Nitric Oxide Production by Necrotrophic Pathogen Macrophomina phaseolina and the Host Plant in Charcoal Rot Disease of Jute: Complexity of the Interplay between Necrotroph–Host Plant Interactions

M. phaseolina, a global devastating necrotrophic fungal pathogen causes charcoal rot disease in more than 500 host plants. With the aim of understanding the plant-necrotrophic pathogen interaction associated with charcoal rot disease of jute, biochemical approach was attempted to study cellular nitric oxide production under diseased condition. This is the first report on M. phaseolina infection in Corchorus capsularis (jute) plants which resulted in elevated nitric oxide, reactive nitrogen species and S nitrosothiols production in infected tissues. Time dependent nitric oxide production was also assessed with 4-Amino-5-Methylamino-2′,7′-Difluorofluorescein Diacetate using single leaf experiment both in presence of M. phaseolina and xylanases obtained from fungal secretome. Cellular redox status and redox active enzymes were also assessed during plant fungal interaction. Interestingly, M. phaseolina was found to produce nitric oxide which was detected in vitro inside the mycelium and in the surrounding medium. Addition of mammalian nitric oxide synthase inhibitor could block the nitric oxide production in M. phaseolina. Bioinformatics analysis revealed nitric oxide synthase like sequence with conserved amino acid sequences in M. phaseolina genome sequence. In conclusion, the production of nitric oxide and reactive nitrogen species may have important physiological significance in necrotrophic host pathogen interaction.     

Potential of Lariophagus distinguendus (Förster) (Hymenoptera: Pteromalidae) to suppress the maize weevil Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) in bagged and bulk stored maize

Grains are often stored in jute bags in developing countries, especially in Africa, as well as in small quantities in bulk. Parasitoids suitable for biological control of stored-product pests should be able to find their hosts in bulk grain or in jute bags over a certain distance in a warehouse containing stacks of bagged grain. The potential of using Lariophagus distinguendus for the biological control of Sitophilus zeamais was assessed in maize stored in jute bags and bulk grain. The ability of the parasitoid to penetrate the jute cloth and the grain mass and parasitize its host was studied under controlled conditions of 25 ± 1 °C and 65 ± 5% RH. Experiments were carried out in small 5-kg jute bags containing 28 d old S. zeamais larvae within infested maize kernels, and in cylinders filled with maize grains and containing caged hosts at different depths. L. distinguendus parasitized S. zeamais in the jute bags and in the storage cylinders at various depths. In the jute bag experiment, out of the 60 L. distinguendus adults released, a mean ± SD of 7.03 ± 1.78% and 6.34 ± 1.01% of the 40 females and of the 20 males released, respectively, entered the jute bags. Significantly, no differences were found between the female and male L. distinguendus that entered the bags. Mean reduction of S. zeamais in the jute bags by parasitoids was 81%. The parasitic wasps also significantly reduced the emergence of S. zeamais in bulk maize. At depths of 20–45 cm from the grain surface, mean reduction of S. zeamais was 74%, while from 95 to 100 cm, mean reduction was 34%. When results from depths lower than 50 cm were pooled and compared with pooled data from depths higher than 90 cm, there was a significant reduction in parasitism at depths of more than 90 cm. For depths below 50 cm a mean of 5.3 L. distinguendus adult offspring per cage emerged compared with a mean of 2.6 at depths of more than 90 cm. These results support the approach to utilize L. distinguendus as a component in the integrated control of S. zeamais in bagged or bulk stored maize.     

