High Antagonistic Activity Research Articles

Antibacterial Effect Of Bacillus species Biomolecules (Metabolites, Silver Nanoparticles, And Functionalized Silver Nanoparticles) Against Multi-Drug Resistance Listeria species

The study investigated the anti-listeria activity of Bacillus subtilis (BsaM), Bacillus licheniformis (BlbM) and Bacillus megaterium (BmcM) against seven Listeria species. Metabolites from Bacillus spp. were used for silver nanoparticles (BsaSNPs, BlbSNPs, and BmcSNPs) biosynthesis. The Silver Nanoparticles (SNPs) characterization was done using visual observation, UV-Visible Spectrophotometric, FTIR, SEM, and EDXRF. The anti-listeria synergistic potential of the biosynthesized SNPs and functionalized SNPs against Multidrug resistance (MDR) Listeria strains was evaluated. The BsaM, BlbM, and BmcM had a varied anti- listeria effect against the Listeria spp. 71.43, 100, and 5.71 % of the Listeria spp. were susceptible to the BsaM, BlbM, and BmcM metabolites respectively. The metabolite bio-actively reduced AgNO3 for BsaSNPs, BlbSNPs, and BmcSNPs production. Surface Plasma Resonance (SPR) peaks of 600 nm, 400 and 600 nm, and 600 nm were recorded for BsaSNPs, BlbSNPs, and BmcSNPs. The shape of the SNPs were aggregated, rods, and crystalline. The functional groups present in the nanoparticles were carboxylic acid, amino acid, alcohol, esters, and aldehydes had the highest intensity. BsaSNPs, BlbSNPs, and BmcSNPs had the highest anti-listeria potential of 20.0, 16.0 and 22.0 mm against Listeria innocua LA22A and Listeria ivanovii LA6. Functionalized BsaSNPs, BlbSNPs, and BmcSNPs had the highest anti-listeria activity compared to the metabolites and commercial antibiotics. Ciprofloxacin-BsaSNPs, Erythromycin-FBlbSNPs, and Erythromycin-FBmcSNPS had the highest antagonistic activity (28, 26, and 27 mm) against Listeria ivanovii LA6 respectively. The Bacillus strains’ metabolites, the SNPs, and functionalized SNPs exhibit antimicrobial activities against Multidrug resistance Listeria species. Functionalization improved the anti-listerial ability of the antibiotics. Keywords: Bacillus species, Anti-listeria activity, SNPs, MDR Listeria species, Functionalization.

Bacillus safensis isolated from white-leg shrimp, Penaeus vannamei in Taiwan with antagonistic activity against common Vibrio pathogens

Pathogenic Vibrio species have been notified as the main reason for mass mortality in marine aquaculture. In recent decades, the application of beneficial bacteria with antagonistic activity as probiotics has been chosen to be a promising disease prevention method which contributes significantly to antibiotic use reduction. In the present study, ten shrimps from a co-culture pond with red tilapia were collected to screen for Bacillus bacteria with antagonistic activity against four main Vibrio pathogenic species namely Vibrio parahaemolyticus, V. harveyi, V. alginolyticus, and V. vulnificus. Within 398 isolates, only 8 isolates showed clear activity against all four tested Vibrio spp. All 8 isolates were identified and confirmed to be the same species Bacillus safensis by using both 16S-rRNA and gyrB gene sequences and the BLAST tool. The most potent isolate VQV8 with the highest antagonistic activity was selected for studying common biological characteristics, growth in different conditions, hemolytic activity and antibiotic assessment activity. The results indicated that Bacillus safensis VQV8 grew better at the temperature of 25–37 °C. It could tolerate wide ranges of salinity and pH. This strain was found with high antagonistic activity on the tested Vibrio spp. Bacillus safensis strain VQV8 was found to be a non-hemolytic strain and highly sensitive to most tested antibiotics. Hence, our results suggested that Bacillus safensis strain VQV8 is a safe strain for probiotic purposes and a potent candidate for Vibrio disease prevention in white-leg shrimp culture.

