Antimicrobial Properties of Copper in Gram-Negative and Gram-Positive Bacteria

For centuries humans have used the antimicrobial properties of copper to their advantage. Yet, after all these years the underlying mechanisms of copper mediated cell death in various microbes remain unclear. We had explored the hypothesis that copper mediated increased levels of lipid peroxidation in the membrane fatty acids is responsible for increased killing in Escherichia coli. In this study we show that in both gram positive (Staphylococcus aureus) and gram negative (Pseudomonas aeruginosa) bacteria there is a strong correlation between copper mediated cell death and increased levels of lipid peroxidation. Interestingly, the non-spore forming gram positive bacteria as well as gram negative bacteria show similar patterns of cell death, increased levels of lipid peroxidation, as well as genomic DNA degradation, however there is some difference in loss in membrane integrity upon exposure to copper alloy surface.

Biodegradation of Malathion by Acinetobacter baumannii Strain AFA Isolated from Domestic Sewage in Egypt

Bacterial strains capable of degradation of malathion from the domestic sewage were isolated by an enrichment culture technique. Three bacterial strains were screened and identified as Acinetobacter baumannii (AFA), Pseudomonas aeruginosa (PS1), and Pseudomonas mendocina (PS2) based on morphological, biochemical identification and 16S rRNA sequence analysis. Acinetobacter baumannii AFA was the most efficient malathion degrading bacterium, so used for further biodegradation study. AFA was able to grow in mineral salt medium (MSM) supplemented with malathion (100 mg/l) as a sole carbon source, and within 14 days, 84% of the initial dose was degraded by the isolate measured by high performance liquid chromatography. Strain AFA could also degrade other organophosphorus compounds including diazinon, chlorpyrifos and fenitrothion. The effect of different culture conditions on the degradation of malathion like inoculum density, other carbon or nitrogen sources, temperature and shaking were examined. Degradation of malathion and bacterial cell growth were accelerated when culture media were supplemented with yeast extract, glucose and citrate. The optimum conditions for malathion degradation by strain AFA were; an inoculum density of 1.5x 10^12CFU/ml at 30°C with shaking. A specific polymerase chain reaction primers were designed manually using multiple sequence alignment of the corresponding carboxylesterase enzymes of Acinetobacter species. Sequencing result of amplified PCR product and phylogenetic analysis showed low degree of homology with the other carboxylesterase enzymes of Acinetobacter strains, so we suggested that this enzyme is a novel esterase enzyme. Isolated bacterial strains may have potential role for use in bioremediation of malathion contaminated.

Antimicrobial Potentials of Flavonoids Isolated from Tagetes erecta

In this study, we are interested in a species of the family of Asteraceae (Tagetes erecta). This family is considered as a source of antimicrobial extracts with strong capacity. The extraction of the flavonoids is carried out by the method of liquid/liquid with the use of successive solvents. Afterwards, we evaluated the biological activity of the flavonoids on five pathogenic bacterial stocks such as Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus and two stocks of yeasts to knowing Candida albicans) and Saccharomyces cerevisiae, by employing the method of the aromatogramme starting from a solid disc. The result of the antimicrobial activity shows an action and a variable degree of sensitivity according to bacterial stocks tested. It will be noted that the flavonoids have an inhibiting effect on E. coli, B. subtilis, K. pneumoniae and S. aureus. But a resistance with respect to the extract by P. aeruginosa, C. albicans and S. cerevisiae is to be mentioned.

Antibacterial and Antifungal Activity of Essential Oil of Eucalyptus camendulensis on a Few Bacteria and Fungi

Red River Gum (Eucalyptus camaldulensis) is a tree of the genus Eucalyptus widely distributed in Algeria and in the world. The value of its aromatic secondary metabolites offers new perspectives in the pharmaceutical industry. This strategy can contribute to the sustainable development of our country. Preliminary tests performed on the essential oil of Eucalyptus camendulensis showed that this oil has antibacterial activity vis-à-vis the bacterial strains (Enterococcus feacalis, Enterobacter cloaceai, Proteus microsilis, Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa) and antifungic (Fusarium sporotrichioide and Fusarium graminearum). The culture medium used was nutrient broth Muller Hinton. The interaction between the bacteria and the essential oil is expressed by a zone of inhibition with diameters of MIC indirectly expression of. And we used the PDA medium to determine the fungal activity. The extraction of the aromatic fraction (essentially oilhydrolat) of the fresh aerian part of the Eucalyptus camendulensis was performed by hydrodistillation. The average essential oil yield is 0.99%. The antimicrobial and fungal study of the essential oil and hydrosol showed a high inhibitory effect on the growth of pathogens.