Fungal deterioration of melon seeds stored in jute sacks and polyethylene bags in Ago-Iwoye,southwestern Nigeria

Laboratory studies were carried out in the Department of Biological Sciences, Ogun State University, Ago-Iwoye, southwestern Nigeria, to determine the extent of fungal deterioration of melon seeds stored in two types of storage bags viz; jute and polyethylene bags. Melon seeds of varieties Tc139 and V2 were stored in jute and polyethylene bags under ambient conditions using the 2 × 2 factorial design (variety vs type of bag) for 12 months. The moisture content (mc), incidence of visible mouldiness (ivm) and germinability of the stored seeds were determined monthly. The mc of Tc139 ranged from 6.1 to 6.7% in jute and 6.2 to 6.5% in polyethylene bags. The ivm which was initially 2.1% increased to 10.7% and 5.5% in jute and polyethylene bags respectively, after 12 months in storage. The germination percentage decreased from 96.3% to 28.7% and 45.3% in jute and polyethylene bags, respectively. The mc of V2 stored in jute and polyethylene bags varied from 5.9 to 6.4%, and 5.8 to 6.2%, respectively. The ivm increased from 1.8% before storage to 8.9% and 4.8% in jute and polyethylene bags, respectively, after 12 months. The percentage seed germination declined from 98.0%to 37.3% in jute and 48.7% in polyethylene bags after 12 months. Decreased incidence of field fungi namely: Alternaria, Botryodiplodia theobromae, Cladosporium, Fusarium and Macrophomina phaseolina was accompanied by simultaneous increase in storage fungi viz: Aspergillus, Penicillium, and Rhizopus with prolonged storage. This revised version was published online in June 2006 with corrections to the Cover Date.     

Lateral flow immunoassay for rapid detection of potato ring rot caused by Clavibacter michiganensis subsp. sepedonicus

A lateral flow immunoassay for the rapid detection of Clavibacter michiganensis subsp. sepedonicus bacteria causing potato ring rot was developed. Multimembrane composites (test strips) containing polyclonal antibodies against the bacteria and gold nanoparticle-antibody conjugates were used for the analysis. The test strips are suitable for the analysis of potato tuber and leaf extracts within 10 min; the detection limit of bacteria is 4 × 10⁵ cells/mL. No cross-reactivity with strains of Clavibacter michiganensis subsp. michiganensis, Pectobacterium carotovorum subsp. carotovorum and saprophytes of healthy potato plants was detected. The results of analysis of 26 potato samples by the developed tests were compared with those obtained by the PCR method and using the commercial enzyme immunoassay kits. The results of lateral flow immunoassay were confirmed in 96.2% of cases, which supports the high correlation with other analytical approaches. The developed immunoassay may be considered as a promising means of phytosanitary control.     

Ciprofloxacin Residues in Municipal Biosolid Compost Do Not Selectively Enrich Populations of Resistant Bacteria

Biosolids and livestock manure are valuable high-carbon soil amendments, but they commonly contain antibiotic residues that might persist after land application. While composting reduces the concentration of extractable antibiotics in these materials, if the starting concentration is sufficiently high then remaining residues could impact microbial communities in the compost and soil to which these materials are applied. To examine this issue, ciprofloxacin was added to biosolid compost feedstock to achieve a total concentration of 19 ppm, approximately 5-fold higher than that normally detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (1 to 3.5 ppm). This feedstock was placed into mesh bags that were buried in aerated compost bays. Once a week, a set of bags was removed and analyzed (treated and untreated, three replicates of each; 4 weeks). Addition of ciprofloxacin had no effect on the recovery of resistant bacteria at any time point (P = 0.86), and a separate bioassay showed that aqueous extractions from materials with an estimated 59 ppm ciprofloxacin had no effect on the growth of a susceptible strain of Escherichia coli (P = 0.28). Regression analysis showed that growth of the susceptible strain of E. coli can be reduced given a sufficiently high concentration of ciprofloxacin (P < 0.007), a result that is consistent with adsorption being the primary mechanism of sequestration. While analytical methods detected biologically significant concentrations of ciprofloxacin in the materials tested here, the culture-based methods were consistent with the materials having sufficient adsorptive capacity to prevent typical concentrations of ciprofloxacin residues from selectively enriching populations of resistant bacteria.     

