Comparative study of fungal stability between Metarhizium strains after successive subculture

Background Metarhizium species are considered one of the most outstanding powerful biological control agents that have been commercialized as biopesticides against various agricultural pests. Fungal stability with successive in vitro cultivation is a desirable trait for a large-scale production of fungal biopesticide. Main body The new Egyptian strain Metarhizium anisopliae AUMC 3262 exhibited auspicious results when compared to Metarhizium brunneum ARSEF 4556 and M. brunneum V275 based on the variations of fungal characteristics, and essential quality control parameters (radial growth rate, conidial yield, viability, and virulence) after repeated in vitro subculturing. Changes in morphological characteristics were noted at both AUMC 3262 and ARSEF 4556. Following the 5th subculture, decreased conidial yield was noted, though radial growth remained stable, confirming that there is a non-positive correlation between conidial yield and radial growth rate for these species. In contrast, V275 showed a high morphological stability, conidial yield, and radial growth rate after repeated subculture. The three tested strains manifested high viability up to 100% and displayed the same pattern of Pr1 production. A slight variation was recorded in the median lethal time (LT50) values against the great wax moth, Galleria mellonella (L.), larvae between different subcultures of the tested Metarhizium strains. Conclusion The new Egyptian strain AUMC 3262 showed a high stability with a slight difference in some parameters after the successive subculture compared to both ARSEF4556 and V275.

Degenerated Virulence and Irregular Development of Fusarium oxysporum f. sp. niveum Induced by Successive Subculture

Successive cultivation of fungi on artificial media has been reported to cause the sectorization, which leads to degeneration of developmental phenotype, and virulence. Fusarium oxysporum f. sp. niveum (Fon), the causal agent of watermelon Fusarium wilt, forms degenerated sectors after successive cultivation. In the present research, we demonstrated that subculture with aged mycelia increased the incidence of degenerations. To further investigate the differences between the Fon wild type (sporodochial type, ST) and variants (MT: mycelial type and PT: pionnotal type), developmental phenotypes and pathogenicity to watermelon were examined. Results in variants (PT2, PT3, PT11, and MT6) were different from ST with mycelia growth, conidia production and chlamydospore formation. Virulence of degenerated variants on susceptible watermelon Grand Baby (GB) cultivar was determined after inoculation with Fon variants and Fon ST. In root dipping methods, Fon variants showed no significant differences in disease progress compared with ST. Fon variants showed a significant decrease in disease progression compared with ST through infested soil inoculation. The contrasting results of two inoculation methods suggest that the degenerative changes due to repeated successive cultivation may lead to the loss of pathogen virulence-related factors of the early stage of Fon infection process. Therefore, cell wall-degrading enzymes (CWDEs; cellulase, pectinase, and xylanase) activities of different variants were analyzed. All Fon degenerated variants demonstrated significant decreases of CWDEs activities compared with ST. Additionally, transcript levels of 9 virulence-related genes (fmk1, fgb1, pacC, xlnR, pl1, rho1, gas1, wc1, and fow1) were assessed in normal state. The degenerated variants demonstrated a significantly low level of tested virulence-related gene transcripts except for fmk1, xlnR, and fow1. In summary, the degeneration of Fon is triggered with successive subculture through aged mycelia. The degeneration showed significant impacts on virulence to watermelon, which was correlated with the reduction of CWDEs activities and declining expression of a set of virulence-related genes.

Degenerated virulence and irregular development of Fusarium oxysporum f. sp. niveum induced by successive subculture

Successive cultivation of fungus has been reported to cause the sectorization, which leads to degeneration of developmental phenotype, and virulence. Fusarium oxysporum f. sp. niveum (Fon), the causal agent of watermelon Fusarium wilt, demonstrated that successive cultivation formed the degenerated sectors. In the present research, we demonstrated that subculture with aged mycelium increased the incidence of degenerations. To further investigate the differences between the Fon wild type (sporodochial type, ST) and variants (MT: mycelium type and PT: pionnotal type), developmental phenotypes and pathogenicity to watermelon were examined. Results have shown that degeneration variants (PT2, PT3, PT11 and MT6) were different from ST with mycelium growth, conidia production and chlamydospore formation. Virulence of degenerated variants on susceptible watermelon Grand Baby (GB) cultivar was determined after inoculated with Fon variants and Fon ST. In root dipping methods, all Fon variants showed slightly increased disease severity than ST. Conversely, all Fon variants showed a significant decrease in disease progression compared with ST through infested soil inoculation. The contrary results of two inoculation methods suggest that the changes of successive cultural degeneration may lead to the loss of pathogen virulence-related factors of the early stage of Fon infection process. Therefore, Cell wall-degrading enzymes (CWDEs; cellulase, pectinase, and xylanase) activities of different variants were analysed. All Fon degenerated variants demonstrated significantly decreased of CWDEs activities compared with ST. Additionally, transcripts level of 9 virulence-related genes (fmk1, fgb1, pacC, xlnR, pl1, rho1, gas1, wc1 and fow1) were assessed in normal state. The degenerated variants demonstrated a significantly low level of tested virulence-related genes transcripts except for fmk1, xlnR and fow1. In summary, the degeneration of Fon is triggered with successive subculture through aged mycelium. The degeneration showed significant impacts on virulence to watermelon, which caused by the reduction of CWDEs activities and declining expression of a set of virulence-related genes.