The Co-application of Plant Growth Promoting Rhizobacteria and Inoculation with Rhizobium Bacteria on Grain Yield and Its Components of Mungbean (Vigna radiate L.) in Ilam Province, Iran

In order to investigate the effect of Plant Growth Promoting Rhizobacteria (PGPR) and rhizobium bacteria on grain yield and some agronomic traits of mungbean (Vigna radiate L.), an experiment was carried out based on randomized complete block design with three replications in Malekshahi, Ilam province, Iran during 2012-2013 cropping season. Experimental treatments consisted of control treatment, inoculation with rhizobium bacteria, rhizobium bacteria and Azotobacter, rhizobium bacteria and Azospirillum, rhizobium bacteria and Pseudomonas, rhizobium bacteria, Azotobacter and Azospirillum, rhizobium bacteria, Azotobacter and Pseudomonas, rhizobium bacteria, Azospirillum and Pseudomonas and rhizobium bacteria, Azotobacter, Azospirillum and Pseudomonas. The results showed that the effect of PGPR and rhizobium bacteria were significant affect on grain and its components in mungbean plant. Grain yield significantly increased by PGPR and rhizobium bacteria, so that the maximum grain yield was obtained from rhizobium bacteria + Azospirillum + Pseudomonas with the amount of 2287 kg.ha-1 as compared to control treatment. Excessive application of chemical fertilizers causes environmental and economic problems. That is, the overfertilization of P and N leads to pollution due to soil erosion and runoff water, so the use of PGPR and rhizobium bacteria can be justified due to reduce input costs, increase in grain yield and environmental friendly.

Bacteria Flora in the Gut and Respiratory Organs of Clarias gariepinus in Fresh and Brackish Water Habitats of Ondo State, South/West Nigeria

Bacteria flora of Clarias gariepinus collected from two natural habitats namely Owena River (freshwater) and Igbokoda lagoon (brackish water) were examined using standard microbiological procedures. Thirteen bacterial species were identified. The result indicated that from the identified bacteria isolated, Vibrio sp, Proteus sp. Shigella sp. and E. coli were present in both habitats (fresh and brackish waters). Others were habitat-selective such as Salmonella sp., Pseudomonas sp, Enterococcus sp, Staphylococcus sp. that were found only in freshwater habitat. While Branhamella sp, Streptococcus sp. and Micrococcus sp. were found in brackish water habitat. Bacteria load from Owena river (freshwater) was found to be the highest load recorded at 6.21 x 104cfu. T-test analysis also revealed that there was a marked significant difference between bacterial load in guts of sampled Clarias from fresh water and brackish water habitats.

In vitro Biological Activity of Some Synthesized Monoazo Heterocycles Based On Thiophene and Thiazolyl-Thiophene Analogues

Potential synthesis of a series of 3-amino-4-arylazothiophene derivatives from reaction of 2-cyano-2-phenylthiocarbamoyl acetamide and the appropriate α-halogenated reagents, followed by coupling with different aryl diazonium salts (Japp-Klingemann reaction), and another series of 5-arylazo-thiazol-2-ylcarbamoyl-thiophene derivatives from base-catalyzed intramolecular condensation of 5-arylazo-2-(N-chloroacetyl)amino-thiazole with selected b-keto compounds (Thorpe-Ziegler reaction) was performed. The biological activity of the two series was studied in vitro. Their versatility for pharmaceutical purposes was reported, where they displayed remarkable activities against selected pathogenic microorganisms; Bacillus subtilis, Staphylococcus aureus (Gram positive bacteria), Escherichia coli, Pseudomonas aeruginosa (Gram negative bacteria), and Aspergillus flavus, Candida albicans (fungi) with various degrees related to their chemical structures.

