Impact of commonly used agrochemicals on bacterial diversity in cultivated soils

The effects of three selected agrochemicals on bacterial diversity in cultivated soil have been studied. The selected agrochemicals are Cerox (an insecticide), Ceresate and Paraquat (both herbicides). The effect on bacterial population was studied by looking at the total heterotrophic bacteria presence and the effect of the agrochemicals on some selected soil microbes. The soil type used was loamy with pH of 6.0–7.0. The soil was placed in opaque pots and bambara bean (Vigna subterranean) seeds cultivated in them. The agrochemicals were applied two weeks after germination of seeds at concentrations based on manufacturer’s recommendation. Plant growth was assessed by weekly measurement of plant height, foliage appearance and number of nodules formed after one month. The results indicated that the diversity index (Di) among the bacteria populations in untreated soil and that of Cerox-treated soils were high with mean diversity index above 0.95. Mean Di for Ceresate-treated soil was 0.88, and that for Paraquattreated soil was 0.85 indicating low bacterial populations in these treatment-type soils. The study also showed that application of the agrochemicals caused reduction in the number of total heterotrophic bacteria population sizes in the soil. Ceresate caused 82.50% reduction in bacteria number from a mean of 40 × 10⁵ cfu g⁻¹ of soil sample to 70 × 10⁴ cfu g⁻¹. Paraquat-treated soil showed 92.86% reduction, from a mean of 56 × 10⁵ cfu g⁻¹ to 40 × 10⁴ cfu g⁻¹. Application of Cerox to the soil did not have any remarkable reduction in bacterial population number. Total viable cell count studies using Congo red yeast-extract mannitol agar indicated reduction in the number of Rhizobium spp. after application of the agrochemicals. Mean number of Rhizobium population numbers per gram of soil was 180 × 10⁴ for the untreated soil. Cerox-treated soil recorded mean number of 138 × 10⁴ rhizobial cfu g⁻¹ of soil, a 23.33% reduction. Ceresate- and Paraquat-treated soils recorded 20 × 10⁴ and 12 × 10⁴ cfu g⁻¹ of soil, respectively, representing 88.89% and 93.33% reduction in Rhizobium population numbers. Correspondingly, the mean number of nodules per plant was 44 for the growth in untreated soil, 30 for the plant in the Cerox-treated soil, 8 for the plant in Paraquat-treated soil and 3 for the plant in Ceresate-treated soil. The study has confirmed detrimental effect of insecticide on bacterial populations in the soil. Total heterotrophic counts, rhizobial counts as well as the number of nodules of all samples taken from the chemically treated soils were all low as compared to values obtained for the untreated soil. However, the effect of the insecticide was minimal in all cases as compared to the effects of the herbicides on the soil fauna. Indiscriminate use of agrochemicals on farms can therefore affect soil flora and subsequently food production.     

Gut microbial community response to herbicide exposure in a ground beetle

Gut microbiota plays a key role in physiological processes of insects, including nutritional metabolism, development, immunity and detoxification. Environmental stressors such as herbicides, used to optimize and improve crop yields, may interfere with the mutualistic relationships causing negative consequences for the host health. Dinitroaniline herbicides, for example pendimethalin, are used worldwide in pre-emergence application to control grass and some broadleaf weeds. They target microtubules to arrest cell division and inhibit the development of roots and shoots. Effects of a pendimethalin-based herbicide were assessed on the gut microbial community of Pterostichus melas italicus Dejean, 1828 (Coleoptera, Carabidae). The exposure effect was tested in vivo by using a recommended field rate (4 L per ha, 330 gL⁻¹ of active ingredient) and evaluating the variability of responses in 21 days, corresponding to the half-life of pendimethalin. The 16S rRNA sequencing data showed that the gut lumen was dominated by Proteobacteria, Firmicutes, Fusobacteria, Tenericutes and Bacteroidetes. The exposure interfered with the bacterial community richness and diversity associated with the gut from 2 days after the treatment. The differential abundance analyses highlighted a shift involving Lactobacillaceae, Streptomycetaceae, Neisseriaceae, Ruminococcaceae and Enterobacteriaceae. An increase in species such as Enterobacter sp., Pseudomonas sp., Pantoea sp and Paracoccus sp. involved in the herbicide degradation was also recorded after 21 days of exposure. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis indicated that the exposure has effects on the most predicted functional categories of gut microbiota related to metabolic function including carbohydrate, amino acid and lipid metabolism. These results demonstrate that pendimethalin can impact microbial communities associated with generalist predators inhabiting croplands leading to severe implications for the species’ ecological role. Understanding the effects of herbicides such as pendimethalin on ground beetles may help to protect beneficial soil insects that have a crucial role in the ecosystem services.     

