Endophytic Bacteria as Effective Agents of New-Generation Biopesticides (Review)

In this review, the data on endophytic bacteria with protective properties are covered, and their characteristics and species composition are discussed. The mechanisms of fungicidal and insecticidal activities, including the synthesis of lipopeptides, siderophores, and hydrolases, systemic resistance induction, and the production of insecto-toxic compounds, are analyzed. Possible methods for the production of complex biological preparations are considered; the preparations are based on endophytic microorganisms and would protect plants from both pathogens and pests. The means of extending their activity spectrum via the combination of different strains or the construction of new recombinant endophytic strains by genetic engineering techniques are also considered.     

Hrp- Mutants of Pseudomonas solanacearum as Potential Biocontrol Agents of Tomato Bacterial Wilt

There have been many attempts to control bacterial wilt with antagonistic bacteria or spontaneous nonpathogenic mutants of Pseudomonas solanacearum that lack the ability to colonize the host, but they have met with limited success. Since a large gene cluster (hrp) is involved in the pathogenicity of P. solanacearum, we developed a biological control strategy using genetically engineered Hrp^- mutants of P. solanacearum. Three pathogenic strains collected in Guadeloupe (French West Indies) were rendered nonpathogenic by insertion of an ω-Km interposon within the hrp gene cluster of each strain. The resulting Hrp^- mutants were tested for their ability to control bacterial wilt in challenge inoculation experiments conducted either under growth chamber conditions or under greenhouse conditions in Guadeloupe. Compared with the colonization by a pathogenic strain which spread throughout the tomato plant, colonization by the mutants was restricted to the roots and the lower part of the stems. The mutants did not reach the fruit. Moreover, the presence of the mutants did not affect fruit production. When the plants were challenge inoculated with a pathogenic strain, the presence of Hrp^- mutants within the plants was correlated with a reduction in disease severity, although pathogenic bacteria colonized the stem tissue at a higher density than the nonpathogenic bacteria. Challenge inoculation experiments conducted under growth chamber conditions led, in some cases, to exclusion of the pathogenic strain from the aerial part of the plant, resulting in high protection rates. Furthermore, there was evidence that one of the pathogenic strains used for the challenge inoculations produced a bacteriocin that inhibited the in vitro growth of the nonpathogenic mutants.     

Hrp Mutants of Pseudomonas solanacearum as Potential Biocontrol Agents of Tomato Bacterial Wilt

There have been many attempts to control bacterial wilt with antagonistic bacteria or spontaneous nonpathogenic mutants of Pseudomonas solanacearum that lack the ability to colonize the host, but they have met with limited success. Since a large gene cluster (hrp) is involved in the pathogenicity of P. solanacearum, we developed a biological control strategy using genetically engineered Hrp mutants of P. solanacearum. Three pathogenic strains collected in Guadeloupe (French West Indies) were rendered nonpathogenic by insertion of an omega-Km interposon within the hrp gene cluster of each strain. The resulting Hrp mutants were tested for their ability to control bacterial wilt in challenge inoculation experiments conducted either under growth chamber conditions or under greenhouse conditions in Guadeloupe. Compared with the colonization by a pathogenic strain which spread throughout the tomato plant, colonization by the mutants was restricted to the roots and the lower part of the stems. The mutants did not reach the fruit. Moreover, the presence of the mutants did not affect fruit production. When the plants were challenge inoculated with a pathogenic strain, the presence of Hrp mutants within the plants was correlated with a reduction in disease severity, although pathogenic bacteria colonized the stem tissue at a higher density than the nonpathogenic bacteria. Challenge inoculation experiments conducted under growth chamber conditions led, in some cases, to exclusion of the pathogenic strain from the aerial part of the plant, resulting in high protection rates. Furthermore, there was evidence that one of the pathogenic strains used for the challenge inoculations produced a bacteriocin that inhibited the in vitro growth of the nonpathogenic mutants.     