Plants and Microorganisms for Phytoremediation of Soils Polluted with Organochlorine Pesticides

The goal of presented work is the development phytoremediation method targeted to cleaning environment polluted with organochlorine pesticides, based on joint application of plants and microorganisms. For this aim the selection of plants and microorganisms with corresponding capabilities towards three organochlorine pesticides (Lindane, DDT and PCP) has been carried out. The tolerance of plants to tested pesticides and induction degree of plant detoxification enzymes by these compounds have been used as main criteria for estimating the applicability of plants in proposed technology. Obtained results show that alfalfa, maize and soybean among tested six plant species have highest tolerance to pesticides. As a result of screening, more than 30 strains from genera Pseudomonas have been selected. As a result of GC analysis of incubation area, 11 active cultures for investigated pesticides are carefully chosen.

The Antibacterial Efficacy of Gold Nanoparticles Derived from Gomphrena celosioides and Prunus amygdalus (Almond) Leaves on Selected Bacterial Pathogens

Gold nanoparticles (AuNPs) have gained increasing interest in recent times. This is greatly due to their special features, which include unusual optical and electronic properties, high stability and biological compatibility, controllable morphology and size dispersion, and easy surface functionalization. In typical synthesis, AuNPs were produced by reduction of gold salt AuCl4 in an appropriate solvent. A stabilizing agent was added to prevent the particles from aggregating. The antibacterial activity of different sizes of gold nanoparticles was investigated against Staphylococcus aureus, Salmonella typhi and Pseudomonas pneumonia using the disk diffusion method in a Müeller–Hinton Agar. The Au-NPs were effective against all bacteria tested. That the Au-NPs were successfully synthesized in suspension and were used to study the antibacterial activity of the two medicinal plants against some bacterial pathogens suggests that Au-NPs can be employed as an effective bacteria inhibitor and may be an effective tool in medical field. The study clearly showed that the Au-NPs exhibiting inhibition towards the tested pathogenic bacteria in vitro could have the same effects in vivo and thus may be useful in the medical field if well researched into.

Exploratory Tests of Crude Bacteriocins from Autochthonous Lactic Acid Bacteria against Food-Borne Pathogens and Spoilage Bacteria

The aim of the present work was to test in vitro inhibition of food pathogens and spoilage bacteria by crude bacteriocins from autochthonous lactic acid bacteria. Thirty autochthonous lactic acid bacteria isolated previously, belonging to the genera: Lactobacillus, Carnobacterium, Lactococcus, Vagococcus, Streptococcus, and Pediococcus, have been screened by an agar spot test and a well diffusion assay against Gram-positive and Gram-negative harmful bacteria: Bacillus cereus, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 8739, Salmonella typhimurium ATCC 14028, Staphylococcus aureus ATCC 6538, and Pseudomonas aeruginosa under conditions means to reduce lactic acid and hydrogen peroxide effect to select bacteria with high bacteriocinogenic potential. Furthermore, crude bacteriocins semiquantification and heat sensitivity to different temperatures (80, 95, 110°C, and 121°C) were performed. Another exploratory test concerning the response of St. aureus ATCC 6538 to the presence of crude bacteriocins was realized. It has been observed by the agar spot test that fifteen candidates were active toward Gram-positive targets strains. The secondary screening demonstrated an antagonistic activity oriented only against St. aureus ATCC 6538, leading to the selection of five isolates: Lm14, Lm21, Lm23, Lm24, and Lm25 with a larger inhibition zone compared to the others. The ANOVA statistical analysis reveals a small variation of repeatability: Lm21: 0.56%, Lm23: 0%, Lm25: 1.67%, Lm14: 1.88%, Lm24: 2.14%. Conversely, slight variation was reported in terms of inhibition diameters: 9.58± 0.40, 9.83± 0.46 and 10.16± 0.24 8.5 ± 0.40 10 mm for, Lm21, Lm23, Lm25, Lm14and Lm24, indicating that the observed potential showed a heterogeneous distribution (BMS = 0.383, WMS = 0.117). The repeatability coefficient calculated displayed 7.35%. As for the bacteriocins semiquantification, the five samples exhibited production amounts about 4.16 for Lm21, Lm23, Lm25 and 2.08 AU/ml for Lm14, Lm24. Concerning the sensitivity the crude bacteriocins were fully insensitive to heat inactivation, until 121°C, they preserved the same inhibition diameter. As to, kinetic of growth , the µmax showed reductions in pathogens load for Lm21, Lm23, Lm25, Lm14, Lm24 of about 42.92%, 84.12%, 88.55%, 54.95%, 29.97% in the second trails. Inversely, this pathogen growth after five hours displayed differences of 79.45%, 12.64%, 11.82%, 87.88%, 85.66% in the second trails, compared to the control. This study showed potential inhibition to the growth of this food pathogen, suggesting the possibility to improve the hygienic food quality.

