George G. Khachatourians

Production, formulation and application of the entomopathogenic fungus Beauveria bassiana for insect control: current status

This review summarizes the progress and achievements made in the last decade in mass production formulation and application technology of the entomopathogenic fungus Beauveria bassiana. Reports published on relevant research from Belgium, Canada, China, Cuba, Czechoslovakia (former), France, Germany, Great Britain, Philippines, Poland, Switzerland, USA and USSR (former) regarding this topic have been covered. Much of the non‐English language literature, particularly that from Eastern European and Chinese sources, has not been translated and is inaccessible to most English or other western language readers. We have done this translation and through this review provide technological details about mass production of B. bassiana in China. Various aspects of B. bassiana growth, substrate use, production of mycelia, conidiospore and blastospores, process technologies associated with separation, drying and milling, formulation, storage and ‘shelf‐life’, and field efficacy are reviewed. Data are presented on: a m...

Identification of Beauveria bassiana extracellular protease as a virulence factor in pathogenicity toward the migratory grasshopper, Melanoplus sanguinipes

Abstract Beauveria bassiana extracellular protease was shown to hydrolyze protein in cuticle of the migratory grasshopper, Melanoplus sanguinipes. Protease-proficient and protease-deficient strains of B. bassiana were assessed for production of extracellular protease in liquid culture and their virulence, as measured by LT50, toward M. sanguinipes. A uv irradiation-derived protease-deficient mutant strain produced less than 10% of protease when compared to the protease-proficient strains when grown in 1% (w/v) gelatin liquid medium or 1% (w/v) ground M. sanguinipes cuticle liquid medium. The protease-proficient strains showed the lowest LT50 (5.71–6.80 days) against the migratory grasshopper, while the protease-deficient strain had the highest LT50 (11.34 days). There was a negative relationship between protease production after 2 days of growth in liquid culture by the various strains and their associated LT50. We conclude that, in this study, protease production by the B. bassiana protease-deficient strain was low enough so that it extended the time course of pathogenesis toward M. sanguinipes.

Production and use of biological pest control agents

Abstract Biological pest control agents are gaining prominence for the control of insect pests in agriculture and forestry. The shift from chemical control has been due to environmental concerns and recent innovations in biotechnology. Production and use of biological insect control agents is the challenge of the future for pest management.

Development of the entomopathogenic fungus Beauveria bassiana in liquid cultures

Growth and development of B. bassiana was followed in four liquid media: peptone, peptone-glucose, glucose and glucose-peptone-yeast extract. Six developmental stages were defined: (I) the unswollen conidium, (II) the swollen conidium, (III) emergence of the germ tube, (IV) elongation of the germ tube and formation of the first septum, (V) polar and bipolar elongation (growth) of the resulting mycelium and initiation of a blastospore and, (VI) seccession of that blastospore. Conidia of B. bassiana produced germ tubes in all liquid media. Blastospores were produced in all liquid media except glucose. In peptone-glucose, the yield of blastospores was four-fold higher than in glucose-peptone-yeast extract. However, biomass production was highest in peptone-glucose-yeast extract.

A comparison of the virulence, stability and cell-wall-surface characteristics of three spore types produced by the entomopathogenic fungus Beauveria bassiana

SummaryBeauveria bassiana can produce three spore types; aerial conidia, submerged conidia and blastospores. We have examined the spore surface characteristics (hydrophobicity and cell-wall surface lectins), thermal inactivation and the virulence towards the migratory grasshopper, Melanoplus sanguinipes, of each of the three spore types. The hydrophobicities of the aerial and submerged conidia were quite similar. Blastospores were less hydrophobic than either of the two types of conidia. Hydrophobic interactions are thought to play a significant role in attachment of the spore to the host organism. However, the less hydrophobic blastospores were slightly more virulent (LT50 of 6.50 days) when compared to the aerial and submerged conidia (7.12 and 7.24 days), respectively. The lectin-binding characteristics of the aerial and submerged conidia were very similar but differed from that of blastospores. Growth of blastospores on a variety of carbohydrates did not affect their lectin-binding characteristics. Spore viability measurements showed that aerial and submerged conidia retained their viability for a longer period than blastospores. The similarity in hydrophobicity, stability, virulence and lectin-binding of aerial and submerged conidia make the latter an ideal candidate for mycoinsecticide production since they can be recovered after growth on inexpensive substrates.

High pH during trisodium phosphate treatment causes membrane damage and destruction of Salmonella enterica serovar Enteritidis

