A study of the traits associated with the biocontrol activity of Phlebiopsis gigantea

Phlebiopsis gigantea has routinely been used for the biocontrol of the conifer pathogen, Heterobasidion annosum s.l. but the mechanism for the biocontrol action has not been properly understood. In the present work, 64 isolates of P. gigantea were screened for traits important for the biocontrol of H. annosum. Growth rate and the interaction patterns of H. annosum s.l. and P.gigantea were studied in both carbon rich (Hagem) and low carbon media (Norkrans). Laccase assay and wood degradation capabilities were performed on the 64 isolates. Data was analyzed with multiple regression and principal component analysis. Results showed a significant effect of culture composition on the outcome of the interaction, 90 % of the isolates were able to displace H. annosum s.s. in sawdust media after 20 days, as compared with only 4% recorded in the glucose rich Hagem media. High growth rate on sawdust, a lignified carbon source, correlated with high growth rate in ferulic acid, a lignin precursor (P = 0.078), high growth rate in xylan, a hemicellulose (P = 0.001) and percentage weight loss in pine (P = 0.01). Interaction in sawdust correlated with high wood degradation capability in pine and spruce with P-values (P = 0.01, P = 0.03) respectively, high growth rate in xylan (P = 0.01), laccase production (P = 0.08), interaction in Hagem (P = 0.01) and mean growth rate at 10 °C (P = 0.001). Additionally, the role of hydrophobin in the competitive interaction was further investigated. The genomic sequence of Phlebiopsis gigantea hydrophobins 1 and 2 (Pgh1 and Pgh2) from a subset of isolates selected on the basis of geographical origins and antagonistic abilities was investigated. Similarly, the expression of Pgh1 and Pgh2 under different substrate conditions was also studied using quantitative PCR. Sequence analysis was performed with Clustal W and inspected with Megalign (DNA Star). Expression data was analyzed using the relative quantification method- 2-ΔΔCt and tested for effects of isolates, genes and culture conditions using the general linear model (GLM) procedure in SAS. There was a close sequence similarity between hydrophobin genes of isolates having different antagonistic capabilities and from different geographical sources. Higher transcript levels of Pgh1 and Pgh2 were recorded in submerged cultures compared with aerial conditions. The effect of substrate on the expression of the two genes (Pgh1 and Pgh2) was statistically significant (P = 0.0001). Differences in transcript levels of Pgh1 and Pgh2 were also observed among isolates belonging to different antagonistic categories. Overall, the results suggests that the antagonistic and competitive advantage of P. gigantea, hinged on the ability of the isolates to degrade the different structural components of wood. A significant correlation was also found between some high antagonistic isolates and the expression of hydrophobin genes (Pgh1 and Pgh2). The significance of these results in the biological control is discussed

Aflatoxin contamination of poultry feeds in Nigerian feed mills and the effect on the performance of Abor Acre broilers

The present study investigated the effect of aflatoxin contamination of poultry feedsfrom different commercial feed sources on the performance of broiler chicks. Seventy five 3 weeks old Abor acre broiler chicks were randomly divided into five groups (A – E), replicated thrice with each replicate having 5 birds. Each replicate constituted an experimental unit and the birds were separately housed in a 1 x 1 m2 sized pen. The birds in each treatment group were fed feeds from 5 companies, A – E. Feed intake and body weights were measured in all the treatment groups. Blood samples from the birds were analyzed for different hematological parameters. Feed samples were analyzed for aflatoxin concentration using reverse phase HPLC. The total aflatoxin content (AFB1 + AFB2 + FG1 + AFG2) of the feeds ranged from 1.4 ± 0.4 - 224.2 ± 74.7 μg/kg. The total aflatoxin level in feed samples C and E were above the European community regulatory limit for poultry feeds (ECRL, 20 μg/kg). There was no statistically significant difference between the haematological values of the birds fed feeds from the different source (P ˃ 0.005). Lymphocyte count was relatively low in all the treatment groups. However, the mixed blood populations were scantly detected in all the groups. Feed intake was positively correlated with body weight in the two treatment groups, A and E (P = 0.0001, R = 1). The high concentration of aflatoxin in some of the feeds caused reduced feed intake and poor body weight among the treated birds.Keywords: Aflatoxin, Poultry feed contamination, Haematology, Body weight,  Reverse phase HPL