Synthesis and Antibacterial Activity Evaluation of Biphenyl and Dibenzofuran Derivatives as Potential Antimicrobial Agents against Antibiotic-Resistant Bacteria

The escalating prevalence of antibiotic-resistant bacteria has led to a serious global public health problem; therefore, there is an urgent need for the development of structurally innovative antibacterial agents. In our study, a series of biphenyl and dibenzofuran derivatives were designed and synthesized by Suzuki-coupling and demethylation reactions in moderate to excellent yields (51–94% yield). Eleven compounds exhibited potent antibacterial activities against the prevalent antibiotic-resistant Gram-positive and Gram-negative pathogens, among which compounds 4′-(trifluoromethyl)-[1,1′-biphenyl]-3,4,5-triol (6i) and 5-(9H-carbazol-2-yl) benzene-1,2,3-triol (6m) showed the most potent inhibitory activities against methicillin-resistant Staphylococcus aureus and multidrug-resistant Enterococcus faecalis with MIC (minimum inhibitory concentration) values as low as 3.13 and 6.25 μg/mL, respectively. Compounds 3′,5′-dimethyl-[1,1′-biphenyl]-3,4,4′,5-tetraol (6e), 4′-fluoro-[1,1′-biphenyl]-3,4,5-triol (6g), and 4′-(trifluoromethyl)-[1,1′-biphenyl]-3,4,5-triol (6i) showed comparable inhibitory activities with ciprofloxacin to Gram-negative bacterium carbapenems-resistant Acinetobacter baumannii. Study of the structure–activity relationship indicated that a strong electron-withdrawing group on the A ring and hydroxyl groups on the B ring of biphenyls were beneficial to their antibacterial activities, and for benzo-heterocycles, N-heterocycle exhibited optimal antibacterial activity. These results can provide novel structures of antibacterial drugs chemically different from currently known antibiotics and broaden prospects for the development of effective antibiotics against antibiotic-resistant bacteria.     

Culturable Leaf-Associated Bacteria on Tomato Plants and Their Potential as Biological Control Agents

Culturable leaf-associated bacteria inhabiting a plant have been considered as promising biological control agent (BCA) candidates because they can survive on the plant. We investigated the relationship between bacterial groups of culturable leaf-associated bacteria on greenhouse- and field-grown tomato leaves and their antifungal activities against tomato diseases in vitro and in vivo. In addition, the isolated bacteria were analyzed for N-acyl-homoserine lactone (AHL) and indole-3-acetic acid (IAA) production, which have been reported to associate with bacterial colonization, and resistance to a tomato alkaloid (α-tomatine). Leaf washings and subsequent leaf macerates were used to estimate the population size of epiphytic and more internal bacteria. Bacterial population sizes on leaves at the same position increased as the leaves aged under both greenhouse and field conditions. Field-grown tomatoes had significantly larger population sizes than greenhouse-grown tomatoes. Analysis of 16S rRNA gene (rDNA) sequencing using 887 culturable leaf-associated bacteria revealed a predominance of the Bacillus and Pseudomonas culturable leaf-associated bacterial groups on greenhouse- and field-grown tomatoes, respectively. Curtobacterium and Sphingomonas were frequently recovered from both locations. From the 2138 bacterial strains tested, we selected several strains having in vitro antifungal activity against three fungal pathogens of tomato: Botrytis cinerea, Fulvia fulva, and Alternaria solani. Among bacterial strains with strong in vitro antifungal activities, Bacillus and Pantoea tended to show strong antifungal activities, whereas Curtobacterium and Sphingomonas were not effective. The results indicated the differences in antifungal activity among predominant bacterial groups. Analysis of α-tomatine resistance revealed that most bacterial strains in the dominant groups exhibited moderate or high resistance to α-tomatine in growth medium. Furthermore, some Sphingomonas and Pantoea strains showed AHL and IAA production activities. Strain 125NP12 (Pantoea ananatis) showed particular α-tomatine resistance, and AHL and IAA production had the highest protective value (91.7) against gray mold. Thus, the differences of these physiological properties among dominant bacteria may be associated with the disease suppression ability of BCAs on tomato plants.     

Endophytic Bacteria Induce Growth Promotion and Wilt Disease Suppression in Oilseed Rape and Tomato

To determine whether bacteria isolated from within plant tissue can have plant growth-promotion potential and provide biological control against soilborne diseases, seeds and young plants of oilseed rape (Brassica napus L. cv. Casino) and tomato (Lycopersicon lycopersicum L. cv. Dansk export) were inoculated with individual bacterial isolates or mixtures of bacteria that originated from symptomless oilseed rape, wild and cultivated. They were isolated after surface sterilization of living roots and stems. The effects of these isolates on plant growth and soilborne diseases for oilseed rape and tomato were evaluated in greenhouse experiments. We found isolates that not only significantly improved seed germination, seedling length, and plant growth of oilseed rape and tomato but also, when used for seed treatment, significantly reduced disease symptoms caused by their vascular wilt pathogens Verticillium dahliae Kleb and Fusarium oxysporum f. sp. lycopersici (Sacc.), respectively.     