ABSTRACT Trisodium phosphate (TSP) is now widely used during the processing of poultry and red meats, but the mechanism whereby it inactivates gram-negative bacteria such Salmonella spp. remains unclear. Thus, Salmonella enterica serovar Enteritidis (ATCC 4931) cells were treated with different concentrations of TSP (1.5, 2.0, and 2.5% [wt/vol]) and compared with (i) cells treated with the same pH as the TSP treatments (pH 10.0, 10.5, and 11.0, respectively) and (ii) cells treated with different concentrations of TSP (1.5, 2.0, and 2.5% [wt/vol]) adjusted to a pH of 7.0 ± 0.2 (mean ± standard deviation). Cell viability, loss of membrane integrity, cellular leakage, release of lipopolysaccharides, and cell morphology were accordingly examined and quantified under the above treatment conditions. Exposure of serovar Enteritidis cells to TSP or equivalent alkaline pH resulted in the loss of cell viability and membrane integrity in a TSP concentration- or alkaline pH-dependent manner. In contrast, cells treated with different concentrations of TSP whose pH was adjusted to 7.0 did not show any loss of cell viability or membrane integrity. A 30-min pretreatment with 1.0 mM EDTA significantly enhanced the loss of membrane integrity only when followed by TSP or alkaline pH treatments. Measuring the absorbance at 260 nm, agarose gel electrophoresis, Bradford assay, and Tricine-sodium dodecyl sulfate gel electrophoresis of filtrates of treated cell suspensions revealed considerable release of DNA, proteins, and lipopolysaccharides compared to controls and pH 7.0 TSP treatments. Electron microscopic examination of TSP- or alkaline pH-treated cells showed disfigured cell surface topology and wrinkled appearance and showed evidence of a TSP concentration- and pH-dependent disruption of the cytoplasmic and outer membranes. These results demonstrate that TSP treatment permeabilizes and disrupts the cytoplasmic and outer membranes of serovar Enteritidis cells because of the alkaline pH, which in turn leads to release of intracellular contents and eventual cell death.

The impact of biotechnology on hyphomycetous fungal insect biocontrol agents

The potential for the control of insect pests by entomopathogenic fungi has been touted for decades, if not centuries. Only recently have advances in biotechnology provided the tools for indepth analysis of the mechanisms involved in pathogenesis and host death at the molecular level. This review outlines the current state of knowledge regarding the mode of infection and targets several key components that are amenable to improvement via biotechnology. Realization of the considerable economic potential of fungal bioinsecticides can occur only through a combined and coordinated effort involving fundamental science, formulation technology and field applications.

Schwanniomyces: SCP and ethanol from starch

SummaryApplication of Schwanniomyces yeasts to single cell protein or alcohol production is feasible based on cell yields of 60% aerobically, and ethanol yields of 86% of theoretical in associative fermentation.

Treatment of Salmonella enterica Serovar Enteritidis with a Sublethal Concentration of Trisodium Phosphate or Alkaline pH Induces Thermotolerance

ABSTRACT The responses of Salmonella enterica serovar Enteritidis to a sublethal dose of trisodium phosphate (TSP) and its equivalent alkaline pH made with NaOH were examined. Pretreatment of S. enterica serovar Enteritidis cells with 1.5% TSP or pH 10.0 solutions resulted in a significant increase in thermotolerance, resistance to 2.5% TSP, resistance to high pH, and sensitivity to acid and H2O2. Protein inhibition studies with chloramphenicol revealed that thermotolerance, unlike resistance to high pH, was dependent on de novo protein synthesis. Two-dimensional polyacrylamide gel electrophoresis (PAGE) of total cellular proteins from untreated control cells resolved as many as 232 proteins, of which 22 and 15% were absent in TSP- or alkaline pH-pretreated cells, respectively. More than 50% of the proteins that were either up- or down-regulated by TSP pretreatment were also up- or down-regulated by alkaline pH pretreatment. Sodium dodecyl sulfate-PAGE analysis of detergent-insoluble outer membrane proteins revealed the up-regulation of at least four proteins. Mass spectrometric analysis showed the up-regulated proteins to include those involved in the transport of small hydrophilic molecules across the cytoplasmic membrane and those that act as chaperones and aid in the export of newly synthesized proteins by keeping them in open conformation. Other up-regulated proteins included common housekeeping proteins like those involved in amino acid biosynthesis, nucleotide metabolism, and aminoacyl-tRNA biosynthesis. In addition to the differential expression of proteins following TSP or alkaline pH treatment, changes in membrane fatty acid composition were also observed. Alkaline pH- or TSP-pretreated cells showed a higher saturated and cyclic to unsaturated fatty acid ratio than did the untreated control cells. These results suggest that the cytoplasmic membrane could play a significant role in the induction of thermotolerance and resistance to other stresses following TSP or alkaline pH treatment.

Regulation of extracellular protease in the entomopathogenic fungus Beauveria bassiana

Factors which affect the production, activity, and stability of the extracellular protease of Beauveria bassiana have been investigated. During growth in liquid culture B. bassiana produced one extracellular protease that released free amino-nitrogen from gelatin. Unbuffered cultures (pH 7.0) containing gelatin and glucose showed a drop in pH to 2.5 which was inhibitory to protease activity as measured by an Azocoll assay. Protease activity was maximum at pH 8.5. The protease was stable for 24 h between pH 5 and 12, but was unstable at pH values below 5. In cultures buffered to pH 7.0, containing gelatin, synthesis of B. bassiana protease is repressed in the presence of ammonium and glucose, glycerol, trehalose, or mannitol but not with maltose. Partial repression of extracellular protease was observed in cultures buffered to pH 7.0 containing gelatin and glucose. Protease was not produced in cultures containing glucose and/or ammonium without gelatin. These results suggest that B. bassiana synthesizes and secretes an extracellular protease into its growth medium when an exogenous protein (gelatin) serves as its principal source of nitrogen and carbon. The regulation of extracellular protease synthesis in B. bassiana is consistent with a multiple control model.