Physiological and molecular analysis of the interaction between the conifer pathogen, Heterobasidion annosum s.l. and Phlebiopsis gigantea

Prophylactic stump treatment with Phlebiopsis gigantea has been used for a long time in the biological control of the conifer pathogen, Heterobasidion annosum sensu lato. However, the mechanisms underlying the biocontrol process are poorly understood. A total of 64 isolates of P. gigantea from different geographical regions were screened for fungal traits (growth rate, enzyme production, wood decay capabilities and antagonism) important for the biocontrol properties. The genomic sequence and the expression profile of P. gigantea hydrophobin encoding genes 1 and 2 in a selected set of isolates of the biocontrol fungus was analysed. Additionally, the distribution and the regulatory patterns of different hydrophobin encoding genes in the ecologically important fungi, P. gigantea and H. irregulare were investigated alongside the evolutionary forces driving hydrophobin gene evolution. The results show that different growth media had a huge influence on the outcome of the interaction between P. gigantea and H. annosum s.s. and the ability to degrade the different structural components of wood could partly explain the higher competitive advantage over H. annosum s.s. The antagonistic ability of P. gigantea also correlated positively with the transcript levels of hydrophobin 1 and 2 encoding genes (Pgh1 and Pgh2) but there was no relationship between the antagonistic ability and expression of either Pgh1 or Pgh2. Furthermore, the regulatory patterns of Pgh1 and Pgh2 suggest a role in the formation of aerial hyphea during the growth and development of P. gigantea. Hydrophobin encoding genes are redundant in both fungal species studied in the thesis research . However, in H. annosum s.s., each gene coding for hydrophobins seemed to be regulated by different environmental factors. Hydrophobin encoding genes were found to have witnessed a considerable expansion in both P. gigantea and H. annosum s.l. as well as in other basidiomycetes while a massive contraction of the hydrophobin encoding genes has occurred in the ascomycetes. Evidence of positive selection was also observed in P. gigantea and H. annosum s.l. hydrophobins and the hydrophobins from other fungal species. Finally, to understand the effect of secondary metabolites produced by P. gigantea on the gene expression profile of H. annosum s.s., transcriptomics analysis was carried out using microarray expression method. Also macroarray analysis was used to compare the transcriptomics profile of different strains of P. gigantea (competitively effective wild type, less competitively effective wild type and their progeny) against the commercial isolate, Rotstop F® when cultivated on artificial growth medium. The metabolites from P. gigantea effectively repressed some genes involved in diverse metabolic pathways in H. annosum s.s. Aditionally, several genes were found to be uniquely expressed in the progeny strain of P. gigantea, an indication that breeding could serve as an alternative for improving the P. gigantea isolates for a better biocontrol application. Generally, the results reported in this study have highlighted some of the physiological, biochemical and molecular mechanisms governing the biological control of the conifer pathogen, H. annosum s.l., by P. gigantea.Ei saatavill

