Isolation Of Bacterial Endophytes Research Articles

Endophytic Bacillus spp. from native chilli cultivars and their effect against fruit rot pathogen of Bhut Jolokia (Capsicum chinense Jacq.)

BackgroundFruit rot disease is one of the most important factors limiting the production potential of Bhut Jolokia (Capsicum chinense Jacq.), which is known as one of the hottest chillies in the world. The management strategies are highly dependent on synthetic chemicals which are causing a detrimental impact on the environment. Considering the factors, this study focuses on exploring potential endophytic microflora from native chilli cultivars which can suppress the fruit rot pathogen.ResultsEndophytic microflora occurs ubiquitously in plants that possess various plant-benefiting abilities. A total of 34 endophytic isolates were obtained from different chilli cultivars. These endophytic isolates were subjected to screening in vitro for their potential to suppress the incitant pathogen Colletotrichumgloeosporioides (the causal agent of chilli fruit rot), which was confirmed based on cultural, morphological, pathogenicity and molecular studies. The preliminary screening yielded four bacterial endophytic isolates capable of suppressing the pathogen which was found non-pathogenic to Bhut Jolokia plant. On the basis of morphological, biochemical and molecular identification, the four most promising isolates were identified as Bacillus velezensis, B. mycoides, B. altitudinis and B. cereus, respectively, and used for further in vitro tests. B. velezensis showed the highest inhibition (68.67%) on mycelial growth of C. gloeosporioides, followed by B. mycoides (65.33%), B. altitudinis (52.89%) and B. cereus (45.33%). Among the compatible combination, the highest efficacy (56.00%) was found in the combination of B. velezensis and B. altitudinis.ConclusionFrom the present study, it can be concluded that B. velezensis and B. mycoides alone and in combination can be used as potential biocontrol agent in managing the fruit rot of Bhut Jolokia considering their performance in field conditions.

Harnessing the Antifungal Potential of Seed Bacteriome against Major Seed and Soil-borne Pathogens in Blackgram

Blackgram is an important pulse crop in India. One of the major constraints on blackgram production is the attack of pathogens, which leads to yield loss. Nowadays, with the understanding of harmful effects of chemical fungicides, biocontrol methods are gaining more importance. One such method is biopriming with endophytes to control pathogens and enhance yield. In the present study, the endophytes of blackgram seeds was explored and tested for its biocontrol potential against major seed- and soil-borne pathogens and growth promotion in blackgram. A total of 14 bacterial endophytic isolates were obtained and screened against Macropomina phaseolina and Fusarium oxysporum. Among them, BSE5 isolate was proven to be more efficient in inhibiting the mycelial growth of M. phaseolina and F. oxysporum, with per cent inhibition of 22.5 and 50.6, respectively, following BSE9. Both the isolates showed improved plant growth parameters like germination percentage, root length, shoot length, dry matter production and vigour index. Molecular characterisation of BSE5 and BSE9 confirmed that the isolates are Pseudomonas aeruginosa and Bacillus xiamenensis.

Maize Apoplastic Fluid Bacteria Alter Feeding Characteristics of Herbivore (Spodoptera frugiperda) in Maize.

Maize is an important cereal crop which is severely affected by Spodoptera frugiperda. The study aims to identify endophytic bacteria of maize root and leaf apoplastic fluid with bioprotective traits against S. frugiperda and plant growth promoting properties. Among 15 bacterial endophytic isolates, two strains—namely, RAF5 and LAF5—were selected and identified as Alcaligenes sp. MZ895490 and Bacillus amyloliquefaciens MZ895491, respectively. The bioprotective potential of B. amyloliquefaciens was evaluated through bioassays. In a no-choice bioassay, second instar larvae of S. frugiperda fed on B. amyloliquefaciens treated leaves (B+) recorded comparatively lesser growth (1.10 ± 0.19 mg mg−1 day−1) and consumptive (7.16 ± 3.48 mg mg−1 day−1) rates. In larval dip and choice bioassay, the same trend was observed. In detached leaf experiment, leaf feeding deterrence of S. frugiperda was found to be greater due to inoculation with B. amyloliquefaciens than Alcaligenes sp. The phenolics content of B. amyloliquefaciens inoculated plant was also found to be greater (3.06 ± 0.09 mg gallic acid g−1). However, plant biomass production was more in Alcaligenes sp inoculated treatment. The study thus demonstrates the potential utility of Alcaligenes sp. and B. amyloliquefaciens for improving growth and biotic (S. frugiperda) stress tolerance in maize.