Antimicrobial Effect of Essential Oil of Plant Schinus molle on Some Bacteria Pathogens

Humans use plants for thousands of years to treat various ailments, in many developing countries; much of the population relies on traditional doctors and their collections of medicinal plants to cure them. Essential oils have many therapeutic properties. In herbal medicine, they are used for their antiseptic properties against infectious diseases of fungal origin, against dermatophytes, those of bacterial origin. The aim of our study is to determine the antimicrobial effect of essential oils of the plant Schinus molle on some pathogenic bacteria. It is a medicinal plant used in traditional therapy. Essential oils have many therapeutic properties. In herbal medicine, they are used for their antiseptic properties against infectious diseases of fungal origin, against dermatophytes, those of bacterial origin. The test adopted, is based on the diffusion method on solid medium (Antibiogram), this method allows to determine the susceptibility or resistance of an organism according to the sample studied. Our study reveals that the essential oil of the plant Schinus molle has a different effect on the resistance of germs: for Pseudomonas aeruginosa strain is a moderately sensitive with an inhibition zone of 10mm, further Enterobacter, Escherichia coli and Proteus are strains that represent a high sensitivity, a zone of inhibition equal to 14.66 mm.

Bio-Surfactant Production and Its Application in Microbial EOR

There are various sources of energies available worldwide and among them, crude oil plays a vital role. Oil recovery is achieved using conventional primary and secondary recovery methods. In-order to recover the remaining residual oil, technologies like Enhanced Oil Recovery (EOR) are utilized which is also known as tertiary recovery. Among EOR, Microbial enhanced oil recovery (MEOR) is a technique which enables the improvement of oil recovery by injection of bio-surfactant produced by microorganisms. Bio-surfactant can retrieve unrecoverable oil from the cap rock which is held by high capillary force. Bio-surfactant is a surface active agent which can reduce the interfacial tension and reduce viscosity of oil and thereby oil can be recovered to the surface as the mobility of the oil is increased. Research in this area has shown promising results besides the method is echo-friendly and cost effective compared with other EOR techniques. In our research, on laboratory scale we produced bio-surfactant using the strain Pseudomonas putida (MTCC 2467) and injected into designed simple sand packed column which resembles actual petroleum reservoir. The experiment was conducted in order to determine the efficiency of produced bio-surfactant in oil recovery. The column was made of plastic material with 10 cm in length. The diameter was 2.5 cm. The column was packed with fine sand material. Sand was saturated with brine initially followed by oil saturation. Water flooding followed by bio-surfactant injection was done to determine the amount of oil recovered. Further, the injection of bio-surfactant volume was varied and checked how effectively oil recovery can be achieved. A comparative study was also done by injecting Triton X 100 which is one of the chemical surfactant. Since, bio-surfactant reduced surface and interfacial tension oil can be easily recovered from the porous sand packed column.