Decolorization of Azo Dye (Orange MR) by an Autochthonous Bacterium, <Emphasis Type="Italic">Micrococcus</Emphasis> sp. DBS 2

Soil and sediment samples obtained from Orange MR dye contaminated habitat were screened for heterotrophic bacterial population. The heterotrophic bacterial density of dye-contaminated soil was 2.14 × 10⁶ CFU/g. The generic composition of heterotrophic bacterial population was primarily composed of 10% of Proteus sp., 15% Aeromonas sp., 20% Bacillus sp., 25% Pseudomonas sp. and 30% Micrococcus sp. The bacterial strain that decolorized the azo dye Orange MR up to 900 ppm was identified as Micrococcus sp. The optimum inoculum load, pH and temperature were found to be 5%, 6 and 35°C, respectively. The rate of decolorization was assessed using spectrophotometer at 530 nm and the percentage of decolorization was ascertained. The autochthonous bacterial isolate was able to utilize the dye as both nitrogen and carbon source.     

Toxicity of Crude Extracellular Products of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> on Rohu, <Emphasis Type="Italic">Labeo rohita</Emphasis> (Ham.)

In the present study the haemolytic and proteolytic activity of extracellular products (ECP) secreted from Aeromonas hydrophila (CAHH14 strain) were studied with respect to temperature and different time of incubation as well as its lethal toxicity on rohu, Labeo rohita. The strain was isolated from Catla catla (showing abdominal dropsy symptom) collected from the pond of Central Institute of Freshwater Aquaculture (CIFA), Bhubaneswar, India and was characterized on the basis of biochemical tests. The highest production of haemolysin was achieved when the bacteria was grown at 35°C for 30 h. The proteolytic activity was found to be highest when the bacterium was grown at 30°C for 36 h. The haemolytic and proteolytic toxin produced by Aeromonas hydrophila was found to be lethal to rohu (LD₅₀ 1.7 × 10⁴ cfu/ml). The lethality of ECP was decreased by heating and completely inactivated by boiling at 100°C for 10 min. This indicates that protease activity and haemolytic activity of A. hydrophila ECP was temperature dependant.     

Resistance to glyphosate in the cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> as result of pre-selective mutations

Adaptation of Microcystis aeruginosa (Cyanobacteria) to resist the herbicide glyphosate was analysed by using an experimental model. Growth of wild-type, glyphosate-sensitive (G^s) cells was inhibited when they were cultured with 120 ppm glyphosate, but after further incubation for several weeks, occasionally the growth of rare cells resistant (G^r) to the herbicide was found. A fluctuation analysis was carried out to distinguish between resistant cells arising from rare spontaneous mutations and resistant cells arising from other mechanisms of adaptation. Resistant cells arose by rare spontaneous mutations prior to the addition of glyphosate, with a rate ranging from 3.1 × 10⁻⁷ to 3.6 × 10⁻⁷ mutants per cell per generation in two strains of M. aeruginosa; the frequency of the G^r allele ranged from 6.14 × 10⁻⁴ to 6.54 × 10⁻⁴. The G^r mutants are slightly elliptical in outline, whereas the G^s cells are spherical. Since G^r mutants have a diminished growth rate, they may be maintained in uncontaminated waters as the result of a balance between new resistants arising from spontaneous mutation and resistants eliminated by natural selection. Thus, rare spontaneous pre-selective mutations may allow the survival of M. aeruginosa in glyphosate-polluted waters via G^r clone selection.     

Production of Exo-polysaccharide by <Emphasis Type="Italic">Rhizobium</Emphasis> sp.