Activity of bacterial seed endophytes of landrace durum wheat for control of Fusarium foot rot

Five bacterial endophytic isolates obtained from durum wheat seeds (landrace “Timilia reste nere”) and identified as belonging to Pantoea (isolates A1, F7, F15 and G1) and Paenibacillus (isolate B) genera on the basis of 16S rDNA gene sequences, were assayed in vitro and in vivo for their ability to inhibit Fusarium culmorum growth and the disease (Fusarium foot rot) it causes in durum wheat. All isolates significantly reduced in vitro growth of F. culmorum in comparison with the control. After 120 hours of incubation, isolates B and G1 showed the greatest mycelial growth inhibition, i.e., respectively, 76 and 74%. When durum wheat “Simeto” seeds were treated with bacterial isolates singly or in combinations and then inoculated with F. culmorum, all treatments with endophytes showed increased, but not statistically significant, seed germination. Except for isolate A1, all bacterial isolates stimulated vegetative parameters of durum wheat seedlings. Mixture of isolates F7 + F15 was the most effective in improving shoot height (+94%), root length (+47%) and vigour index (+81%). Mixture of isolates A1 + B reduced Fusarium foot rot incidence (-21%) and severity (-30%), and isolate A1 reduced incidence (-15%) and severity (-16%) of the disease. These results indicate potential of bacterial seed endophytes, identified in this study, for control of Fusarium foot rot and suggest that bacterial seed endophytes may provide a new biocontrol agent for an environmentally sustainable durum wheat disease management programme.

Effect of the endophytic plant growth promoting Enterobacter ludwigii EB4B on tomato growth

Summary This study aims to develop a biocontrol agent against Fusarium oxysporum f.sp. radicis-lycopersici (FORL) in tomato. For this, a set of 23 bacterial endophytic isolates has been screened for their ability to inhibit in vitro the growth of FORL using the dual plate assay. Three isolates with the most sound antagonistic activity to FORL have been qualitatively screened for siderophore production, phosphates solubilization and indolic acetic acid (IAA) synthesis as growth promotion traits. Antagonistic values of the three candidates against FORL were respectively: 51.51 % (EB4B), 51.18 % (EB22K) and 41.40 % (EB2A). Based on 16S rRNA gene sequence analysis, the isolates EB4B and EB22K were closely related to Enterobacter ludwigii EN-119, while the strain EB2A has been assigned to Leclercia adecarboxylata NBRC 102595. The promotion of tomato growth has been assessed in vitro using the strains EB2A, EB4B and EB22K in presence of the phytopathogen FORL. The treatments with the selected isolates increased significantly the root length and dry weight. Best results were observed in isolate EB4B in terms of growth promotion in the absence of FORL, improving 326.60 % of the root length and 142.70 % of plant dry weight if compared with untreated controls. In the presence of FORL, the strain EB4B improved both root length (180.81 %) and plant dry weight (202.15 %). These results encourage further characterization of the observed beneficial effect of Enterobacter sp. EB4B for a possible use as biofertilizer and biocontrol agent against FORL.

Characterization of Antifungal Volatile Organic Compounds Produced by Bacterial Endophytes against Fusarium oxysporum through GC-MS Analysis

Banana (Musa spp.) is one of the most important fruit crops cultivated throughout the world. Fusarium wilt caused by the fungus, Fusarium oxysporum f. sp. cubense (E.F. Smith) Snyder & Hansen (Foc) causes significant threats to banana production. Endophytic bacteria play a significant role in plant protection against soil borne plant pathogens and promote overall productivity of agricultural ecosystems. Secondary metabolites produced by several species of Bacillus have been found to possess antibacterial or antifungal activity against different isolates of Fusarium oxysporum f. sp. cubense. These were identified by crude tests using agar gel diffusion and GC-MS analysis of the fungal crude extracts. Understanding of the metabolites produced by endophytes is also important in the context of biological control of soil-borne diseases of banana. GC-MS analysis of crude antibiotic extracts of bacterial endophytic isolates viz., GNBS3, PVBS3, NPBS4, KVBS4, BS1, revealed DL-Proline,5 Oxo as the major compound. Having both antifungal and antibacterial activity.

