nonstreptomycete actinomycetes, and 12 outgroups consisting of gram-positive and -negative species. Most of the primers were useful in identifying unique DNA polymorphisms of all strains tested. We have used RAPD techniques to develop a genus-specific probe, one not necessarily targeting the ribosomal gene, for Streptomyces, and a strain-specific probe for the biological control agent Streptomyces lydicus WYEC108. In the course of these investigations, small-scale DNA isolations were also developed for efficiently isolating actinomycete DNA. Various modifications of isolation procedures for soil DNA were compared, and the reliability and specificity of the RAPD methodology were tested by specifically detecting the S. lydicus WYEC108 in DNA isolated from soil.

The potential for the biological control of cavity‐spot disease of carrots, caused by Pythium coloratum, by streptomycete and non‐streptomycete actinomycetes

Actinomycetes isolated from carrot rhizosphere were screened for their in vitro and in vivo antagonism to Pythium coloration Vaartaja, a causal agent of cavity-spot disease of carrots (Daucus carata L.). Forty-five streptomycete and non-streptomycete actinomycete isolates were screened for in vitro antagonism in a carrot bioassay. Of these, seven which reduced or prevented lesion formation were identified using cultural, morphological, physiological, biochemical and cell wall characteristics as Streptomyces janthimts, S. cinerochromogenes, Streptoverticilium netropsis, Actinomadura rubra, Actinoplanes philippinensis, Muromonospora carbonaceae, and Streptosporangium albidum. All seven isolates tested produced non-volatile antifungal metabolites, but failed to produce inhibitory volatile compounds. Actinoplanef philippincnsis and M. carbonacea grew epiphytically on the hyphae and oospores of P. eoloratum. The external surface of the oospores of the pathogen was heavily colonized by both hyperparasites, their hyphae were found to coil tightly around the oospore wall, and frequently caused cytoplasmic collapse of oospores. Sporangia of A. philippinensis were often seen to emerge from the colonized hyphae and oospores of P. eoloratum. None of the other actinomycete isolates showed hyperparasitism. All seven isolates significantly reduced the incidence of cavity spot in soil artificially infested with the pathogen in the glasshouse. Streptomyces janlhinus and Stfepto. albidum were the most effective in reducing the disease in inoculated plants. In addition, all the actinomycetes species except Ac. rubra and M. carbonacea, in the presence or absence of the pathogen, significantly (P < 0.05) increased mean fresh root weight compared to the treatment which included P. thorium only. This study shows that these actinomycetes have considerable potential for future use as biocontrol agents of cavity spot under natural field conditions. This is the first report of cavity-spot disease of carrots being controlled by microbial antagonists, and is the first report of non-streptomycete actinomycetes to control a Pythium disease.

Microbiological differences between limed and unlimed soils and their relationship with cavity spot disease of carrots (Daucus carota L.) caused by Pythium coloratum in Western Australia

Application of lime (4000 kg ha-1) to a soil used for commercial carrot production (pH 6.9) significantly (p<0.05) reduced the incidence of cavity spot disease of carrots compared to unlimed soil (pH 5.1). It significantly (p<0.01) increased soil microbial activity as measured by the hydrolysis of fluorescein diacetate and arginine ammonification. The application of lime resulted in a significant (p<0.01) increase in the total numbers of colony forming units (efu) of aerobic bacteria, fluorescent pseudomonads, Gram negative bacteria, actinomycetes and a significant (p<0.01) decrease in the cfu of filamentous fungi and yeasts compared to unlimed soil. Liming also increased the cfu of non-streptomycete actinomycetes rarely reported in similar studies. These non-streptomycete actinomycetes were estimated and isolated using polyvalent Streptomyces phages and the dry heat technique to reduce the dominance of streptomycetes on isolation plates. The non-streptomycete actinomycetes isolated included species of Actinoplanes, Micromonospora, Streptoverticillium, Nocardia, Rhodococcus, Microbispora, Actinomadura, Dactylosporangium and Streptosporangium. The numbers of actinomycetes antagonistic to Pythium coloratum, a causal agent of cavity spot disease of carrots increased in soil amended with lime. Application of lime also reduced the isolation frequency of P. coloratum from asymptomatic carrot roots grown in soil artificially infested with the pathogen, 3, 4 and 5 weeks after sowing.

A super lytic actinophage system as a pre-treatment in the isolation of non-streptomycete actinomycetes from soil

P.F. LONG AND G.E. AMPHLETT. 1996. An increase in the abundance and diversity of non‐streptomycete actinomycetes isolated from soils was achieved after the number of streptomycetes was reduced by pre‐incubating the soil with streptomycete‐specific actinophage. This method is described as a super lytic actinophage system.