Mixtures of Quaternary Ammonium Compounds and Long-chain Fatty Acids as Antifungal Agents

The influence of undecylenic acid on the fungistatic effect of phenoxyethyldimethyldodecylammonium bromide (Domiphen bromide) against Trichophyton mentagrophytes was investigated. The unsaturated fatty acid was found to enhance the fungistatic activity of Domiphen bromide against this organism. The ratio of concentrations of these agents has a marked influence on the results of in vitro tests for antifungal action resulting in a completely different effect than heretofore noted in combination experiments against bacteria. The enhancing phenomenon is not particular to T. mentagrophytes, it was observed also with Candida albicans.     

New Type of Antimicrobial Protein Produced by the Plant Pathogen Clavibacter michiganensis subsp. michiganensis

It has previously been shown that the tomato pathogen Clavibacter michiganensis subsp. michiganensis secretes a 14-kDa protein, C. michiganensis subsp. michiganensis AMP-I (CmmAMP-I), that inhibits growth of Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato. Using sequences obtained from tryptic fragments, we have identified the gene encoding CmmAMP-I and we have recombinantly produced the protein with an N-terminal intein tag. The gene sequence showed that CmmAMP-I contains a typical N-terminal signal peptide for Sec-dependent secretion. The recombinant protein was highly active, with 50% growth inhibition (IC₅₀) of approximately 10 pmol, but was not toxic to potato leaves or tubers. CmmAMP-I does not resemble any known protein and thus represents a completely new type of bacteriocin. Due to its high antimicrobial activity and its very narrow inhibitory spectrum, CmmAMP-1 may be of interest in combating potato ring rot disease.     

Cytotoxicity and anticancer studies of Bacillus cereus and Bacillus pumilus metabolites targeting human cancer cells

The metabolites of bacteria Bacillus cereus and Bacillus pumilus isolated from soil samples in Shimoga region, Karnataka (India) were tested for cytotoxicity and anticancer properties. The various solvent extract fractions obtained from the metabolites of the two bacteria were tested for their cytotoxicity against normal human liver cell lines and 2 cancer cell lines by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay. The two fractions obtained from B. cereus showed high cytotoxicity. These two fractions were further screened for anticancer activity by nuclear staining studies and DNA fragmentation analysis. Both the fractions demonstrated significant activity by membrane blebbing during nuclear staining and caused the damage the DNA patterns during DNA fragmentation analysis. On the other hand, the metabolites of B. pumilus revealed toxic effect against cancer cells as well as normal ones.     

The adaptation responses of bacterial cytoplasmic membrane fluidity in the presence of environmental stress factors — polychlorinated biphenyls and 3-chlorobenzoic acid

Only bacteria sufficiently resistant to the toxic compounds in their environment can be used for the efficient biodegradation process in order to eliminate a widespread contamination by polychlorinated biphenyls (PCBs). The presence of PCBs results in bacterial controlled rigidification of cytoplasmic membrane. The four bacterial isolates from long-term PCB-contaminated soil (Alcaligenes xylosoxidans, Pseudomonas stutzeri) and sediment (Ochrobactrum anthropi, Pseudomonas veronii) have been used to select the strain most adapted to the PCBs, i.e. with efficient changes in the membrane phospholipid fatty acids. PCBs and their toxic degradation products — the 3-chlorobenzoic acids (3-CBA as the most toxic one) — were added separately to the liquid medium with glucose in two experimental sets: at lag phase and in stationary phase of bacterial growth in order to evaluate the effects of chemicals to cytoplasmic membrane. The main parameter — the changes in fatty acids composition (in the total lipids and the main membrane phospholipid phosphatidyletanolamine) were studied. 3-CBA caused growth inhibition when added at lag phase. However, when added during the stationary growth, inhibition was not observed. Similarly, after addition of PCBs to the stationary growth culture, inhibition of growth was not observed with all tested strains (except for P. stutzeri). This fact indicates the importance of time contact of bacteria during growth phase with xenobiotics. O. anthropi and A. xylosoxidans appeared to be the most adapted to the presence of PCBs (with sufficient membrane adaptation), active under the adverse conditions, and able to survive in the contaminated environment.     