Trichoderma asperellum GDFS1009 ‐mediated maize resistance against Fusarium graminearum stalk rot and mycotoxin degradation

Maize stalk rot caused by Fusarium graminearum is a major soil-borne maize disease, and biocontrol strategies are the best possible and eco-friendly solution to disease control and maize yield enhancement. In the past several years, the disease was successfully controlled with Trichoderma in China. However, little knowledge is available regarding the mechanism of the disease control and detoxification of Fusarium mycotoxin. Therefore, in the present study, we used physiological, biochemical, and microbiological methods to screen Trichoderma strains with high antagonistic activity to pathogens and studied the changes in major defence response genes, signal transduction, and mycotoxin degradation in maize through Trichoderma asperellum GDFS1009 against stalk rot diseases in both greenhouse and field conditions. T. asperellum significantly inhibited the growth of F. graminearum in the roots and stems of greenhouse and field plants. In addition, the plant height and fresh weight were also significantly increased. The expression of defence-related genes and signal transduction in maize plants showed that T. asperellum could upregulate the expression of the class III plant peroxidase (POD) gene, salicylic acid (SA) pathway-related gene pathogenesis-related protein 5(PR5), and jasmonic acid (JA)-related gene 12-oxo-phytodienoic acid reductase (OPR7) in roots, stems, and leaves of 5–6 leaf stage seedlings. During the tasseling period, Trichoderma upregulated the expression of PR5 in maize basal and middle stems and leaves, while OPR7 and allene oxide synthase (ACO) were upregulated in the basal stem segments. T. asperellum effectively reduced F. graminearum infection and degraded mycotoxins through upregulation of the UDP-glucuronosyltransferase (UGT) gene in the microbes and basal stem of maize plants, eventually increasing maize yield in both greenhouse and field trials.

Designing a synthetic microbial community devoted to biological control: The case study of Fusarium wilt of banana.

Fusarium oxysporum f. sp. cubense (Foc) tropical race 4 (TR4) is threatening banana production because of its increasing spread. Biological control approaches have been widely studied and constitute interesting complementary measures to integrated disease management strategies. They have been based mainly on the use of single biological control agents (BCAs). In this study, we moved a step forward by designing a synthetic microbial community (SynCom) for the control of Fusarium wilt of banana (FWB). Ninety-six isolates of Pseudomonas spp., Bacillus spp., Streptomyces spp., and Trichoderma spp. were obtained from the banana rhizosphere and selected in vitro for the antagonism against Foc TR4. In pot experiments, a large community such as SynCom 1.0 (44 isolates with moderate to high antagonistic activity) or a small one such as SynCom 1.1 (seven highly effective isolates) provided similar disease control (35% symptom severity reduction). An in vitro study of the interactions among SynCom 1.1 isolates and between them and Foc revealed that beneficial microorganisms not only antagonized the pathogen but also some of the SynCom constituents. Furthermore, Foc defended itself by antagonizing the beneficial microbes. We also demonstrated that fusaric acid, known as one of the secondary metabolites of Fusarium species, might be involved in such an interaction. With this knowledge, SynCom 1.2 was then designed with three isolates: Pseudomonas chlororaphis subsp. piscium PS5, Bacillus velezensis BN8.2, and Trichoderma virens T2C1.4. A non-simultaneous soil application of these isolates (to diminish cross-inhibition) delayed FWB progress over time, with significant reductions in incidence and severity. SynCom 1.2 also performed better than two commercial BCAs, BioPak® and T-Gro. Eventually, SynCom 1.2 isolates were characterized for several biocontrol traits and their genome was sequenced. Our data showed that assembling a SynCom for biocontrol is not an easy task. The mere mixtures of antagonists (e.g., SynCom 1.0 and 1.1) might provide effective biocontrol, but an accurate investigation of the interactions among beneficial microorganisms is needed to improve the results (e.g., SynCom 1.2). SynCom 1.2 is a valuable tool to be further developed for the biological control of FWB.

Bacteriocin-Producing Escherichia coli Isolated from the Gastrointestinal Tract of Farm Animals: Prevalence, Molecular Characterization and Potential for Application.