Study the Efficacies of Green Manure Application as Chickpea Pre Plant

In order to Study the efficacy application of green manure as chickpea pre plant, field experiments were carried out in 2007 and 2008 growing seasons. In this research the effects of different strategies for soil fertilization were investigated on grain yield and yield component, minerals, organic compounds and cooking time of chickpea. Experimental units were arranged in splitsplit plots based on randomized complete blocks with three replications. Main plots consisted of (G1): establishing a mixed vegetation of Vicia panunica and Hordeum vulgare and (G2): control, as green manure levels. Also, five strategies for obtaining the base fertilizer requirement including (N1): 20 t.ha-1 farmyard manure; (N2): 10 t.ha-1 compost; (N3): 75 kg.ha-1 triple super phosphate; (N4): 10 t.ha-1 farmyard manure + 5 t.ha-1 compost and (N5): 10 t.ha-1 farmyard manure + 5 t.ha-1 compost + 50 kg.ha-1 triple super phosphate were considered in sub plots. Furthermoree four levels of biofertilizers consisted of (B1): Bacillus lentus + Pseudomonas putida; (B2): Trichoderma harzianum; (B3): Bacillus lentus + Pseudomonas putida + Trichoderma harzianum; and (B4): control (without biofertilizers) were arranged in sub-sub plots. Results showed that integrating biofertilizers (B3) and green manure (G1) produced the highest grain yield. The highest amounts of yield were obtained in G1×N5 interaction. Comparison of all 2-way and 3-way interactions showed that G1N5B3 was determined as the superior treatment. Significant increasing of N, P2O5, K2O, Fe and Mg content in leaves and grains emphasized on superiority of mentioned treatment because each one of these nutrients has an approved role in chlorophyll synthesis and photosynthesis abilities of the crops. The combined application of compost, farmyard manure and chemical phosphorus (N5) in addition to having the highest yield, had the best grain quality due to high protein, starch and total sugar contents, low crude fiber and reduced cooking time.

Promotion of Growth and Modulation of As- Induced Stress Ethylene in Maize by As- Tolerant ACC Deaminase Producing Bacteria

One of the major pollutants in the environment is arsenic (As). Due to the toxic effects of As to all organisms, its remediation is necessary. Conventional technologies used in the remediation of As contaminated soils are expensive and may even compromise the structure of the soil. An attractive alternative is phytoremediation, which is the use of plants which can take up the contaminant in their tissues. Plant growth promoting bacteria (PGPB) has been known to enhance growth of plants through several mechanisms such as phytohormone production, phosphate solubilization, siderophore production and 1-aminocyclopropane-1- carboxylate (ACC) deaminase production, which is an essential trait that aids plants especially under stress conditions such as As stress. Twenty one bacteria were isolated from As-contaminated soils in the vicinity of the Janghang Smelter in Chungnam Province, South Korea. These exhibited high tolerance to either arsenite (As III) or arsenate (As V) or both. Most of these isolates possess several plant growth promoting traits which can be potentially exploited to increase phytoremediation efficiency. Among the identified isolates is Pseudomonas sp. JS1215, which produces ACC deaminase, indole acetic acid (IAA), and siderophore. It also has the ability to solubilize phosphate. Inoculation of JS1215 significantly enhanced root and shoot length and biomass accumulation of maize under normal conditions. In the presence of As, particularly in lower As level, inoculation of JS1215 slightly increased root length and biomass. Ethylene increased with increasing As concentration, but was reduced by JS1215 inoculation. JS1215 can be a potential bioinoculant for increasing phytoremediation efficiency.

Study the Biological Activities of Tribulus Terrestris Extracts

In this study the extracts of the Iraqi herb Tribulus terrestris (Al-Hassage or Al-Kutub) was done by using of polar and non polar solvents, then the biological activity of these extractants was studied in three fields, First, the antibacterial activity (in vitro) on gram positive bacteria (Staphylococcus aureus), and gram negative bacteria (E. coli, Proteus vulgaris, Pseudomonas aerugiuosa, and Klebsiella), all extracts showed considerable activity against all bacteria. Second, the effect of extracts on free serum testosterone level in male mice (in vivo), the alcoholic, and acetonitrilic extracts showed significant (P < 0.05) increase in free serum testosterone level, and we found that the extracts contained compounds with less genotoxic effects in mice germ cells. 3rd, was to study the effect of methanolic extract of T. terrestris in diabetes management.

Preparation and Antibacterial Properties of Ag+-Exchanged Tobermorite-Chitosan Films

Silver-exchanged zeolites and clays are used in polymer composites to confer broad-spectrum antimicrobial properties on a range of functional materials. Tobermorite is a layer lattice mineral whose potential as a carrier for Ag+ ions in antibacterial composites has not yet been investigated. Accordingly, in this study, synthetic tobermorite was ion-exchanged with 10 wt% silver ions and the resulting material was incorporated into a composite film with chitosan. Chitosan is a biocompatible, biodegradable derivative of chitin, a polysaccharide obtained from the shells of crustaceans. The solvent-cast Ag+-exchanged tobermorite-chitosan films were found to exhibit antimicrobial action against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.