Non rhizobial endophytic bacteria from chickpea (Cicer arietinum L.) tissues and their antagonistic traits

Bacteria that colonize plant tissues other than rhizobia and are beneficial for plant growth referred to non rhizobial plant growth-promoting endophytic bacteria (PGPEB). This study was designed to assay the biocontrol activity of plant growth promoting endophytic bacterial isolates those found positive for P. solubilization, ACC deaminase, Indole acetic acid and Gibberelic acid production. These bacterial isolates were obtained from chickpea (Cicer arietinum L.) tissues (roots and nodules). In a previous study a total of 263 non rhizobial endophytic bacterial isolates were isolated. Out of 263 isolates, 64.5% and 34.5% were Gram positive and negative, respectively. Further for biochemical characterization, catalase, oxidase, citrate utilization, nitrate reduction, methyl red and Voges Proskauer’s tests, were performed. On the basis of P solubilization, ACC deaminase, Indole acetic acid and Gibberelic acid production 75 potential isolates were selected and screened for their biocontrol activity viz. (production of cell wall degrading enzymes, production of HCN and fluorescent pigment). Out of 75 isolates, only 29 isolates produced cellulase, 64 isolates were able to produce protease and 28 were positive for both cellulose and protease. Of 75 endophytic isolates 12 isolates (7 from root tissue and 5 from nodules tissue, respectively) were positive for HCN production and 16 isolates were found to be fluorescent pigment producer under µv ligh. As chemical fertilizers and pesticides have detrimental effects on the environment. So these bacterial endophytic isolates will be used not only as a biofertilizer because of their plant growth promotional activities but also used as an alternative of synthetic chemicals for control of several plant diseases.

Isolation of Bacterial Endophytes from Phalaris arundinacea and their Potential in Diclofenac and Sulfamethoxazole Degradation.

Diclofenac (DCF), a non-steroidal anti-inflammatory drug (NSAID) and sulfamethoxazole (SMX), an antimicrobial agent, are in common use and can be often detected in the environment. The constructed wetland systems (CWs) are one of the technologies to remove them from the aquatic environment. The final effect of the treatment processes depends on many factors, including the interaction between plants and the plant-associated microorganisms present in the system. Bacteria living inside the plant as endophytes are exposed to secondary metabolites in the tissues. Therefore, they can possess the potential to degrade aromatic structures, including residues of pharmaceuticals. The endophytic strain MG7 identified as Microbacterium sp., obtained from root tissues of Phalaris arundinacea exposed to DCF and SMX was tested for the ability to remove 2 mg/l of SMX and DCF in monosubstrate cultures and in the presence of phenol as an additional carbon source. The MG7 strain was able to remove approximately 15% of DCF and 9% of SMX after 20 days of monosubstrate culture. However, a decrease in the optical density of the MG7 strain cultures was observed, caused by an insufficient carbon source for bacterial growth and proliferation. The adsorption of pharmaceuticals onto autoclaved cells was negligible, which confirmed that the tested strain was directly involved in the removal of DCF and SMX. In the presence of phenol as the additional carbon source, the MG7 strain was able to remove approximately 35% of DCF and 61% of SMX, while an increase in the optical density of the cultures was noted. The higher removal efficiency can be explained by adaptive mechanisms in microorganisms exposed to phenol (i.e. changes in the composition of membrane lipids) and by a co-metabolic mechanism, where non-growth substrates can be transformed by non-specific enzymes. The presence of both DCF and SMX and the influence of the supply frequency of CWs with the contaminated wastewater on the diversity of whole endophytic bacterial communities were demonstrated. The results of this study suggest the capability of the MG7 strain to degrade DCF and SMX. This finding deserves further investigations to improve wastewater treatment in CWs with the possible use of pharmaceuticals-degrading endophytes.