Due to the spread of antibiotic-resistant bacteria, new alternatives to antibiotics and ways to prevent infections are being sought. Bacteriocin-producing bacteria are therefore attracting attention due to their probiotic potential as a safe alternative to antimicrobial drugs. The aim of this work was to determine the prevalence of bacteriocin-encoded genes among Escherichia coli strains from healthy farm animals and to characterize the presence of virulence-associated genes, the possibility of prophage induction, and hemolytic and bacterial antagonistic activity of the bacteriocin-producing E. coli in order to reveal their potential for application. It was found that 17 of 72 E. coli strains (23.6%) produced bacteriocins. Among them, 18 out of 30 bacteriocin genes were detected: the most prevalent genes were those for microcin M (58.8%), colicin E1 (52.9%), and colicin M (35.3%). Colicin Ia (29.4%), colicin E9, colicin Ib, colicin B (23.5%), and colicin E9 (17.7%) genes were also frequent, while the prevalence of genes encoding microcins V, B17, and H47 and colicins E3, K, N, U, Y, 5, and 10 did not exceed 11.8%. At least two different bacteriocin genes were detected in all 17 bacteriocinogenic strains; the highest number of different bacteriocin genes detected in one strain was seven genes. E. coli strains with combinations of colicin E1 and E or microcin M and colicin E1 genes were more prevalent than others (17.7%). Among the 17 bacteriocin-producing E. coli strains, 5.9% were hemolytic, 47.1% contained prophages, and 58.8% carried genes encoding toxins. Cell-free supernatants of bacteriocin-producing strains were shown to inhibit the growth of pathogenic E. coli strains belonging to the APEC, STEC, and ETEC pathotypes. Thus, among the studied bacteriocin-producing E. coli isolated from the gastrointestinal tract of farm animals, three strains with high antagonistic bacterial activity and the absence of pathogenicity genes, prophages, and hemolytic activity were identified and therefore have potential for application.

Variation in Anti-inflammatory, Anti-arthritic, and Antimicrobial Activities of Different Extracts of Common Egyptian Seaweeds with an Emphasis on Their Phytochemical and Heavy Metal Contents

The anti-inflammatory, anti-arthritic, and antimicrobial activities of some common Egyptian seaweeds in addition to their phytochemical and heavy metal contents were investigated. Phytochemical screening of the seaweed extracts showed the presence of different primary and secondary metabolites with different concentrations according to their species and the used solvent. The ethanolic extract of Colpmenia sinuosa (CSBE2) exhibited the maximum anti-inflammatory and anti-arthritic activity at 1000 μg/ml concentration compared to other seaweed extracts. The dichloromethane extract of Corallina officinalis (CORM) exerted the highest antimicrobial activity with an average inhibition zone diameter (AV) = 15.29 mm and activity index (AI) = 1.53 and with the highest antagonistic activity against Escherichia coli (28 mm). It is followed by Ulva linza ethanolic extract (ULGE2) which recorded (AV) of 14.71 mm and (AI) of 1.30 with the highest antifungal activity against Candida albicans (30 mm). The collected seaweeds would therefore be a very promising source for treating inflammatory, arthritic, and microbial diseases. Moreover, the investigated seaweeds showed variable concentrations of heavy metals among various species. The mean concentrations of the heavy metals took the following order: Fe > Zn > Mn > Ba > Cu > As > Cr > Ni > Pb > V > Cd > Se > Co > Mo. Based on the permissible limits set by the WHO and CEVA, Pb and Ni in the studied seaweeds were found to be within the permissible limits, whereas Cd and Zn contents were at the borderline. Significant correlations were observed between studied parameters. The estimated daily intakes for most heavy metals were lower than the recommended daily intakes.

Juvenile hormone antagonistic activity of secondary metabolites from Streptomyces lactacystinicus and their insecticidal activity against Plutella xylostella

• IMBL-1412 isolate showed high JHAN and insecticidal activity against P. xylostella . • IMBL-1823 isolate showed high JHAN and insecticidal activity against P. xylostella . • Both IMBL-1412 and IMBL-1823 were identified as Streptomyces lactacystinicus . • Ethyl acetate fraction of them showed high JHAN and insecticidal activities. Due to their specificity to target insects and low toxicity to non-target organisms, insect growth regulators (IGRs) have been promising alternatives to neurotoxic insecticides. Actinobacteria produce a wide range of secondary metabolites with insecticidal and insect growth regulatory activities. In this study, the culture media of 25 actinobacteria isolates showing high juvenile hormone antagonist (JHAN) activity were assessed for their insecticidal activity to identify novel IGR compounds toxic to Plutella xylostella . Among them, four isolates exhibited high insecticidal activity against 3rd instar larvae of P. xylostella . Two isolates of IMBL-1412 and IMBL-1823 showing relatively high insecticidal activities (greater than90% mortality) were identified as Streptomyces lactacystinicus based on colony color on various International Streptomyces Project (ISP) media and nucleotide sequences of the 16S rRNA gene. The ethyl acetate fractions of both isolates showed high JHAN and insecticidal activities against P. xylostella larvae at a concentration of 100 ppm when the culture media of these two isolates were extracted sequentially using hexane, ethyl acetate, and butyl alcohol. These results suggested that secondary metabolites of these actinobacterial isolates could be efficiently applied to develop novel IGR insecticides for the control of P. xylostella .