Augmentative on-farm delivery methods for the parasitoid Habrobracon hebetor Say (Hymenoptera: Braconidae) to control the millet head miner Heliocheilus albipunctella (de Joannis) (Lepidoptera: Noctuidae) in Burkina Faso and Niger

Augmentative on-farm delivery methods for the parasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae) to control the millet head miner (MHM) Heliocheilus albipunctella (de Joannis) (Lepidoptera: Noctuidae) were investigated in Burkina Faso from 2011 to 2012 and in Niger in 2012. Our findings indicate that 7 cm × 10 cm jute bags containing 50 g of millet grains, 30 g of millet flour, 25 Corcyra cephalonica larvae and two mated H. hebetor females are the most effective option for on-farm delivery of the parasitoid. The parasitoid progeny started emerging from the bags eight days after confinement and 57–71 parasitoid adults emerged from each bag. Using the methods we developed, over 90 % parasitism of MHM larvae was achieved in millet farms. The implications of these findings for a large extension of MHM biocontrol program are discussed.     

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, an Inhibitor from Zea mays with Differential Activity against Soft Rotting Erwinia Species

[2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one] DIMBOA was extracted with ethyl acetate from acidified water homogenates of corn (Zea mays L.) seedlings. Pure DIMBOA or ethyl acetate extracts of corn tissue were added to bacterial growth medium at five concentrations (measured as hydroxamates). DIMBOA and corn extracts were more inhibitory to soft rot bacteria (Erwinia spp.) that are nonpathogenic to corn than to soft rot bacteria that are corn pathogens. The inhibitory activity of DIMBOA was similar to that of the ethyl acetate extracts. Both corn extracts and DIMBOA prolonged the lag phase of bacterial growth without significantly changing log phase growth rates. At various concentrations of the inhibitor, 50 to 100% of the activity of corn extracts inhibitory to different bacterial isolates was attributable to DIMBOA. Extracts of DIMBOA-deficient plants (genotype bxbx) were not inhibitory to Erwinia spp. It was concluded that DIMBOA is the major active component in those corn extracts which are inhibitory to soft rot Erwinia species.     

The usefulness of histopathology in aquatic toxicity studies

• 1.1. A survey was carried out to examine the usefulness of histopathology for the identification of toxic effects of environmental contaminants in fish.
• 2.2. Two small fish species, Poecilia reticulata (guppy) and Oryzias latipes (medaka) were used, and two exposure periods (1 and 3 months) were chosen.
• 3.3. The following compounds were studied: β-hexachlorocyclohexane, bis(tri-n-tributyltin)oxide, di-n-butyltindichloride, sodium bromide, methyl bromide, and methylmercury chloride.
• 4.4. The following is concluded: histopathology provides useful data in characterizing toxic effects in fish; there is a slight advantage for Poecilia reticulata over Oryzias latipes; there is no advantage for 3 months exposure vs 1 month.

     

Evaluation of Disinfection Techniques for, and Their Effects on, Rectal Thermocouple Catheters

The antibacterial activities of an iodophor (Wescodyne G), a quaternary ammonium compound (Roccal), and an iodine tincture as agents for the cold disinfection of rectal catheters contaminated in vitro were determined. Following thorough cleaning with an alcoholic solution of soft soap, each of the three disinfectants tested showed satisfactory results (100% kill) in 5 min against the enteric test bacteria (Escherichia coli and Salmonella typhosa) as well as a test species of the genus Pseudomonas, among the bacteria most resistant to surface-active agents.

An aqueous solution of Wescodyne G containing 75 ppm available iodine was used both as a wiping solution and for subsequent disinfection of rectal catheters contaminated in vivo. Total bacterial destruction was found to follow a 60-min soak preceded by the wiping procedure.

Rectal catheters subjected to prolonged immersion in each of the test disinfectants were found to be essentially unaffected, retaining their initial calibrations within a permissible tolerance. Neither Roccal nor Wescodyne G solutions were found to measurably attack bare thermocouples. Alcoholic iodine 0.5% did, however, exert a deteriorating effect on bare thermocouples in a short time, as measured by change in resistance characteristics.