The Effect of Plant Growth Promoting Rhizobacteria (PGPR) on Germination, Seedling Growth and Yield of Maize

The effect of plant growth-promoting rhizobacteria (PGPR) on seed germination, seedling growth and yield of field grown maize were evaluated in three experiments. In these experiments six bacterial strains include P.putida strain R-168, P.fluorescens strain R-93, P.fluorescens DSM 50090, P.putida DSM291, A.lipoferum DSM 1691, A.brasilense DSM 1690 were used. Results of first study showed seed Inoculation significantly enhanced seed germination and seedling vigour of maize. In second experiment, leaf and shoot dry weight and also leaf surface area significantly were increased by bacterial inoculation in both sterile and non-sterile soil. The results showed that inoculation with bacterial treatments had a more stimulating effect on growth and development of plants in nonsterile than sterile soil. In the third experiment, Inoculation of maize seeds with all bacterial strains significantly increased plant height, 100 seed weight, number of seed per ear and leaf area .The results also showed significant increase in ear and shoot dry weight of maize.

A New blaVIM Gene in a Pseudomonas putida Isolated from ENT Units in Sulaimani Hospitals

A total of twenty tensile biopsies were collected from children undergoing tonsillectomy from teaching hospital ENT department and Kurdistan private hospital in sulaimani city. All biopsies were homogenized and cultured; the obtained bacterial isolates were purified and identified by biochemical tests and VITEK 2 compact system. Among the twenty studied samples, only one Pseudomonas putida with probability of 99% was isolated. Antimicrobial susceptibility was carried out by disk diffusion method, Pseudomonas putida showed resistance to all antibiotics used except vancomycin. The isolate further subjected to PCR and DNA sequence analysis of blaVIM gene using different set of primers for different regions of VIM gene. The results were found to be PCR positive for the blaVIM gene. To determine the sequence of blaVIM gene, DNA sequencing performed. Sequence alignment of blaVIM gene with previously recorded blaVIM gene in NCBI- database showed that P. putida isolate have different blaVIM gene.

A Mathematical Modelling to Predict Rhamnolipid Production by Pseudomonas aeruginosa under Nitrogen Limiting Fed-Batch Fermentation

In this study, a mathematical model was proposed and the accuracy of this model was assessed to predict the growth of Pseudomonas aeruginosa and rhamnolipid production under nitrogen limiting (sodium nitrate) fed-batch fermentation. All of the parameters used in this model were achieved individually without using any data from the literature. The overall growth kinetic of the strain was evaluated using a dual-parallel substrate Monod equation which was described by several batch experimental data. Fed-batch data under different glycerol (as the sole carbon source, C/N=10) concentrations and feed flow rates were used to describe the proposed fed-batch model and other parameters. In order to verify the accuracy of the proposed model several verification experiments were performed in a vast range of initial glycerol concentrations. While the results showed an acceptable prediction for rhamnolipid production (less than 10% error), in case of biomass prediction the errors were less than 23%. It was also found that the rhamnolipid production by P. aeruginosa was more sensitive at low glycerol concentrations. Based on the findings of this work, it was concluded that the proposed model could effectively be employed for rhamnolipid production by this strain under fed-batch fermentation on up to 80 g l- 1 glycerol.

The Effect of Seed Inoculation (Pseudomonas putida+Bacillus lentus) and Different Levels of Fertilizers on Yield and Yield Components of Wheat (Triticum aestivum L.) Cultivars

In order to study of The Effect of seed inoculation with Pseudomonas putida+Bacillus lentus on yield and yield components of wheat (Triticum aestivum L.) cultivars, an experiment was carried out as factorial based on Randomized Complete Block Design (RCBD) in Agricultural Research Station of Shahrood University of Technology. Results showed that inoculation with Pseudomonas putida+Bacillus lentus promoted seed germination. Also, inoculation with Pseudomonas putida+Bacillus lentus significantly affected grain yield, Number of spikes per m2, Number of grain per spike and 1000-seed weight and There was not statistically significant difference between Chamran and Pishtaz cultivars . Finally, the dosages of chemical fertilizers currently applied in commercial wheat field in Iran (Shahrood region) could be reduced through proper combination of Pseudomonas putida+Bacillus lentus inoculation plus fertilization.