Faecal characteristics and production of dairy cows in early lactation

Mgbeahurike . A. C. Faecal characteristics and production of dairy cows in early lactation. Master of Science Thesis. No. 62 ISSN 1403-2201 There is limited information available on the effects of different forages on faecal characteristics in early-lactation dairy cows. In addition, information regarding relationships among dietary variables, production and faecal characteristics is limited. The aim of the present investigation includes 1) determination of the effects of diets, when differing in forage source, on faecal particle size distribution, consistency, pH and nutrient content as well as on production and body condition scores of early-lactation dairy cows; 2) investigation of potential correlations among dietary, production and faecal variables; and 3) understanding the principle involved in the use of image analysis to characterize particles from faeces of early lactating dairy cows. Twenty-six herds in Sweden comprising dairy cows (37.4±17.9 days in milk) of Swedish Red and Swedish Holstein breeds were used in the study. Nine herds used grassclover silage (GCS) as the sole forage, eight herds used GCS combined with whole crop cereal silage (WCCS), four herds used GCS combined with whole-crop maize silage (MS) and five herds used GCS combined with ensiled pressed beet pulp (EPBP). The chemical composition of forages, nutrient contents of the diets and the formulated intakes were registered. Data were analysed by ANOVA using the GLM procedure of SAS (2001) with herd as the experimental unit. Visit was treated as repeated measure on a herd level. Because no significant treatment by visit interactions were found, data were analyzed as a mean over the two visits. Pearson partial correlation coefficients, with consideration to treatment effects, were estimated among dietary, production and faecal variables on a herd level basis (n=26) using the CORR procedure of SAS. Cows fed GCS alone or GCS combined with EPBP tended to have a higher yield of energy-corrected milk (ECM) than cows fed WCCS (P<0.059). For cows fed EPBP in addition to GCS, the higher ECM yield can be related to their higher formulated DM intake (P<0.033) and their improved feed conversion (P<0.040. Cows fed GCS combined with MS or WCCS had a higher faecal NDF concentration in dry matter than cows fed GCS alone or in combination with EPBP (P<0.034). The higher faecal NDF concentration in cows fed GCS and MS was associated with the improved faecal consistency score value by cows fed this diet (P<0.016). Increased formulated starch intake from concentrate and 10 diet increased the number of long faecal particles (r=0.439* and r=0.390†, respectively). Increased formulated DM intake of forage was associated with decreased faecal DM concentration (r=-0.541**) whereas increasing formulated DM intake of concentrate increased faecal DM concentration (r=0.630**). The strong positive linear correlation between number of grain kernels in the faeces and faecal starch concentration could be used as a tool to predict faecal starch content (r=0.827***). For the particle size determination by image analysis, gamma distribution curves(density plots) and accumulated distribution curves described and characterized the different particle dimensions in faeces into arithmetic mean particle length, arithmetic mean particle width, mode particle length, mode particle width, median particle length, median particle width, geometric mean particle length, geometric mean particle width and a 95-percentile fraction. The median length and width values, as well as the 95% length and width values were estimated using cumulative distribution function CDF in SAS. The 95-percentile fraction shows the maximum length or width for 95% of the faecal particles and describes the capacity of rumen to retain large particles. Image analysis can be used for measuring the characteristics, the length and width dimension values, of washed faecal particles

Evaluation of the Medicinal Uses and Antimicrobial Activity of Piper guineense Schumach &Thonn