Inhibitors of Renin as Potential Therapeutic Agents

Abstract

The reaction of usual (U) and atypical (A) cholinesterase phenotypes was studied with six organophosphorus compounds, two pyridinium oximes (HI-6 and PAM-2) and with 4–4-bipyridine (4,4-BP). No difference in the inhibition rate constants for the two phenotypes was found with the progressive inhibitors tabun, sarin, paraoxon and soman. The other two progressive inhibitors, VX and the positively charged phosphostigmine, inhibited the U phenotype more strongly than the A phenotype.

The positively charged reversible inhibitor HI-6 showed a higher affinity for the U than for the A phenotype, while PAM-2 and the non-charged 4,4′-BP did not show a significant difference in their affinity towards the two enzymes.

Both phenotypes phosphylated by VX or sarin were reactivatable by HI-6 and PAM-2, and the A phenotype was always reactivated more slowly than the U phenotype. The paraoxon-inhibited phenotypes were reactivated at equal rates with PAM-2 but were not reactivated with HI-6. The phosphylated phenotypes did not reactivate spontaneously during one hour.

The effect of reversible inhibitors upon the rate of phosphylation (protection) was tested with HI-6 (for inhibition by soman, tabun and paraoxon) and with 4,4′-BP (for inhibition by soman). By applying the concentrations of the protectors equal to their enzyme/inhibitor dissociation constants, a better protection of the U than of the A phenotype was achieved by HI-6, but equal protection was given by 4,4′-BP.     

抗感青枯病番茄的内生细菌数量动态分析及其对青枯病的生物防治

对番茄内生细菌数量动态及其对青枯病的生物防治研究结果表明:番茄内生细菌可来源于种子内部。番茄不同生育期,内生细菌数量最多在成株期,其中抗病品种根、茎分别为24.3×104CFU/g鲜重和22.9×104CFU/g鲜重,感病品种根、茎分别为9.8×104CFU/g鲜重和13.4×104CFU/g鲜重。抗病品种中具有拮抗青枯菌的内生细菌菌株为17个,感病品种中7个。部分内生细菌具促进番茄种子萌发和防治番茄青枯病的作用,其中5R和3R内生菌株的防病效果分别达91.7%和81.3%。     

Biological Evaluation of Endophytic Fungus, Chaetomium globosum JN711454, as Potential Candidate for Improving Drug Discovery

The main objective of this research work focused on investigating the biological and chemical aspects of endophytic fungus Chaetomium globosum, for pharmaceutical purposes to improve the drug discovery process. The endophytic C. globosum was isolated from healthy leaves of Egyptian medicinal plant Adiantum capillus-veneris collected from Saint Katherine Protectorate, Sinai, Egypt. The identification of C. globosum was on the basis of classical and molecular taxonomy. Gene encoding for 18S rRNA was partially sequenced, submitted to the GenBank and got the accession number JN711454, to resolve the phylogenetic relations with fungal ancestor using phylogenetic tree. To explore the biosynthetic power of endophytic C. globosum JN711454, the fungus was cultivated over five different media, oatmeal, rice, yeast malt glucose, potato dextrose agar (PDA) and Czapek’s dox media, for 3 weeks at 30 °C, followed by extraction with different solvents, ethyl acetate (EA), and methanol. The ethyl acetate extract of C. globosum cultivated on PDA medium was the most potent extract. It showed strong antioxidant activity with EC₅₀ 11.5 μg/ml, potent anticancer activity with 55 % toxicity toward HepG-2 cells at 100 μg/ml and 66 % cytotoxicity to FGC₄ cells at 250 μg/ml, promising butyrylcholinesterase inhibitory activities (>85 %), and moderate antimicrobial and stopped the attachment of HSV-2 virus to VERO cells. The metabolomic profiling of PDA–EA extract using LC–MS revealed the presence of several metabolites to which the observed bioactivities could be attributed. Here we report for the first time inhibitory activity of endophytic C. globosum JN711454 secondary metabolites to butyrylcholinesterase, one of neuro hydrolase enzymes that play a major role in development of Alzheimer’s disease.     