Mixtures of suppressive bacteria enhance biological control of tomato bacterial wilt

Tomato bacterial wilt is one of the most destructive soil-borne plant diseases in the world, causing considerable economic losses. Biological control is an effective and environmentally friendly strategy to reduce disease incidence and yield loss. In this study, six Streptomyces-like strains, NEAU-Z2, NEAU-D18, NEAU-88, NEAU-174, NEAU-C12 and NEAU-66 exhibiting high antagonistic activities against Ralstonia solanacearum were isolated from tomato rhizosphere soil and used for further study. Of which strains NEAU-D18 and NEAU-88 were compatible with each other and the mixed strain (NEAU-D18 + NEAU-88) showing the strongest antibacterial activity against R. solanacearum were prepared. Hydroponic experiment at seedling stage showed that strains NEAU-D18 and NEAU-88 had the best control efficacy at the spore concentration of 108 cfu ml−1, which were 66.6% and 80%, respectively. Therefore, strains NEAU-D18 and NEAU-88 were inoculated into tomato seedling roots with different proportions (i.e., 1:1, 1:2, and 2:1) at the spore concentration of 108 cfu ml−1, and the ratio of 1:1 showed the best control efficacy (100%) on tomato bacterial wilt. Pot experiment also indicated that the mixed strain (NEAU-D18 + NEAU-88) had better biocontrol efficacy than that of single strains. Moreover, the mixed strain and R. solanacearum could utilize 31 of 51 tested compounds of tomato root exudates, indicating the mixed strain was more competitive than the single strains (27, 29) in theory. Furthermore, in vitro nutritional competition experiment showed that the incidence of tomato bacterial wilt with the addition of compounds that could not be utilized by R. solanacearum and mixed strain was equal to that of the control group (CK4-1), but the biocontrol efficacy was great different after adding six compounds that could be utilized by R. solanacearum and mixed strain, which confirmed that the nutritional competition occurred between the mixed strain and R. solanacearum. In addition, solid-state fermentation was used to prepare bioorganic fertilizer of mixed strain. The application of bioorganic fertilizer could effectively suppress the occurrence of tomato bacterial wilt, and the incidence was only 21.67 at 14 days post-challenge inoculation, while 85% of tomato plants developed bacterial wilt with organic fertilizer treatment. In conclusion, this study demonstrated that the mixed strain (NEAU-D18 + NEAU-88) showed better control efficacy than that of single strains and was a potential biocontrol agent for controlling tomato bacterial wilt.

Corybungines A−K: Isoquinoline alkaloids from Corydalis bungeana with dopamine D2 receptor activity

Eleven undescribed isoquinoline alkaloids corybungines A−K including a protoberberine-type alkaloid, an isoquinoline alkaloid with a unique 6-norprotoberberine skeleton, one 13,14-seco-protoberberine-type alkaloid, two 1a,14-seco-protoberberine-type alkaloids with a 4-(hydroxymethyl)phenoxy moiety and six aporphine alkaloids, together with seven known alkaloids, have been isolated from the whole herb extract of Corydalis bungeana Turcz. Their structures and absolute configurations were elucidated based on an analysis of spectroscopic data and electronic circular dichroism (ECD) spectra. (R)-stephanine displayed high antagonistic activity against the dopamine D2 receptor with an IC50 value of 0.85 ± 0.09 μM in CHO-D2 cells. Additionally, corybungines D, F, H, (R)-roemerine, (R)-vireakine and (R)-tuduranine showed moderate D2 antagonism (IC50 5.20–26.07 μM). The preliminary structure-activity relationships (SARs) of aporphine alkaloids were discussed.