Two fold increase in the yield of glucose and maltose containing exo-polysaccharide (EPS) by Rhizobium sp. was observed during its growth in modified YEMB. EPS production, plant growth promotion activity and root colonization of Rhizobium sp. studies showed enhanced EPS synthesis, more seed germination and over all improvement in plant growth over control and R. meliloti treatment. Groundnut seeds bacterized with Rhizobium sp. resulted in 69.75% more root length, 49.51% more shoot height, 13.75% more number of branches and 13.60% more number of pods over the control and R. meliloti treatment. Bacterization of wheat seeds increased the dry matter yield of roots (1.7-fold), and roots adhering soil (RAS) (1.5) and shoot mass (1.9-fold). Rhizobium sp. inoculation also increased the population density of EPS-producing bacteria on the rhizoplane. Roots of plants inoculated with Rhizobium sp. maintained a higher K⁺/Na⁺ ratio and K⁺–Na⁺ selectivity.     

CpG‐related SNPs in the MS4A region have a dose‐dependent effect on risk of late–onset Alzheimer disease

CpG‐related single nucleotide polymorphisms (CGS) have the potential to perturb DNA methylation; however, their effects on Alzheimer disease (AD) risk have not been evaluated systematically. We conducted a genome‐wide association study using a sliding‐window approach to measure the combined effects of CGSes on AD risk in a discovery sample of 24 European ancestry cohorts (12,181 cases, 12,601 controls) from the Alzheimer's Disease Genetics Consortium (ADGC) and replication sample of seven European ancestry cohorts (7,554 cases, 27,382 controls) from the International Genomics of Alzheimer's Project (IGAP). The potential functional relevance of significant associations was evaluated by analysis of methylation and expression levels in brain tissue of the Religious Orders Study and the Rush Memory and Aging Project (ROSMAP), and in whole blood of Framingham Heart Study participants (FHS). Genome‐wide significant (p < 5 × 10^?8) associations were identified with 171 1.0 kb‐length windows spanning 932 kb in the APOE region (top p < 2.2 × 10^?308), five windows at BIN1 (top p = 1.3 × 10^?13), two windows at MS4A6A (top p = 2.7 × 10^?10), two windows near MS4A4A (top p = 6.4 × 10^?10), and one window at PICALM (p = 6.3 × 10^‐9). The total number of CGS‐derived CpG dinucleotides in the window near MS4A4A was associated with AD risk (p = 2.67 × 10^?10), brain DNA methylation (p = 2.15 × 10^?10), and gene expression in brain (p = 0.03) and blood (p = 2.53 × 10^?4). Pathway analysis of the genes responsive to changes in the methylation quantitative trait locus signal at MS4A4A (cg14750746) showed an enrichment of methyltransferase functions. We confirm the importance of CGS in AD and the potential for creating a functional CpG dosage‐derived genetic score to predict AD risk.     

Flow‐injection determination of cysteine in pharmaceuticals based on luminol–persulphate chemiluminescence detection

A flow injection (FI) method is reported for the determination of l‐ cysteine, based on its enhancement on chemiluminescence (CL) emission of luminol oxidized by sodium persulphate in alkaline solution. The calibration graph was linear over the range 1.0 × 10^–9–5.0 × 10^–7 mol/L (r² = 0.9992), with relative standard deviations (RSDs) in the range 1.1–2.3% (n = 4). The limit of detection (3σ blank) was 5.0 × 10^–10 mol/L with a sample throughput of 120/h. The method was applied to pharmaceuticals and the results obtained were in reasonable agreement with the amount labelled. The proposed method was also applied to cysteine in synthetic amino acid mixtures. Calibration graphs of N‐acetylcysteine and glutathione over the range 1.0–50 × 10^–8 and 0.5–7.5 × 10^–7 mol/L were also established (r² = 0.998 and 0.9986) with RSDs in the range 1.0–2.0% (n = 4), and the limits of detection (3σ blank) were 5.0 × 10^–9 and 1.0 × 10^–8 mol/L, respectively. Copyright © 2008 John Wiley & Sons, Ltd.     