Biocontrol of Fusarium Wilt and Growth Promotion of Tomato Plants Using Endophytic Bacteria Isolated from Solanum elaeagnifolium Stems

Seven culturable bacterial isolates, obtained from the internal stem tissues of Solanum elaeagnifolium and successfully colonizing the internal stem tissues of tomato cv. Rio Grande, were screened for their in vivo antifungal activity against Fusarium oxysporum f.sp. lycopersici (FOL) and their growth‐promoting potential on tomato plants. SV101 and SV104 isolates, assessed on pathogen‐challenged tomato plants led to a significant decrease (77–83%) in Fusarium wilt severity and vascular browning extent (76%), as compared to the inoculated and untreated control. Isolates enhanced growth parameters on pathogen‐challenged and unchallenged tomato plants. SV104 and SV101 isolates were most effective in suppressing disease and enhancing plant growth. These two isolates were identified as Bacillus sp. str. SV101 ( KU043040 ) and B. tequilensis str. SV104 ( KU976970 ). They displayed antifungal activity against FOL; pathogen growth was inhibited by 64% and an inhibition zone (11.50 and 19.75 mm) against FOL could be formed using whole cell suspensions. SV101 and SV104 extracellular metabolites also inhibited FOL growth by 20 and 55%, respectively, as compared to control. B. tequilensis str. SV104 was shown to produce protease, chitinase, pectinase, IAA and siderophores. Bacillus sp. str. SV101 displayed pectinase activity and was found to be an IAA‐producing and phosphate‐solubilizing agent. To our knowledge, this is the first study reporting on S. elaeagnifolium use as a potential source of potent biocontrol and plant growth‐promoting agents.     

Oral Bacteria as Potential Probiotics for the Pharyngeal Mucosa

The research described here was aimed at the selection of oral bacteria that displayed properties compatible with their potential use as probiotics for the pharyngeal mucosa. We included in the study 56 bacteria newly isolated from the pharynges of healthy donors, which were identified at the intraspecies level and characterized in vitro for their probiotic potential. The experiments led us to select two potential probiotic bacterial strains (Streptococcus salivarius RS1 and ST3) and to compare them with the prototype oral probiotic S. salivarius strain K12. All three strains efficiently bound to FaDu human epithelial pharyngeal cells and thereby antagonized Streptococcus pyogenes adhesion and growth. All were sensitive to a variety of antibiotics routinely used for the control of upper respiratory tract infections. Immunological in vitro testing on a FaDu layer revealed different responses to RS1, ST3, and K12. RS1 and ST3 modulated NF-κB activation and biased proinflammatory cytokines at baseline and after interleukin-1β (IL-1β) induction. In conclusion, we suggest that the selected commensal streptococci represent potential pharyngeal probiotic candidates. They could display a good degree of adaptation to the host and possess potential immunomodulatory and anti-inflammatory properties.Metagenomics and functional molecular immunology substantiate the interpretation of humans as holobionts, in the sense of functional superorganisms, combining the self and microbes acting in concert to produce phenomena governed by the collective (25, 42). The association between host and symbionts affects the fitness of the holobiont within its environment, and it often governs the physiological homeostasis of the narrow balance between host well being and dysfunction (13, 35).The mechanisms underlying the cross talk between a human host and microbes are only marginally understood. Their elucidation at a molecular level could supply the theoretical bases to develop strategies for preventing or treating several human dysfunctions, such as autoimmune diseases, through the reconstitution of a proper human-microbe mutualism.The probiotic approach, in its widest sense, falls into this context, since it consists of the modification of a human microbiota by exogenous administration of microbial cells (or cell components), aimed at benefiting the host''s health. A most commonly accepted definition comes from FAO/WHO, which states that probiotics are “live microorganisms which when administered in adequate amounts confer a health benefit on the host” (17).So far, probiotics have been most predominantly investigated for and applied to the intestinal tract. Nevertheless, a few applications beyond the gut have proposed the potential beneficial role of probiotics for the stomach (23), vaginal mucosa (36), urinary tract (6), skin (27), and oral cavity (39). With respect to oral probiotics, particularly noticeable are the studies done by J. R. Tagg and coworkers of Streptococcus salivarius strain K12. Tagg and others, in fact, showed that, following oral administration, the bacterial strain K12 can colonize the oral mucosae of infants and adults (20, 34), downregulate the innate immune responses of human epithelial cells (11), and reduce oral volatile sulfur compound levels (8). Strain K12 was also revealed to produce two plasmid-encoded lantibiotic peptides (22, 38) that are active against Streptococcus pyogenes, the main etiological agent of bacterial pharyngitis. These investigations demonstrated the potential effectiveness of the probiotic intervention in the oropharyngeal tract.Encouraged by the promising results obtained in J. R. Tagg''s experiments, in the present study, we screened oral bacteria for their potential use as probiotics in the pharyngeal mucosa. We tested the ability of bacteria, which were newly isolated from the pharynges of healthy volunteers, to adhere to a human pharyngeal cell layer and to antagonize S. pyogenes on two different epithelial cell lines. The study allowed the selection of two bacterial strains, which were further investigated from an immunological point of view for their ability to cross talk with human epithelial cells in vitro.     