Isolation of endophytic bacterial isolates from healthy banana trees and determination of their in vitro antagonistic activities against crown rot disease agent Fusarium verticillioides

Aims: Fungal diseases are one of the most important biotic factors causing serious losses in banana cultivation in field or greenhouses during cultivation, harvest, storage and transportation periods. Crown rot, caused by Fusarium verticillioides, is considered one of the most important postharvest fungal disease of banana fruits. In this study, endophytic bacterial isolates were obtained from fruits, branches and leaves of healthy banana trees and their antagonistic potentials were investigated against F. verticillioides as a biological control agent (BCA) in vitro conditions.Methods and Results: A total of 23 putative endophytic bacterial isolates were obtained from fruits, stems and leaves of healthy banana trees. All bacterial isolates were identified by MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry) analysis. In vitro antagonistic efficiencies of 12 different bacterial isolates, from eight different species (Bacillus subtilis ssp. spizizenii (3), Bacillus amyloliquefaciens (2), Bacillus subtilis ssp. subtilis (2), Bacillus mojavensis, Enterococcus faecium, Enterobacter cloacae, Enterobacter ludwigii and Pseudomonas stutzeri), were tested on inhibiting mycelial growth by using dual culture tests. Among bacterial isolates, the highest antagonistic activity was displayed by Bacillus mojavensis BEn3 isolate which significantly inhibited the mycelial growth by 50.83%. Enterobacter cloacae BEn1, Enterobacter ludwigii BEn2, Enterobacter faecium BEn7, however, failed to inhibit the mycelial growth of fungi.Conclusions: Significant suppression in the mycelial growth caused by endophytic bacterial isolates indicates that Bacillus mojavensis BEn3 isolate could be considered as possible BCA against crown rot disease agent.Significance and Impact of the Study: Our findings suggest that B. mojavensis Ben3, as the most successful endophyte bacterial isolate that suppresses the growth of F. verticillioides, can be used as a promising biological control agent as an alternative to chemical control against crown rot disease.

Production of <em>Aspergillus quadrilineatus</em> MT083999 Chitinase, β-1,3-Glucanase and Nano-silver Important for Biocontrol of <em>Fusarium</em> spp<em>.</em> Infecting Crops

In our search for an active biocontrol agent against F. solani and F. oxysporum , the major species causing wilt, root rot and damping-off in plants affecting crops. The fungal isolate genetically identified as Aspergillus quadrilineatus MT083999 displayed high antagonistic activities against the tested root-rot fungi, and presented valuable chitinase and β-glucanase activities of 235.484 and 508.953 U/gds, respectively. Solid state fermentation conditions were optimized by applying Three–factor–five–level central composite design (CCD) with twenty experiments and two responses of chitinase R1 and β-glucanase R2 activities, respectively. The optimum activities of chitinase (386.70 U/gds) and β-glucanase (1094.70 U/gds) were obtained in trial 4 and 2 respectively, with about 1.64-fold increase in chitinase activity and 2.15-fold increase in β-glucanase activity when compared with initial fermentation. Biosynthesis of nano-silver from A. quadrilineatus was evaluated in 10 experiments of another design of 2–factors–5–levels. The antifungal effects of each biosynthesized nano-silver against F. solani (R1) and F. oxysporum (R2) were evaluated as Inhibition zone diameter (IZD). The high fungicide nano-silver against both tested phytopathogens was characterized by UV–visible spectrophotometry, transmission electron microscopy (TEM), Zeta dynamic light scatter (DLS) and Fourier transform infrared spectroscopy (FTIR). The results indicated that A. quadrilineatus MT083999 chitinolytic enzymes and nano-silver can be applied as a novel candidate in biocontrol of Fusarium wilt infecting crops worldwide.

MOLECULAR, MORPHOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF ACTINOMYCETES AND THEIR ANTAGONISTIC ACTIVITY AGAINST PHYTOPATHOGENIC FUNGI