Microbial Inoculants with Multifaceted Traits Suppress Rhizoctonia Populations and Promote Plant Growth in Cotton

The suppressive effects of microbial inoculants on cotton seedling mortality were assessed in Rhizoctonia solani‐infested soil. Per cent mortality ranged from 16 to 32 (60–120 days after sowing, DAS) and significant differences were recorded at 120 DAS, especially after drenching with compost tea of Azotobacter sp. and Anabaena torulosa—Trichoderma viride‐biofilmed formulations. The activity of hydrolytic enzymes was reduced in diseased root tissues due to a majority of the microbially inoculated treatments, compared with healthy root tissues. Per cent changes in the amounts of glomalin‐related soil proteins (GRSPs) were 2 to 85% greater than those of the uninoculated experimental controls. These microbial inoculants altered the rhizosphere bacterial communities as evident from the Denaturing gradient gel electrophoresis (DGGE) banding patterns and, also reduced the population of R. solani. While the copy numbers of the internal transcribed spacer (ITS) gene of R. solani in the uninoculated (infested soil) were approximately 1.47 × 10¹¹ per g soil, they were 1.34–1.42 × 10⁵ per g soil after the application of A. torulosa, Anabaena laxa and A. torulosa–Bacillus sp. Increases in yield (ranging from 3 to 23%) due to various microbial inoculants relative to uninoculated controls illustrated their promise as plant growth‐promoting and disease‐suppressing agents. This study illustrates the modulation of rhizosphere ecology through microbial inoculants as a mechanism of disease suppression and sustaining plant growth.     

Isolation and characterization of three <Emphasis Type="Italic">Sphingobium</Emphasis> sp. strains capable of degrading isoproturon and cloning of the catechol 1,2-dioxygenase gene from these strains

Three strains of bacteria (designated as YBL1, YBL2, YBL3 respectively) capable of degrading isoproturon, 3-(4-isopropylphenyl)-1, 1-dimethylurea, were isolated from the soils of two herbicide plants. Based on the comparative analysis of the 16S rRNA gene, and phenotypic and biochemical characterization, these strains were identified as Sphingobium sp. The optimum conditions for isoproturon degradation by these strains were pH 7.0, and temperature 30°C. Mg²⁺ (1 mM) enhanced the isoproturon degradation rate, while Ni²⁺ and Cu²⁺ (1 mmol l⁻¹) inhibited isoproturon degradation significantly. These three strains also showed the ability to remove the residues of other phenylurea herbicides such as chlorotoluron, diuron and fluometuron in mineral salt culture medium. The N-demethylation was the first step of degradation of dimethylurea-substituted herbicides. Strain YBL1 was found capable of degrading both dimethylurea-substituted herbicides and methoxymethylphenyl-urea herbicides i.e. linuron (3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea). Using the PCR method, partial sequences of the catechol 1,2-dioxygenase gene were obtained from these strains.     

Establishment of functional primary cultures of heart cells from the clam <Emphasis Type="Italic">Ruditapes decussatus</Emphasis>

Heart cells from the clam Ruditapes decussatus were routinely cultured with a high level of reproducibility in sea water based medium. Three cell types attached to the plastic after 2 days and could be maintained in vitro for at least 1 month: epithelial-like cells, round cells and fibroblastic cells. Fibroblastic cells were identified as functional cardiomyocytes due to their spontaneous beating, their ultrastructural characteristics and their reactivity with antibodies against sarcomeric α-actinin, sarcomeric tropomyosin, myosin and troponin T-C. Patch clamp measurements allowed the identification of ionic currents characteristic of cardiomyocytes: a delayed potassium current (I _K slow) strongly suppressed (95%) by tetraethylammonium (1 mM), a fast inactivating potassium current (I _K fast) inhibited (50%) by 4 amino-pyridine at 1 mM and, at a lower level (34%) by TEA, a calcium dependent potassium current (I _KCa) activated by strong depolarization. Three inward voltage activated currents were also characterized in some cardiomyocytes: L-type calcium current (I _Ca) inhibited by verapamil at 5 × 10⁻⁴ M, T-type Ca²⁺ current, rapidly activated and inactivated, and sodium current (I _Na) observed in only a few cells after strong hyperpolarization. These two currents did not seem to be physiologically essential in the initiation of the beatings of cardiomyocytes. Potassium currents were partially inhibited by tributyltin (TBT) (1 μM) but not by okadaic acid (two marine pollutants). DNA synthesis was also demonstrated in few cultured cells using BrdU (bromo-2′-deoxyuridine). Observed effects of okadaic acid and TBT demonstrated that cultured heart cells from clam Ruditapes decussatus can be used as an experimental model in marine toxicology.     