Microwave-assisted Synthesis of Novel Thiazolocarbazoles and Evaluation as Potential Anticancer Agents. Part III

Novel 6-substituted thiazolocarbazole derivatives have been synthesized under microwave irradiation via the corresponding imino-1,2,3-dithiazoles. In vitro antitumor potential of these polyheterocyclic compounds was evaluated. Among all the tested thiazolocarbazoles, compound 10 is the most effective in inhibiting cell growth.     

Nitric Oxide Donors as Potential Antitumor Agents

Biophysics - The role of nitric oxide as one of the universal regulators of metabolic processes in living organisms is considered. The results of experimental studies of NO association with cancer...     

一株拮抗香蕉枯萎病的内生细菌的分离及其几丁质酶基因信号肽的分泌活性分析

目的：旨在分离并选择一株香蕉内生细菌作为内生基因工程生防菌,并克隆其几丁质酶基因的信号肽序列。方法：从香蕉植株杆下部分离并选择了一株拮抗香蕉枯萎病且具有分泌几丁质酶能力的内生细菌,对该菌株进行了形态观察、生理生化测定和16S rDNA序列分析,克隆了其几丁质酶基因的编码序列并预测了其信号肽,构建了含有信号肽和不含信号肽的几丁质酶的表达菌株BL-chi1和BL-chi2。结果：结合形态观察、生理生化特征和16S rDNA序列比对分析确定该菌株为Klebsiella属,将该菌株命名为KKWB 5;BL-chi1和BL-chi2经IPTG诱导后,均表达了与预期蛋白大小一致的蛋白,同时BL-chi1诱导后的培养基上清中出现一条约45kDa的条带,而BL-chi2和空载体的BL-pET22b诱导后的培养基上清中均无此条带;几丁质水解试验发现,BL-chi1诱导后的培养基上清中的蛋白经浓缩和纯化后都能在几丁质平板上形成透明水解圈。结论：该几丁质酶的信号肽能被BL21（DE3）所识别,将几丁质酶分泌到培养基中,并且分泌的几丁质酶具有水解几丁质的生物学活性。内生菌KKWB-5的分离及其几丁质酶分泌信号肽序列的克隆为进一步构建内生工程菌来防治香蕉枯萎病打下了基础。     

Autotrophic, Hydrogen-Oxidizing, Denitrifying Bacteria in Groundwater, Potential Agents for Bioremediation of Nitrate Contamination

Addition of hydrogen or formate significantly enhanced the rate of consumption of nitrate in slurried core samples obtained from an active zone of denitrification in a nitrate-contaminated sand and gravel aquifer (Cape Cod, Mass.). Hydrogen uptake by the core material was immediate and rapid, with an apparent K_m of 0.45 to 0.60 μM and a V_max of 18.7 nmol cm^-3 h^-1 at 30°C. Nine strains of hydrogen-oxidizing denitrifying bacteria were subsequently isolated from the aquifer. Eight of the strains grew autotrophically on hydrogen with either oxygen or nitrate as the electron acceptor. One strain grew mixotrophically. All of the isolates were capable of heterotrophic growth, but none were similar to Paracoccus denitrificans, a well-characterized hydrogen-oxidizing denitrifier. The kinetics for hydrogen uptake during denitrification were determined for each isolate with substrate depletion progress curves; the K_ms ranged from 0.30 to 3.32 μM, with V_maxs of 1.85 to 13.29 fmol cell^-1 h^-1. Because these organisms appear to be common constituents of the in situ population of the aquifer, produce innocuous end products, and could be manipulated to sequentially consume oxygen and then nitrate when both were present, these results suggest that these organisms may have significant potential for in situ bioremediation of nitrate contamination in groundwater.