Plants are susceptible to a wide variety of pathogens that can affect their physiology, growth and cause death. Synthetic fungicides are used for fungal control; however, these chemicals can be harmful to the environment and favor the development of resistance; therefore, as a viable alternative, antagonistic fungi, bacteria and actinomycetes can be used in agriculture as biocontrol agents. A total of 1763 isolates of actinomycete were obtained from soil samples from different agroecosystems in Chihuahua, Mexico. Forty-four isolates were analyzed for their morphology, molecular characteristics, ability to hydrolyze various substrates, ability to use different energy sources and antagonistic activity. The isolated actinomycetes belong to the genera Amycolatopsis, Saccharopolyspora, Nocardioides and Streptomyces, according to molecular identification of the 16S rRNA gene. Streptomyces was the most abundant (90.9 %). Most of the selected isolates produced siderophores (93.2 %) and indole acetic acid (66 %). Isolates preferably used soluble starch among 19 tested energy sources. Confrontation studies of isolates with Fusarium equiseti, F. oxysporum, Alternaria alternata and Sclerotium rolfsii showed inhibition of radial growth (IRG) of 0.3- 92.1 %, 0-98.5 %, 0.4-96.9 %, and 0.2-100 %, respectively. Isolates A07 (Streptomyces cangkringensis CIAD-CA07), A27 (Streptomyces misionensisCIAD-CA27) and A45 (Streptomyces kanamyceticus CIAD-CA45) showed IRG values larger than 50 % against the four fungi. Isolate A27 caused the highest IRG values (92.1- 99.1 %), with S. rolfsii being the most susceptible fungus and F. equiseti the most resistant one. Results demonstrated the wide abundance and diversity of actinomycetes in agroecosystems. Some of the isolated actinomycetes showed high antagonistic activity and might be used as biological control agents of phytopathogenic fungi.

Biocontrol potential of plant growth-promoting rhizobacteria (PGPR) against Sclerotiorum rolfsii diseases on sugar beet (Beta vulgaris L.)

A total of 49 bacterial isolates were collected from the sugar beet rhizosphere in two regions of Morocco (Kenitra and Meknes). These bacteria were tested for their inhibitory activity against Sclerotium rolfsii Sacc., the causative pathogen of damping-off and root rot of sugar beet. Ten isolates showed a high ability to inhibit the mycelial growth of the pathogenic fungus in dual culture bioassays. The molecular identification based on 16S ribosomal DNA gene revealed that these isolates belong to the Bacillus genus. In addition, these bacterial isolates have also been characterized in terms of the production of certain antifungal products as well as for their capacities as plant growth-promoting traits. Under greenhouse conditions, sugar beet seeds treated with the each bacterium showed significant reduction in damping-off disease. Plants from treated seeds showed significant increases in both the length of shoots and roots, and the plant fresh and dry weight. Iinterestingly, these selected bacterial isolates displayed high antagonistic activity against S. rolfsii, and significantly reduced the severity and incidence of root rot disease. Therefore, these results highlighted that the soil harbors antagonistic bacteria offering several plant growth-promoting (PGP) traits, which can be exploited as a powerful growth promoter and/or biological control agent in sugar beet plants.

COMPARATIVE ANALYSIS OF THE CHEMICAL COMPOSITION AND FEATURES OF SURFACE MICROBIAL COMPLEXES OF LICHENS AND THEIR GROWTH SUBSTRATES

The aim of the study was a comparative analysis of the chemical composition and features of the surface microbial complexes of three species of epigeic lichens (Cladonia rangiferina (L.), Cetraria islandica (L.), Peltigera horizontalis (Huds.)), one species of epiphytic lichen (Hypogymni aphysodes (L.) Nyl.) and their growth substrates. It was found that the accumulation of inorganic ions by epigeic lichens exceeds their content in the soil by 4–450 times. Maximum biochemical mobility is characteristic of nutrients (potassium ions, phosphate ions). P. horizontalis and C. islandica were characterized by high biochemical mobility of copper and zinc, and H. physodes of cadmium and lead, and therefore these species of lichens can be considered bioaccumulators of these elements. The epiphytic lichen H. physodes was characterized by a relatively high content of phenolic compounds, which indicates its good antioxidant properties. Different physiological groups made the maximum contribution to the structure of microbial populations on the surface of lichens. Nitrogen-fixing bacteria dominated in the microbial complex in the epiphytic lichen H. physodes, and ammonifiers in the epigeic lichens. There is a direct correlation between the number of ammonifiers and the total number of microorganisms on the surface of epigeic lichens and their number in the soil under lichens. The gram-positive spore bacterium Bacillus polymyxa was isolated from the surface of the leafy lichen C. rangiferina (L.) into a pure culture, for which a high antagonistic activity was established with respect to phytopathogenic fungi pp. Fusarium and Alternaria. In the future, this strain can become the basis for the creation of an environmentally friendly biological product to combat plant diseases.