Inactivation of Bacillus cereus by Na‐chlorophyllin‐based photosensitization on the surface of packaging

Aims: This study was focused on the possibility to inactivate food‐borne pathogen Bacillus cereus by Na‐chlorophyllin (Na‐Chl)‐based photosensitization in vitro and after attachment to the surface of packaging material. Methods and Results: Bacillus cereus in vitro or attached to the packaging was incubated with Na‐Chl (7·5 × 10^?8 to 7·5 × 10^?5 mol l^?1) for 2–60 min in phosphate buffer saline. Photosensitization was performed by illuminating cells under a light with a λ of 400 nm and an energy density of 20 mW cm^?2. The illumination time varied 0–5 min and subsequently the total energy dose was 0–6 J cm^?2. The results show that B. cereus vegetative cells in vitro or attached to the surface of packaging after incubation with 7·5 × 10^?7 mol l^?1 Na‐Chl and following illumination were inactivated by 7 log. The photoinactivation of B. cereus spores in vitro by 4 log required higher (7·5 × 10^?6 mol l^?1) Na‐Chl concentration. Decontamination of packaging material from attached spores by photosensitization reached 5 log at 7·5 × 10^?5 mol l^?1 Na‐Chl concentration. Comparative analysis of different packaging decontamination treatments indicates that washing with water can diminish pathogen population on the surface by <1 log, 100 ppm Na‐hypochlorite reduces the pathogens about 1·7 log and 200 ppm Na‐hypochlorite by 2·2 log. Meanwhile, Na‐Chl‐based photosensitization reduces bacteria on the surface by 4·2 orders of magnitude. Conclusions: Food‐borne pathogen B. cereus could be effectively inactivated (7 log) by Na‐Chl‐based photosensitization in vitro and on the surface of packaging material. Spores are more resistant than vegetative cells to photosensitization‐based inactivation. Comparison of different surface decontamination treatments indicates that Na‐Chl‐based photosensitization is much more effective antibacterial tool than washing with water or 200 ppm Na‐hypochlorite. Significance and Impact of the Study: Our data support the idea that Na‐Chl‐based photosensitization has great potential for future application as an environment‐friendly, nonthermal surface decontamination technique.     

Commercially formulated glyphosate can kill non‐target pollinator bees under laboratory conditions

The use of glyphosate‐based herbicides in agroecosystems has increased over the past few years because of the advent of genetically modified glyphosate‐resistant crops and resistant weeds. This is alarming because of potential damaging effects on non‐target organisms. In sub‐Saharan Africa, for example Ghana, many rural farmers have not received training in the use of glyphosate‐based herbicides, thus tend to apply higher than recommended concentrations on farms. Therefore, this study investigated the effect of glyphosate‐based herbicides on beneficial insects under laboratory conditions, using Apis mellifera L. (Hymenoptera: Apidae, Apini) and Hypotrigona ruspolii (Magretti) (Hymenoptera: Apidae, Meliponini) as models. The bees were put in contact for 24 h with the recommended concentration of Sunphosate 360 SL, a glyphosate‐based herbicide, 2× the recommended concentration, or distilled water as control. The effect of the herbicide on the bees was compared to the effect of a lambda‐cyhalothrin insecticide. Generally, more bees died after contact with plants freshly sprayed with the herbicide than on herbicide‐treated filter paper. In both cases, more bees died after contact with the higher concentration of the herbicide. These findings suggest that beneficial insects, specifically A. mellifera and H. ruspolii, may get killed if they are sprayed upon or come into contact with plants that have been freshly sprayed with (more than) the recommended concentration of glyphosate‐based herbicides. Therefore, it is important to restrict access and use of such herbicides to trained personnel who will comply with spraying guidelines, that is, recommended concentrations and timing of spray. Spraying at a time when insects are flying about may be detrimental to beneficial insects such as pollinator bees, parasitoids, and predators.     