Piper guineense is a medicinal plant that has wide application in African traditional medicine where it is often used in the treatment of bacterial and fungal infections. It is an economic plant with numerous health benefits which is also consumed regularly as a functional food. The fruits, leaves and seeds are used as spices and flavouring agents in commercial food preparations in West Africa. The extracts are also used for the treatment of various diseases ranging from diarrhea, intestinal diseases, rheumatoid arthritis, bronchitis, cough, stomach ache, asthma to febrile convulsions, fever and mental disorders. There is also recent interest on the biological and pharmacological properties of its bioactive compounds such as piperine, the main alkaloid constituents of P. guineense which is responsible for its pungent aroma. Based on these numerous ethnobotanical, traditional and economic uses of this plant, it became interesting to evaluate the bioactive compounds present in the extracts and to further screen the extracts against some selected human pathogenic bacterial and fungal strains so as to ascertain the efficacy of the extracts and its compounds as potent antibacterial and antifungal lead compounds. Microbial resistance to the currently available antibiotics is a global problem that has resulted to a constant search for a new antimicrobial drug with strong efficacy and low cost. There is need to screen the extracts and bioactive compounds from P. guineense for possible lead compounds for antibacterial and antifungal drug discovery. In this study, first, a method was developed for the chemical profiling, qualitative and quantitative analysis of P. guineense extracts and a good mobile phase composition was developed for the high performance liquid chromatography (HPLC) and thin layer chromatographic (TLC) analysis of the extracts. The effect of the chamber type on the separation was also evaluated using unsaturated horizontal chamber in sandwich configuration, horizontal chamber in non-sandwich configuration and twin-trough vertical chamber. Furthermore, the in vitro antibacterial activity of the extracts were evaluated using 8 pathogenic Gram-positive and Gram-negative bacterial strains. An ethnobotanical survey was also conducted on the use of P. guineense extracts in the treatment of fungal infections in West African traditional medicine. The study area was chosen to be Imo state, South Eastern Nigeria were P. guineense is mostly used by traditional healers for the treatment of fungal infections which is often common among those suffering from HIV and AIDS. The aim of the survey was to document the various methods of preparations and administrations of these extracts for the treatment of fungal diseases. From this ethnobotanical approach, the leaves and fruits extracts of the plant was further tested against 5 fungal strains including Cryptococcus neoformans which causes meningitis in immunocompromised individuals. HPLC and TLC methods were developed for the analysis of P. guineense extracts with emphasis on the shortest analysis time and minimal solvent consumption, and the best mobile phase giving favourable resolution of bands was found to be toluene: ethyl acetate (PS 6-4 corresponding to 60:40 % v/v). The result of the TLC analysis showed that the developing chamber conditions does not affect the TLC separation efficacy in the analysis of P. guineense extracts. The extracts were active against the tested bacterial and fungal strains with minimum inhibitory concentration (MIC) values ranging from 19 to 2500 µg/mL

An ethnobotanical survey and antifungal activity of Piper guineense used for the treatment of fungal infections in West-African traditional medicine

Ethnobotanical relevance: Piper guineense occurs commonly in West Africa where it is used for fungal infections instead of the costly and not always accessible conventional antifungals. Fungal, yeast-based diseases are common in West-Africa especially among those living with HIV/AIDS, and thus this study was performed in Imo state, South-Eastern Nigeria, where P. guineense is predominantly used for the treatment of fungal diseases, such as skin rashes, oral thrush and vaginosis. Aim of study: The scarce number of previous studies on the documentation of the traditional uses of P. guineense extracts for the treatment of fungal infections in Nigeria prompted this survey. The investigation focused on how traditional healers recognize and diagnose fungal infections, how P. guineense is collected, on the various parts used for the treatments, methods of preparations, administrations and treatments. In addition, an in vitro anti fungal screening of P. guineense fruit and leaf extracts of various polarities, and piperine and piperlongumine, representing the main constituents in these extracts, were performed. Methods: A house to house ethnobotanical survey was conducted using questionnaires. Twenty traditional medical practitioners (TMP) and herb sellers from ten villages were interviewed. Four human pathogenic strains of yeast and Cryptococcus neoformans, a yeast-like basidiomycete causing meningitis in immunocompromised individuals, were used for the antifungal screening. Results: The traditional medical practitioners (TMP) and herb sellers explained that the leaves and fruits are the most commonly used plant parts for the treatments. The oral intake of the extracts in locally produced bamboo alcohol (Kai-kai) is the most common method of administration. In accordance with these recorded traditional uses, we found that extracts of P. guineense were growth inhibitory against the fungal strains with MIC values ranging from 39 to 2500 g/mL. The lowest MIC value of 39 g/mL was recorded for a methanol fruit extract against Candida albicans, C. glabrata and C. tropicalis. In addition, ethanol and hexane fruit extracts were effective against the growth of C. albicans and C. glabrata, respectively, with a MIC of 78 g/mL. Piperlongumine and piperine were active against C. albicans with MIC values of 39 and 78 g/mL respectively. Conclusion: P. guineense fruit and leaf extracts, as well as their piperamide alkaloid constituents piperine and piperlongumine, have interesting antifungal properties and could have potential as new antifungal scaffolds. Our results warrant further in-depth investigations to isolate and characterize piperamide alkaloids and other compounds responsible for the antifungal activity in the extracts.Peer reviewe