Biological Efficacy of Mycoflora Isolated from Mushroom Substrate Against Pathogenic Fungi

Four fungi isolates, Aspergillus flavus, Aspergillus niger, Trichoderma harzianum, Penicillium sp. were isolated from mushroom substrates and identified. Biological activities these fungi were evaluated against three plant pathogens namely against Fusarium oxysporum, Sclerotium rolfsii, Colletotrichum corchori. Among the fungal isolates, Trichoderma harzianum showed the highest antagonistic activity against all three pathogens, Penicillium sp. showed less significant antagonistic activity than Aspergillus niger and Aspergillus flavus. Trichoderma harzianum showed the highest inhibitory effect in case of Fusarium oxysporum (71.75%) followed by Sclerotium rolfsii (61.64%) and Colletotrichum corchori (57%). SAARC J. Agric., 19(2): 73-84 (2021)

Biocontrol properties from phyllospheric bacteria isolated from Solanum lycopersicum and Lactuca sativa and genome mining of antimicrobial gene clusters

BackgroundBiocontrol agents are sustainable eco-friendly alternatives for chemical pesticides that cause adverse effects in the environment and toxicity in animals including humans. An improved understanding of the phyllosphere microbiology is of vital importance for biocontrol development. Most studies have been directed towards beneficial plant-microbe interactions and ignore the pathogens that might affect humans when consuming vegetables. In this study we extended this perspective and investigated potential biocontrol strains isolated from tomato and lettuce phyllosphere that can promote plant growth and potentially antagonize human pathogens as well as plant pathogens. Subsequently, we mined into their genomes for discovery of antimicrobial biosynthetic gene clusters (BGCs), that will be further characterized.ResultsThe antimicrobial activity of 69 newly isolated strains from a healthy tomato and lettuce phyllosphere against several plant and human pathogens was screened. Three strains with the highest antimicrobial activity were selected and characterized (Bacillus subtilis STRP31, Bacillus velezensis SPL51, and Paenibacillus sp. PL91). All three strains showed a plant growth promotion effect on tomato and lettuce. In addition, genome mining of the selected isolates showed the presence of a large variety of biosynthetic gene clusters. A total of 35 BGCs were identified, of which several are already known, but also some putative novel ones were identified. Further analysis revealed that among the novel BGCs, one previously unidentified NRPS and two bacteriocins are encoded, the gene clusters of which were analyzed in more depth.ConclusionsThree recently isolated strains of the Bacillus genus were identified that have high antagonistic activity against lettuce and tomato plant pathogens. Known and unknown antimicrobial BGCs were identified in these antagonistic bacterial isolates, indicating their potential to be used as biocontrol agents. Our study serves as a strong incentive for subsequent purification and characterization of novel antimicrobial compounds that are important for biocontrol.

Myxococcus xanthus R31 Suppresses Tomato Bacterial Wilt by Inhibiting the Pathogen Ralstonia solanacearum With Secreted Proteins.

The pathogenic bacterium Ralstonia solanacearum caused tomato bacterial wilt (TBW), a destructive soil-borne disease worldwide. There is an urgent need to develop effective control methods. Myxobacteria are microbial predators and are widely distributed in the soil. Compared with other biocontrol bacteria that produce antibacterial substances, the myxobacteria have great potential for biocontrol. This study reports a strain of Myxococcus xanthus R31 that exhibits high antagonistic activity to R. solanacearum. Plate test indicated that the strain R31 efficiently predated R. solanacearum. Pot experiments showed that the biocontrol efficacy of strain R31 against TBW was 81.9%. Further study found that the secreted protein precipitated by ammonium sulfate had significant lytic activity against R. solanacearum cells, whereas the ethyl acetate extract of strain R31 had no inhibitory activity against R. solanacearum. Substrate spectroscopy assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of secreted proteins showed that some peptidases, lipases, and glycoside hydrolases might play important roles and could be potential biocontrol factors involved in predation. The present study reveals for the first time that the use of strain M. xanthus R31 as a potential biocontrol agent could efficiently control TBW by predation and secreting extracellular lyase proteins.