Real‐time PCR quantification of Bemisia tabaci (Homoptera: Aleyrodidae) B‐biotype remains in predator guts

The cotton whitefly, Bemisia tabaci (Gennadius) B‐biotype, is fed on by a wide variety of generalist predators, but there is little information on these predator–prey interactions, especially under field conditions. In this study, a real‐time polymerase chain reaction (PCR) assay was developed to quantify B. tabaci B‐biotype remains in predator gut. The B. tabaci B‐biotype genomic DNA copy number was referred to the actual amount of BT1 isolate, the B. tabaci B‐biotype specific DNA fragment. The numbers of BT1 isolate in one B. tabaci B‐biotype egg, individual adult and a single red‐eyed nymph were 2.56 × 10³, 2.56 × 10⁴, and 1.29 × 10⁴ copies, respectively. When Propylaea japonica adults fed on one, two, four, eight or 16 red‐eyed nymphs, the detected numbers of BT1 isolate ranged from 2.77 × 10⁴ to 4.05 × 10⁵ copies, forming a strong linear relationship (R² = 0.9899). Following the consumption of two red‐eyed nymphs, prey DNA was detectable in 100% of P. japonica at t = 0, decreasing to 80.0% and 60.0% after 1–4 h and 8 h of digestion, respectively, with 3.36 × 10⁴–1.25 × 10³ BT1 isolate copies. The predation by field‐collected predators, 26 larvae of P. japonica, and of Harmonia axyridis each, Chrysopa spp. larvae (Chrysopa pallens and C. formosa, 18 individuals in total), and a single adult of Scymnus hoffmanni, 19 adults of Orius sauteri and nine adult spiders (Erigonnidium graminicolum and Neoscona doenitzi), on B. tabaci B‐biotype were quantified. Of the 99 analysed predator individuals, 3.65 × 10²–4.60 × 10⁵ copies of BT1 isolate, equivalent to 0.8–18.8 red‐eyed nymphs were detected. These results suggest that TaqMan real‐time PCR technology may provide a rapid and sensitive method for quantifying B. tabaci B‐biotype remains in predator guts and will be invaluable in assessing the food web relationship between prey and arthropod predators.     

Nucleopolyhedrovirus infection and/or parasitism by Microplitis pallidipes affected haemolymph titre of 20‐hydroxyecdysone in Spodoptera exigua larvae

This study examined the physiological effects of joint and separate parasitism and infection by the endoparasitoid Microplitis pallidipes Szépligeti and the nucleopolyhedrovirus (NPV), respectively, on haemolymph 20‐hydroxyecdysone (20‐E) titre in Spodoptera exigua (Hübner) larvae. The results indicated that in parasitized larvae, virus‐infected larvae (5.7 × 10³ and 5.7 × 10⁵ OB/ml) and parasitized larvae infected with virus at 5.7 × 10⁵ OB/ml, compared to healthy larvae, the 20‐E all declined during the first 3 days but began to increase from day 4 after treatment, while in jointly parasitized and infected larvae (5.7 × 10³ OB/ml), the 20‐E declined during the first 4 days but began to increase on day 5 after treatment. Meanwhile, compared to parasitized larvae, the 20‐E declined during the first 4 days but significantly increased on day 5 in jointly parasitized and infected larvae (5.7 × 10³ OB/ml), while significantly increased during the first 2 days but began to decrease from day 3 after treatment in jointly parasitized and infected larvae (5.7 × 10⁵ OB/ml). Finally, in larvae that were both parasitized and virus infected (5.7 × 10³ OB/ml), compared to just virus‐infected larvae (5.7 × 10³ OB/ml), the 20‐E was lower on days 3 and 4 but higher on other days after treatment; in larvae that were both parasitized and virus infected (5.7 × 10⁵ OB/ml), compared to just virus‐infected larvae (5.7 × 10⁵ OB/ml), the 20‐E was significantly higher at the first 2 days but lower from day 3 after treatment. Our results revealed that 2nd instar larval M. pallidipes in host bodies may release 20‐E into the haemolymph of S. exigua larvae and that NPV infection may stimulate S. exigua to release more 20‐E during its third to fourth instar larval moulting. We found that this stimulatory effect was greater with higher virus concentrations.     