Label-Free Proteomics Reveals the Molecular Mechanism of Subculture Induced Strain Degeneration and Discovery of Indicative Index for Degeneration in Pleurotus ostreatus

Pleurotus ostreatus is one of the widely cultivated edible fungi across the world. Mycelial subculture is an indispensable part in the process of cultivation and production for all kinds of edible fungi. However, successive subcultures usually lead to strain degeneration. The degenerated strains usually have a decrease in stress resistance, yield, and an alteration in fruiting time, which will subsequently result in tremendous economic loss. Through proteomic analysis, we identified the differentially expressed proteins (DEPs) in the mycelium of Pleurotus ostreatus from different subcultured generations. We found that the DNA damage repair system, especially the double-strand breaks (DSBs), repairs via homologous recombination, was impaired in the subcultured mycelium, and gradual accumulation of the DSBs would lead to the strain degeneration after successive subculture. The TUNEL assay further confirmed our finding about the DNA breaks in the subcultured mycelium. Interestingly, the enzyme activity of laccase, carboxylic ester hydrolase, α-galactosidase, and catalase directly related to passage number could be used as the characteristic index for strain degeneration determination. Our results not only reveal for the first time at the molecular level that genomic instability is the cause of degeneration, but also provide an applicable approach for monitoring strain degeneration in process of edible fungi cultivation and production.

Isolation and characterization of Acetobacter aceti from rotten papaya

The present study was concerned with the isolation and characterization of Acetobacter aceti from rotten papaya for vinegar production. The samples were inoculated in sterilized GYC standard media and then incubated at 30°C for 48 hours. Successive subculture was performed to screen out the strains. In Gram’s staining, the morphology of the isolated bacteria exhibited pink, small rod shaped single, pair and chain in arrangement, in the hanging drops technique, all the isolates revealed motile. Biochemical tests were performed by fermentation of five basic sugars by producing both acid and gas bubbles in Durham tube. All of the isolates were Indole, Voges-Proskauer (VP) and Oxidase negative, Methyl Red (MR) and Catalase positive. The growth rate of isolated strain was optimized by weighing dry cell and turbidity at 600 nm at different concentrations of dextrose (1%, 5% and 10%). Ten (10) percent dextrose solution showed rapid growth and higher cell mass than 5% and 1% solution respectively. Acidity of the media gradually increased from 0.102% to 2.18% from day 0 to day 7 and pH of the media decreased from 6.8 to 5.5 during the period. This protocol was successful for enriching Acetobacter aceti, which was essential for vinegar production.J. Bangladesh Agril. Univ. 13(2): 299-306, December 2015

Method for the selection of Lactococcus lactis mutants producing excess carbon dioxide

One of the characteristics of Roquefort cheese is the presence of irregularly shaped openings. Although many factors affect the development of opening in blue-veined cheeses (Martley & Crow, 1996), the limiting step in the case of Roquefort cheese is the production of CO2 by lactic acid bacteria (J.-P. Reverbel, pers. comm.). Most of the opening occurs after moulding; the process is difficult to control and many manufacturing runs result in cheeses with an insufficient opening. Concentrated suspensions of Leuconostoc strains are used to increase the production of CO2 (Devoyod & Muller, 1969), but it would clearly be useful to have microorganisms that produce larger quantities of the gas. McKay & Baldwin (1974) isolated a spontaneous mutant of Lactococcus lactis that produced more acetoin and CO2 than the parent strain. This mutant was lactate dehydrogenase (LDH)-deficient, which favoured the conversion of pyruvate into end products other than lactate. Following nitrosoguanidine mutagenesis, we isolated three Lc. lactis mutants whose LDH activities were reduced to varying extents and which produced varying amounts of CO2 (Boumerdassi et al. 1997). Subsequent work showed that these mutants were unstable on successive subculture in milk or synthetic broth (El Attar et al. 2000).The aim of our current work was to select a large number of Lc. lactis mutants producing excess CO2. This would increase the probability of selecting stable mutants and also provide a collection of strains with differing gas production activities. Currently available screening methods are, however, unsuitable for processing large numbers of mutants, which is why we have developed an improved screening method.

Properties of the R factor R144drd3 inKlebsiella pneumoniaestrain M5a1

SUMMARYThe R factor, R144drd3, mobilized chromosomal genes inKlebsiella pneumoniaestrain M5a1 at similar frequencies to those observed for this R factor inEscherichia coliK12. In a derivativeK. pneumoniaedonor, strain HF3, R144 persisted in the autonomous state but now gave rise to polarized transfer of the chromosome in the order Ohis…arg2…leu…trp. R144drd3 was unstable inK. pneumoniaeM5a1; after successive subculture drug resistance, colicinogeny and transmissibility were lost. In strain HF3, transmissibility was preferentially lost, but determinants for drug resistance, colicinogeny and superinfection immunity were retained; 10–11 copies per chromosome of R144 were present in log phase cultures of both strains.

In vitro shoot proliferation and development of micropropagation protocol from leaf disc of Gladiolus

The experiment was carried out during the period from July 2008 to March, 2009 at the Biotechnology Laboratory, Department of Biotechnology, Bangladesh Agricultural University, Mymensingh. The present investigation was to be better source for shoot multiplication. The calli derived from leaf discs were cultured on shoot induction media containing different combinations and concentrations of BAP (1.0, 1.5, 3.0 and 6.0 mg/L) and Kn (0.5, 1.0, 1.5 and 2.0 mg/L). The highest percentage of shoot regeneration (91.66%) and the highest shoot length was 4.24 (cm) with a minimum number of days (13.33) was observed in the MS medium supplemented with 3.0 mg/L BAP and 0.5 mg/L Kn. Thus shoot multiplication in successive subculture was possible. The present investigation was carried out to study the in vitro shoot multiplication in gladiolus by using leaf disc as explants Keyword: Shoot proliferation; Micropropagation; BAP: 6-benzylamino purine; Kn: Kinetin; Leaf disc DOI: http://dx.doi.org/10.3329/jbau.v9i1.8739 JBAU 2011; 9(1): 21-26