Isolation of bacterial endophytes from Actinidia chinensis and preliminary studies on their possible use as antagonists against Pseudomonas syringae pv. actinidiae

Isolation of bacterial endophytes from Actinidia chinensis and preliminary studies on their possible use as antagonists against Pseudomonas syringae pv. actinidiae

Antimicrobial Activity of Isolated Bacterial Endophytes from Cichorium intybus L, Pelargonium hortorum, and Portulaca oleracea Against Human Nosocomial Bacterial Pathogens

Background: Bacterial endophytes are colonizers of the inner plant tissues in which they do not normally cause any substantial morphological changes or disease symptoms. Endophytic bacteria are safe microorganisms that reside within the plant hosts and are known to enhance the growth and development of host plants, probably by secreting growth hormones. These bacteria are known to enhance growth and products of plants by fixing atmospheric nitrogen, solubilization of phosphate, production of phytohormones and siderophores, and possession of antagonistic activity, as well as reducing the level of stress ethylene in host plants. Objectives: In this descriptive study, we focused on the isolation of bacterial endophytes from three medicinal plants Cichorium intybus L, Pelargonium hortorum, and Portulaca oleracea and screened them for activities against nosocomial isolates of Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeroginosa, and Acinetobacter baumannii. Methods: Random samples from asymptomatic leaves and branches of three medicinal plants (Cichorium intybus L, Pelargonium hortorum, and Portulaca oleracea) were collected. To isolate the endophytic bacteria, the disinfected portions of the plants were distributed onto the isolation media. To examine endophytic bacterial contents, bioassays were conducted using growing colonies in PA and YEA, inactivating them by chloroform. To test the antibacterial activity of the endophytic bacterial culture broth, filter-sterilized supernatants were poured into cylinders on each bacterial plate. Results: A total of 24 phenotypically distinguishable bacterial endophytes were isolated in pure form from three medicinal plants. In part of the chloroform-inactivated colonies of all 24 isolated endophytes, the most effective herb was C. intybus L, followed by Po. oleracea, and in part of the supernatant culture broth, the most effective herb was Po. oleracea, followed by C. intybus L. Conclusions: Endophytic microorganisms residing in Cichorium intybus L, Portulaca oleracea, and Pelargonium hortorum are a very promising source for production of bioactive compounds. In general, most isolated endophytes had an acceptable effect against indicator bacterial pathogens.

Antimicrobial Activity of Isolated Bacterial Endophytes from Cichorium intybus L, Pelargonium hortorum, and Portulaca oleracea Against Human Nosocomial Bacterial Pathogens

Background: Bacterial endophytes are colonizers of the inner plant tissues in which they do not normally cause any substantial morphological changes or disease symptoms. Endophytic bacteria are safe microorganisms that reside within the plant hosts and are known to enhance the growth and development of host plants, probably by secreting growth hormones. These bacteria are known to enhance growth and products of plants by fixing atmospheric nitrogen, solubilization of phosphate, production of phytohormones and siderophores, and possession of antagonistic activity, as well as reducing the level of stress ethylene in host plants. Objectives: In this descriptive study, we focused on the isolation of bacterial endophytes from three medicinal plants Cichorium intybus L, Pelargonium hortorum, and Portulaca oleracea and screened them for activities against nosocomial isolates of Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeroginosa, and Acinetobacter baumannii. Methods: Random samples from asymptomatic leaves and branches of three medicinal plants (Cichorium intybus L, Pelargonium hortorum, and Portulaca oleracea) were collected. To isolate the endophytic bacteria, the disinfected portions of the plants were distributed onto the isolation media. To examine endophytic bacterial contents, bioassays were conducted using growing colonies in PA and YEA, inactivating them by chloroform. To test the antibacterial activity of the endophytic bacterial culture broth, filter-sterilized supernatants were poured into cylinders on each bacterial plate. Results: A total of 24 phenotypically distinguishable bacterial endophytes were isolated in pure form from three medicinal plants. In part of the chloroform-inactivated colonies of all 24 isolated endophytes, the most effective herb was C. intybus L, followed by Po. oleracea, and in part of the supernatant culture broth, the most effective herb was Po. oleracea, followed by C. intybus L. Conclusions: Endophytic microorganisms residing in Cichorium intybus L, Portulaca oleracea, and Pelargonium hortorum are a very promising source for production of bioactive compounds. In general, most isolated endophytes had an acceptable effect against indicator bacterial pathogens.