Effect of plant stage, <Emphasis Type="Italic">Colletotrichum truncatum</Emphasis> dose,and use of herbicide on control of <Emphasis Type="Italic">Matricaria perforata</Emphasis>

Colletotrichum truncatum (Ct) was examined in a tank mix with the herbicide 2,4-D, clopyralid plus MCPA (Caurtail M^®), or metribuzin (Sencor^®) for control of scentless chamomile at 8- (younger) and 11-leaf stages (older) under controlled conditions. In initial trials, Ct at 7 × 10⁶ spores/ml (200 l/ha) reduced the fresh weight of scentless chamomile only slightly. However, its combinations with herbicides improved the efficacy variably depending on the herbicide used and stage of the weeds. Ct plus 2,4-D reduced the fresh weight by about 50% at both leaf stages of scentless chamomile when compared to untreated controls but no plants were killed. The fungus plus Curtail M consistently killed younger but not older plants, and the efficacy was substantially greater than that of the herbicide alone. The herbicide Sencor was highly effective on younger plants, and adding Ct did not achieve additional benefits. On older plants, however, Ct plus Sencor was substantially more effective than the herbicide alone, causing 76% fresh-weight reduction when compared to controls and killing 9 out of 16 older plants in four trials. Sencor applied alone reduced the fresh weight of older plants by 65%, but no plants were killed. Tested at doses ranging from 2 × 10⁶ to 20 × 10⁶ spores/ml, Ct plus Curtail M was most effective at the highest fungal inoculum dose, consistently killing younger but not older plants. In comparison, Ct at a medium dose (7 × 10⁶ spores/ml) plus Sencor killed the majority of older chamomile plants (7 out of 12), whereas the herbicide alone did not cause plant mortality. Further increasing fungal inoculum dose from this medium level did not enhance the weed control by Ct plus Sencor.     

Mycelial cultivation of <Emphasis Type="Italic">Phellinus linteus</Emphasis> using cheese-processing waste and optimization of bioconversion conditions

A medicinal mushroom, Phellinus linteus, was successfully cultivated using a cheese-processing waste, whey, and the optimal bioconversion conditions for the maximum mycelial growth rate was also estimated through solid-state cultivation experiments. Response surface analysis with a face-centered design (center point replication = 5) was applied to statistically approximate the simultaneous effects of the three variables, i.e., substrate concentration (10–30 g lactose l⁻¹), temperature (20–30°C), and pH (4–6), on the mycelial growth rate of P. linteus. The following is a partial cubic model where η is the mycelial growth rate (K _r ) and x _k is the corresponding variable term (k = substrate concentration, temperature, and pH in order): η = −23.8 + 8.67 × 10⁻² x ₁ + 1.48x ₂ + 1.77x ₃ + 8.00 × 10⁻⁴ x ₁ x ₂ + 7.25 × 10⁻² x ₁ x ₃ + 5.13 × 10⁻² x ₂ x ₃ −1.28 × 10⁻² x ₁² –3.18 × 10⁻² x ₂². −2.64 × 10⁻¹ x ₃² −3.28 × 10⁻³ x ₁ x ₂ x ₃ + 4.68 × 10⁻⁴ x ₁² x ₂. The produced response surface model proved to be significant (r ² > 0.99, P-value <0.0001, coefficient of variation <5%) to describe the explored space. Temperature was found to be the most significant factor of dominant effects on the mycelial growth rate, and other variables such as temperature², pH, pH², and (substrate concentration² × temperature) also showed significant effects on the model output. The maximum mycelial growth rate was predicted to be 2.80 mm d⁻¹ at 29.7 g lactose l⁻¹, 26.2°C, and pH 5. Our results proved a good potential of whey to serve as an alternative growth medium for cultivating P. linteus mycelia. This may provide another potential for managing this nutrient-rich waste in a cost-effective way.     