Isolation and Molecular Identification of a Siderophore Producing Bacterium and its Antagonistic Effect against Fusarium oxysporum f. sp. cubense Tropical Race 4

Bananas are one of the world’s most consumed fruits. Developing countries in the Global South depend on bananas for food security and livelihoods. Still, the banana industry also drives a multinational trade worth billions of US dollars. In addition, banana plants also hold cultural and religious significances in many Asian countries. However, banana production faces several challenges, and one of the major issues is the Fusarium wilt disease caused by the fungus Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4). This disease threatens numerous banana cultivars, including the Cavendish, the most traded banana cultivar. Therefore, the objective of this study was to find effective measures to control the spread of this disease through antagonistic soil bacteria. This study isolated 14 fluorescent, siderophore-producing bacteria with in vitro inhibition rates of 21.73-50.38% against Foc TR4 from the soil surrounding banana plants. Most of the isolates were identified as Pseudomonas spp. via 16S rRNA sequencing, and phylogenetic analysis based on 16s rRNA sequences showed clustering of isolates congruent with the results of similarity searches. Three isolates that exhibited relatively higher antagonistic activity against Fusarium oxysporum f. sp. cubense compared to other isolates were JBAA132 (50.38%), K2B131 (46.28%), and KTP231 (45.38%). Isolate JBAA132 displayed differences in biochemical characteristics compared to its closest match (Pseudomonas aeruginosa type strain DSM50071). It also demonstrated nitrogen-fixing and phosphate solubilising properties common to plant growth promoters. Therefore, isolate JBAA132 may be further explored as a potential biocontrol agent in curbing the spread of Foc TR4.

Bacterial volatile organic compounds induce adverse ultrastructural changes and DNA damage to the sugarcane pathogenic fungus Thielaviopsis ethacetica.

Due to an increasing demand for sustainable agricultural practices, the adoption of microbial volatile organic compounds (VOCs) as antagonists against phytopathogens has emerged as an eco-friendly alternative to the use of agrochemicals. Here, we identified three Pseudomonas strains that were able to inhibit, in vitro, up to 80% of mycelial growth of the phytopathogenic fungus Thielaviopsis ethacetica, the causal agent of pineapple sett rot disease in sugarcane. Using GC/MS, we found that these bacteria produced 62 different VOCs, and further functional validation revealed compounds with high antagonistic activity to T. ethacetica. Transcriptomic analysis of the fungal response to VOCs indicated that these metabolites downregulated genes related to fungal central metabolism, such as those involved in carbohydrate metabolism. Interestingly, genes related to the DNA damage response were upregulated, and micro-FTIR analysis corroborated our hypothesis that VOCs triggered DNA damage. Electron microscopy analysis showed critical morphological changes in mycelia treated with VOCs. Altogether, these results indicated that VOCs hampered fungal growth and could lead to cell death. This study represents the first demonstration of the molecular mechanisms involved in the antagonism of sugarcane phytopathogens by VOCs and reinforces that VOCs can be a sustainable alternative for use in phytopathogen biocontrol.

Anti-Salmonella activity of lactobacilli from different habitats

Lactic acid bacteria (LAB) may contribute to the food safety. In the present study, the antagonistic activity of 45 Bulgarian Lactobacillus homo- and heterofermentative strains of human and dairy origin, and 4 multibacterial formulas against Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) was assessed. In vitro tests were performed in different model systems - milk, soymilk, De Man Rogose Sharp (MRS) medium, to simulate real conditions in the food chain. The highest antagonistic activity was observed with cell-free supernatants of exponential MRS broth cultures of the strains isolated from breast milk, followed by lactobacilli from white brined and green cheese. The detected antimicrobial activity against the pathogen was strain-specific and depended on the culture conditions. Lactobacillus (reclassified as Lactoplantibacillus) plantarum strains, cultivated in skimmed milk and whey protein medium, were able to inhibit S. Typhimurium growth, while a limited inhibitory activity was detected for fermented soymilks. A bacteriocinogenic Ligilactobacillus (the previous Lactobacillus) salivarius strain reduced the number of living pathogenic cells during co-cultivation in whole milk. The inhibition was significant only when L. salivarius was inoculated in predominance. In case of underrepresented LAB number, S. Typhimurium over-growth was observed. Eight lactobacilli in combination as a multibacterial co-culture expressed synergic antagonistic effect against Salmonella and were pre-selected as promising. Further characterisation of their active metabolites, however, is needed before their classification as bio-protective agents.