Antibacterial Activities of Bacterial Endophytes Isolated From Zataria multiflora, Achillea willhelmsii and Calendula officinalis L. Against Some Human Nosocomial Pathogens

Background: Endophytes are microorganisms which live within plant tissues without causing any apparent harm to their host. Endophytes have been found in nearly all plant families and can be a source of metabolites that are potential source of antibacterial drugs. Objectives: In this study, we focused on the isolation of bacterial endophytes from three medicinal plants; Zataria multiflora, Achillea willhelmsii and Calendula officinalis L. and screening them for activities against some human bacterial pathogens isolated from nosocomial infections. Methods: In this descriptive study random samples from asymptomatic leaves and branches of three medicinal plants namely: Zataria multiflora, Achillea willhelmsii and Calendula officinalis L. were collected from Chaharmahal Va Bakhtiari province of Iran in spring 2013. For isolation of endophytic bacteria, the disinfected portions of the plants were distributed onto the isolation media. To examine endophytic bacterial contents, the bioassays were conducted with using growing colonies in peptone agar (PA) and yeast extract agar (YEA) media, then inactivated them by chloroform. To assay antibacterial activity of endophytic bacterial culture broth, filter-sterilized supernatants were poured in cylinders on each bacterial plate. Results: Nine discernible bacterial endophytes were isolated in purification from three medicinal plants. Generally, all isolated endophytes showed acceptable effects against indicator bacteria, however endophytes isolated from Zataria multiflora and Achillea willhelmsii were more effective against Staphylococcus aureus than other endophytes. Conclusions: Endophytes of examined medicinal plants could be a good source of antibacterial substances.

Bakteri Endofit dari Tanaman Kehutanan sebagai Pemacu Pertumbuhan Tanaman Tomat dan Agens Pengendali Meloidogyne sp.

Meloidogyne sp. is one of the main constraints of tomato production in Indonesia. Endophytic bacteria may be considered as biocontrol agents for controlling Meloidogyne sp. The objective of this study was to isolate endophytic bacteria from forestry plants and to evaluate its potential for controling Meloidogyne sp. on tomato. Endophytic bacteria were isolated from roots of mahoni ( Swietenia mahogany ), trambesi ( Albizia saman ), gaharu ( Aquilaria malacensis ), and meranti ( Shorea sp.). Isolation of bacterial endophytes from plant tissue was conducted using surface sterilization method with 70% alcohol, 3% NaOCl and sterile water on medium trypsic soy agar. Endophytic bacteria was separated and purified based on shape and color of the colony. A total of 33 isolates of endophytic bacteria were isolated from roots of mahoni (11 isolates), trambesi (5 isolates), gaharu (7 isolates), and meranti (10 isolates). The bacteria was tested for the hypersensitivity reaction on tobacco plants and the result showed that 22 isolates did not cause necrosis, indicated they are not pathogenic. Ten isolates of endophytic bacteria was selected for further experiment, i.e. to evaluate their potential as biocontrol agent for Meloidogyne sp. and as growth promotor for tomato plants. The experiment was conducted in the screenhouse using seed treatment. The result showed that two isolates of endophytic bacteria, i.e. MSJ1H and AGS1F were able to increase the growth of tomato plants up to 60% and reduce the number of root knot caused by Meloidogyne sp. Endophytic bacteria isolated from forestry plants have the potential as a biocontrol agents to plant parasitic nematode Meloidogyne sp.