The results of this study have led to the recommendation that Wescodyne G containing 75 ppm available iodine be used in standing operating procedures for the initial cleaning and subsequent disinfection of rectal thermocouple catheters.

     

A New Toxic Substance,Teleocidin, Produced by Streptomyces

The production and isolation of a new toxic substance, Teleocidin, and its biological properties were previously reported^1,2). Thereafter it has been found that an other strain of Streptomyces produced such specific toxic substance as Teleocidin in its cultured mycellium. Comparative tests of these two purified crystalline powders showed the new toxic substance resembles Teleocidin closely though differs in certain chemical properties. Therefore, the original Teleocidin is designated Teleocidin A, whereas that produced by a new strain of Streptomyces is named Teleocidin B, which had been tentatively called as the SK-toxic substance.

From the results of the chemical studies of Teleocidin B and its hydrogenated derivative, which was easily obtained as a crystalline form by the catalytic hydrogenation of Teleocidin B with Adam’s catalyst, molecular formula, C₂₈H_39~41N₃O₂ was postulated for Teleocidin B.

It was also recognized that an alcoholic hydroxyl, a lactam ring and a heterocyclic ring like indole or pyrrole structure existed as the functional groups of Teleocidin B.     

Isolation of toxic metal-tolerant bacteria from soil and examination of their bioaugmentation potentiality by pot studies in cadmium- and lead-contaminated soil

Heavy metals, also regarded as toxic metals, are the important environmental pollutants that affect all forms of life. Accumulation of toxic metals in plants results in various biochemical, physiological and structural disturbances, leading to inhibited growth and sometimes plant death. Toxic metal contamination disturbs the soil ecology as well as the agricultural productivity. Several indigenous microbes can withstand the effect of toxic metal and play a vital role in the revival of tarnished soil. In the present study, soil samples were collected from contaminated crop field of Cachar district of Assam, India. Segregation, enumeration and identification of bacteria from soil samples were performed. Among all the tested isolates, very few were able to withstand a high concentration of Cd and Pb in nutrient agar plates. Toxic metal-tolerant bacteria were identified as Pseudomonas aeruginosa and Bacillus cereus. The isolates having a higher tolerance for Cd and Pb were taken into consideration for pot studies. P. aeruginosa strain SN4 and strain SN5 showed significant results at Cd- and Pb-contaminated soil, evidenced by the healthy growth of Oryza sativa seedlings. However, B. cereus strain SN6 showed high tolerance towards Cd and Pb, but pot experimental studies showed adverse effects on seedling germination and shoot growth of O. sativa. P. aeruginosa strains were significantly able to reduce the negative impact of Cd and Pb in the soil, thus finding an alternative in removal, recovery and remediation of toxic metal-contaminated crop field.     

Induction, activation and inhibition of epoxide hydrase: an anomalous prevention of chlorobenzene-induced hepatotoxicity by an inhibitor of epoxide hydrase

Epoxide hydrase activity, measured with [³H]styrene oxide as substrate, is present in mammalian liver, kidney, lung, intestine and skin. The hepatic level of the enzyme, measured in vitro with [³H]styrene oxide, benzene oxide or naphthalene-1,2-oxide, is elevated substantially by pretreatment of rats with phenobarbital and to a lesser extent by pretreatment with 3-methylcholanthrene. Metyrapone and 1-(2-isopropylphenyl)-imidazole, two monooxygenase inhibitors, activate epoxide hydrase in vitro, but have no demonstrable effect on the enzyme in vivo. 3,3,3-Trichloropropene oxide, a potent in vitro inhibitor of epoxide hydrase, has no effect on monooxygenase activity measured in vitro with [³H]benzenesulfonanilide. Trichloropropene oxide is extremely toxic. In sub-lethal dosages, it does not significantly inhibit epoxide hydrase activity in vivo, although it and several other epoxides do react with and thereby reduce hepatic levels of glutathione. Cyclohexane oxide, another potent in vitro inhibitor of epoxide hydrase, reduces hepatic glutathione levels to 10% of control values. This relatively non-toxic substance should potentiate the hepatotoxicity of chlorobenzene by inhibiting further metabolism of the toxic chlorobenzene oxide intermediate through either hydration or conjugation with glutathione. Instead, co-administration of cyclohexene oxide and chlorobenzene significantly reduces the rate of metabolism of [¹⁴C]chlorobenzene and prevents the hepatic centrilobular necrosis caused by chlorobenzene in rats. Arene oxide-mediated hepatotoxicity apparently is dependent upon a variety of factors including both rates of formation and degradation of arene oxides in tissue. The presently known hydrase inhibitors are not sufficiently selective in their effects on liver cells to permit a quantitative assessment of the relative importance of these factors.     