An investigation into soil pollution and remediation of selected polluted sites around the globe

In this study, simulated soil (SS), as well as its constituents (quartz sand [QS], bentonite clay [BC] and peat moss [PM]) were used as a model to study the leaching behaviour of potential toxic elements (PTEs) in a batch process using EDTA as an extractant and comparing it with the extraction capacities of the other extractants EDDS, 2, 4-pentanedione [Hacac], citric acid [CA] and tartaric acid [TA]). Prior to leaching, SS and its constituents QS, BC, and PM were contaminated with single metals (SM) and multi-metals (MM) at different concentrations. EDTA was the most efficient and TA the least efficient extractant. Hacac showed preferential recovery for Cu and Ni. Metal recovery by EDDS in SS was significantly diminished compared to EDTA. Once the leaching behaviour in the batch process had been established, other process configurations (column and heap) were investigated. The effect of three different leaching processes and 4 different leaching agents on the extraction of five metals showed that total metal extraction of up to 50% efficiency could be achieved. EDTA proved to be the best overall extractant when used in a batch leaching process. Different metals showed preferential recoveries with specific processes and extractants. The results obtained from the multi-metal leaching profile of the SS was used to predict the leaching capacities of the three environmental soils sourced from Nigeria (Nig-S) and Romania (Ro-SB and Ro-PH), using column and heap leaching processes. The behaviour of SS compared in practice with the leaching behaviour exhibited by the Ro-SB. The leaching profiles of the base metals from the different soils (both natural and simulated) was studied by using first-order, power function and Elovich equations. The Elovich equation provided the best fit of the experimental data. The rate of metal release was higher for the SS and Ro-SB soils than the Nig-S and Ro-PH soils. Subsequently, the data were analysed to determine the B/C Ratio, NPV, and payback period of the study. The analysis gave a B/C Ratio of 0.2, negative NPV of £ 1797.70 and £ 351.77 (for 50% and 90% recovery of EDTA-Heap process respectively) and payback period of 4 years. These indicators are suggestively below the decision criteria. Thus, the process is not economically viable. However, a sensitivity analysis performed based on 25% and 50% changes in the prices of the various metals and extractants shows that when 90% of the extractants could be recycled, a significant change in the process economics could be achieved. Overall the results suggest that the design of a contaminant-specific leaching process performed in a sequential manner could practically leach all the metals, and offer a potential separation of the metals from the leach liqour

Antimicrobial and Synergistic Effects of Commercial Piperine and Piperlongumine in Combination with Conventional Antimicrobials

Microbial resistance to currently available antibiotics is a public health problem in the fight against infectious diseases. Most antibiotics are characterized by numerous side effects that may be harmful to normal body cells. To improve the efficacy of these antibiotics and to find an alternative way to minimize the adverse effects associated with most conventional antibiotics, piperine and piperlongumine were screened in combination with conventional rifampicin, tetracycline, and itraconazole to evaluate their synergistic, additive, or antagonistic interactions against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. The fractional inhibitory concentration index was used to estimate the synergistic effects of various combination ratios of the piperamides and antibiotics against the bacterial and fungal strains. Both piperine and piperlongumine showed synergistic effects against S. aureus when combined at various ratios with rifampicin. Synergistic interaction was also observed with piperine in combination with tetracycline against S. aureus, while antagonistic interaction was recorded for piperlongumine and tetracycline against S. aureus. All the piperamide/antibacterial combinations tested against P. aeruginosa showed antagonistic effects, with the exception of piperine and rifampicin, which recorded synergistic interaction at a ratio of 9:1 rifampicin/piperine. No synergistic interaction was observed when the commercial compounds were combined with itraconazole and tested against C. albicans. The results showed that piperine and piperlongumine are capable of improving the effectiveness of rifampicin and tetracycline. Dosage combinations of these bioactive compounds with the antibiotics used may be a better option for the treatment of bacterial infections that aims to minimize the adverse effects associated with the use of these conventional antibacterial drugs