Biocontrol potential of endophytes harbored in Radula marginata (liverwort) from the New Zealand ecosystem.

Radula marginata and Cannabis sativa L. are two phylogenetically unrelated plant species containing structurally similar secondary metabolites like cannabinoids. The major objective of our work was the isolation, identification, biocontrol efficacies, biofilm forming potential and anti-biofilm ability of endophytic microbial community of the liverwort R. marginata, as compared to bacterial endophytic isolates harbored in C. sativa plants. A total of 15 endophytic fungal and 4 endophytic bacterial isolates were identified, including the presence of a bacterial endosymbiont within an endophytic fungal isolate. The endosymbiont was visible only when the fungus containing it was challenged with two phytopathogens Botrytis cinerea and Trichothecium roseum, highlighting a tripartite microbe-microbe interaction and biocontrol potency of endophytes under biotic stress. We also observed sixteen types of endophytic fungal-pathogen and twelve types of endophytic bacterial-pathogen interactions coupled to varying degree of growth inhibitions of either the pathogen or endophyte or both. This showed the magnitude of biocontrol efficacies of endophytes in aiding plant fitness benefits under different media (environmental) conditions. Additionally, it was ecologically noteworthy to find the presence of similar endophytic bacterial genera in both Radula and Cannabis plants, which exhibited similar functional traits like biofilm formation and general anti-biofilm activities. Thus far, our work underlines the biocontrol potency and defensive functional traits (in terms of antagonism and biofilm formation) of endophytes harbored in liverwort R. marginata as compared to the endophytic community of phylogenetically unrelated but phytochemically similar plant C. sativa.

Isolation and functional characterization of bacterial endophytes from Carica papaya fruits

To isolate and characterize the endophytes from papaya fruits and to determine the fermentative potential of the strains. Endophytes provide potential sources for novel natural products for the use in agriculture and nutrition. There is very limited information on isolation and characterization of bacterial endophytes from papaya. We describe isolation and characterization of eighteen endophytes of papaya fruit from four economically important papaya varieties viz 'Red lady', 'Solo', 'Coorg Honey' and 'Bangalore'. The phylogenetic analysis based on the 16S rRNA sequence revealed that isolated endophytes are genetically distinct and cluster as discrete clades in the dendrogram. The Bacillus species is a predominant bacterial endophyte across papaya varieties. The seeds and the endocarp of papaya fruits harbour Kocuria, Acinetobacter and Enterobacter species. The Staphylococcus species were detected in the fruit mesocarp of two papaya varieties used in the study. The endophytes isolated from papaya fruits were capable of producing extracellular enzymes like amylase, cellulase, pectinase and xylanase. Three isolates, Bacillus (PE-LR-1 and PE-LR-3) and Kocuria (PE-LR-2), were selected for fruit fermentation, and antioxidant potential of the fermented product was evaluated. PE-LR-3 fermented product has the free radical scavenging activity of 61·2% and a microbial cocktail of PE-LR-3 with Saccharomyces cerevisiae MTCC 2918 enhances the antioxidant potential to 75·7%. These findings suggest that different parts of papaya fruits harbour an array of bacterial endophytes that could be important agents in attributing the high nutritive status to the fruit and can serve as potent microbial cocktails for developing value-added fermented products of this important fruit. This study describes isolation of a bacterial endophyte from papaya fruit that is capable of improving the antioxidant potential of raw papaya after fermentation.

Isolation of bacterial endophytes from germinated maize kernels

The germination of surface-sterilized maize kernels under aseptic conditions proved to be a suitable method for isolation of kernel-associated bacterial endophytes. Bacterial strains identified by partial 16S rRNA gene sequencing as Pantoea sp., Microbacterium sp., Frigoribacterium sp., Bacillus sp., Paenibacillus sp., and Sphingomonas sp. were isolated from kernels of 4 different maize cultivars. Genus Pantoea was associated with a specific maize cultivar. The kernels of this cultivar were often overgrown with the fungus Lecanicillium aphanocladii; however, those exhibiting Pantoea growth were never colonized with it. Furthermore, the isolated bacterium strain inhibited fungal growth in vitro.