Forensic Application of Microbiological Culture Analysis To Identify Mail Intentionally Contaminated with Bacillus anthracis Spores

The discovery of a letter intentionally filled with dried Bacillus anthracis spores in the office of a United States senator prompted the collection and quarantine of all mail in congressional buildings. This mail was subsequently searched for additional intentionally contaminated letters. A microbiological sampling strategy was used to locate heavy contamination within the 642 separate plastic bags containing the mail. Swab sampling identified 20 bags for manual and visual examination. Air sampling within the 20 bags indicated that one bag was orders of magnitude more contaminated than all the others. This bag contained a letter addressed to Senator Patrick Leahy that had been loaded with dried B. anthracis spores. Microbiological sampling of compartmentalized batches of mail proved to be efficient and relatively safe. Efficiency was increased by inoculating culture media in the hot zone rather than transferring swab samples to a laboratory for inoculation. All mail sampling was complete within 4 days with minimal contamination of the sampling environment or personnel. However, physically handling the intentionally contaminated letter proved to be exceptionally hazardous, as did sorting of cross-contaminated mail, which resulted in generation of hazardous aerosol and extensive contamination of protective clothing. Nearly 8 × 10⁶ CFU was removed from the most highly cross-contaminated piece of mail found. Tracking data indicated that this and other heavily contaminated envelopes had been processed through the same mail sorting equipment as, and within 1 s of, two intentionally contaminated letters.     

Isolation of Pathogenic Bacillus circulans from Callus Cultures and Healthy Offshoots of Date Palm (Phoenix dactylifera L.)

Pure cultures of a gram-negative, rod-shaped bacterium, which produced endospores after 3 to 5 days on solid medium, were isolated exclusively from tissue cultures of the date palm Phoenix dactylifera L. Electron microscopic examination of thin sections of the bacteria revealed the bilayer membrane typical of gramnegative bacteria and confirmed the nature of the spores as true endospores. Biochemical and physiological tests indicated that the bacteria were Bacillus circulans. B. circulans was consistently isolated from the internal tissues, including the meristem, of apparently healthy offshoots of date palm. When meristem and embryo callus tissue culture samples were injected with B. circulans isolated from similar tissue culture samples and from offshoots, the majority of the isolates produced a rapid, destructive soft rot of the tissues.     

The influence of physical-chemical characteristics of surface modified copper nanoparticles on E. coli cell population growth suppression and on electrostatic properties of their membranes

The biological activity of copper nanoparticles, able to suppress growth of E. coli cells population under contact interactions, was explored. Three types of samples with oxide layers of various sizes, thickness and composition were used in experiments. It was found out, that an increase in electron density on the external membrane of E. coli correlated with copper nanoparticles suppression capability and with lower activation energy of electron transfer on bacteria. The analysis of experimental data helps to correct conditions for obtaining nanoparticles with certain properties of their surface oxide layers. The character of temperature dependence of electron density reveals the electron type of conductivity in contact area of E. coli and nano-particles. These results help to find approach to understanding the nature of toxic influence of copper nano-particles on E. coli cells under contact interaction.