Confirming therapeutic target of protopine using immobilized β2‐adrenoceptor coupled with site‐directed molecular docking and the target‐drug interaction by frontal analysis and injection amount–dependent method

Drug‐protein interaction analysis is pregnant in designing new leads during drug discovery. We prepared the stationary phase containing immobilized β₂‐adrenoceptor (β ₂‐ AR) by linkage of the receptor on macroporous silica gel surface through N ,N ′‐carbonyldiimidazole method. The stationary phase was applied in identifying antiasthmatic target of protopine guided by the prediction of site‐directed molecular docking. Subsequent application of immobilized β ₂‐ AR in exploring the binding of protopine to the receptor was realized by frontal analysis and injection amount–dependent method. The association constants of protopine to β ₂‐ AR by the 2 methods were (1.00 ± 0.06) × 10⁵M⁻¹ and (1.52 ± 0.14) × 10⁴M⁻¹. The numbers of binding sites were (1.23 ± 0.07) × 10⁻⁷M and (9.09 ± 0.06) × 10⁻⁷M, respectively. These results indicated that β ₂‐ AR is the specific target for therapeutic action of protopine in vivo. The target‐drug binding occurred on Ser¹⁶⁹ in crystal structure of the receptor. Compared with frontal analysis, injection amount–dependent method is advantageous to drug saving, improvement of sampling efficiency, and performing speed. It has grave potential in high‐throughput drug‐receptor interaction analysis.     

Virulence of a South African isolate of the <Emphasis Type="Italic">Cryptophlebia leucotreta</Emphasis> granulovirus to <Emphasis Type="Italic">Thaumatotibia leucotreta</Emphasis> neonate larvae

Surface inoculation dose–response and time–response bioassays and detached fruit bioassays were conducted with a novel South African isolate of the Cryptophlebia leucotreta granulovirus (CrleGV-SA) against Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Noctuidae) neonate larvae. LC₅₀ and LC₉₀ values were estimated to be 4.095 × 10³ and 1.185 × 10⁵ OBs ml⁻¹, respectively. LT₅₀ and LT₉₀ values were estimated to be 4 days 22 h and 7 days 8 h, respectively, categorising the virus as a fast or type 2 granulovirus. There was a conspicuous difference in behaviour between larvae on inoculated diet and untreated diet, resulting in a significant reduction in penetration of diet. Bioassays on detached Navel oranges revealed LC₅₀ and LC₉₀ values of 9.310 × 10⁷ and 1.515 × 10⁹ OBs ml⁻¹, when using data on numbers of larvae per fruit rather than on numbers of infested fruit. Field trials will be conducted.     

Hyperproduction of chitinase influences crystal toxin synthesis and sporulation of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Bacillus thuringiensis HD-73 was transformed with the endochitinase gene chiA74 under the control of a strong promoter (pcytA) and a 5′ mRNA stabilizing (STAB-SD) sequence (HD-73-pEBchiA74). Expression levels were compared with those observed from the wild type strain (HD-73) and the recombinant HD-73 strain expressing chiA74 under the control of its native promoter (HD-73-pEHchiA74). The chitinolytic activity of HD-73-pEBchiA74 was markedly elevated, being ~58- and 362-fold higher than, respectively, HD-73-pEHchiA74 and parental HD-73, representing the highest levels of chitinase expression in recombinant B. thuringiensis reported to date. Parasporal crystals measured under transmission electron microscopy showed that HD-73 produced crystals of 1.235 (±0.214) and 1.356 (±0.247) μm in length when the bacterium was grown in respectively, NBS and NBS with glucose. Otherwise, HD-73-pEBchiA74 synthesized crystals of 1.250 (±0.222) and 1.139 (±0.202) μm in length when cultivated in NBS and NBS with glucose, respectively, values that showed a diminution of ~10 and 20% compared with crystals produced by HD-73-pEHchiA74 grown under the same conditions. Comparison of viable spore counts per ml showed that HD-73-pEBchiA74 produced fewest viable spores (1.5 × 10⁹, 1.3 × 10⁹), compared to HD-73-pEHchiA74 (4.9 × 10⁹, 5.3 × 10⁹) and HD-73 (6.8 × 10⁹, 8.8 × 10⁹) when grown in NBS and NBS supplemented with glucose, respectively. No change in cellular protease activity was observed despite the overproduction of the chitinase.