Ethnopharmacology, Antimicrobial Potency, and Phytochemistry of African Combretum and Pteleopsis Species (Combretaceae): A Review

Bacterial and fungal resistance to antibiotics is of growing global concern. Plants such as the African Combretum and Pteleopsis species, which are used in traditional medicine for the treatment of infections, could be good sources for antimicrobial extracts, drug scaffolds, and/or antibiotic adjuvants. In African countries, plant species are often used in combinations as traditional remedies. It is suggested that the plant species enhance the effects of each other in these combination treatments. Thus, the multi-species-containing herbal medications could have a good antimicrobial potency. In addition, plant extracts and compounds are known to potentiate the effects of antibiotics. The objective of this review is to compile the information on the botany, ethnopharmacology, ethnobotany, and appearance in herbal markets of African species of the genera Combretum and Pteleopsis. With this ethnobotanical information as a background, this review summarizes the information on the phytochemistry and antimicrobial potency of the extracts and their active compounds, as well as their combination effects with conventional antibiotics. The databases used for the literature search were Scopus, Elsevier, EBSCOhost, PubMed, Google Scholar, and SciFinder. In summary, a number of Combretum and Pteleopsis species were reported to display significant in vitro antibacterial and antifungal efficacy. Tannins, terpenes, flavonoids, stilbenes, and alkaloids—some of them with good antimicrobial potential—are known from species of the genera Combretum and Pteleopsis. Among the most potent antimicrobial compounds are arjunglucoside I (MIC 1.9 µg/mL) and imberbic acid (MIC 1.56 µg/mL), found in both genera and in some Combretum species, respectively. The in vitro antimicrobial properties of the extracts and compounds of many Combretum and Pteleopsis species support their traditional medicinal uses

Alkaloid-Rich Crude Extracts, Fractions and Piperamide Alkaloids of Piper guineense Possess Promising Antibacterial Effects

Piper guineense is a food and medicinal plant commonly used to treat infectious diseases in West-African traditional medicine. In a bid to identify new antibacterial compounds due to bacterial resistance to antibiotics, twelve extracts of P. guineense fruits and leaves, obtained by sequential extraction, as well as the piperine and piperlongumine commercial compounds were evaluated for antibacterial activity against human pathogenic bacteria. HPLC-DAD and UHPLC/Q-TOF MS analysis were conducted to characterize and identify the compounds present in the extracts with promising antibacterial activity. The extracts, with the exception of the hot water decoctions and macerations, contained piperamide alkaloids as their main constituents. Piperine, dihydropiperine, piperylin, dihydropiperylin or piperlonguminine, dihydropiperlonguminine, wisanine, dihydrowisanine and derivatives of piperine and piperidine were identified in a hexane extract of the leaf. In addition, some new piperamide alkaloids were identified, such as a piperine and a piperidine alkaloid derivative and two unknown piperamide alkaloids. To the best of our knowledge, there are no piperamides reported in the literature with similar UV absorption maxima and masses. A piperamide alkaloid-rich hexane leaf extract recorded the lowest MIC of 19 mu g/mL against Sarcina sp. and gave promising growth inhibitory effects against S. aureus and E. aerogenes as well, inhibiting the growth of both bacteria with a MIC of 78 mu g/mL. Moreover, this is the first report of the antibacterial activity of P. guineense extracts against Sarcina sp. and E. aerogenes. Marked growth inhibition was also obtained for chloroform extracts of the leaves and fruits against P. aeruginosa with a MIC value of 78 mu g/mL. Piperine and piperlongumine were active against E. aerogenes, S. aureus, E. coli, S. enterica, P. mirabilis and B. cereus with MIC values ranging from 39-1250 mu g/mL. Notably, the water extracts, which were almost devoid of piperamide alkaloids, were not active against the bacterial strains. Our results demonstrate that P. guineense contains antibacterial alkaloids that could be relevant for the discovery of new natural antibiotics.Peer reviewe