Phenylalanine ammonia-lyase activity in callus cultures of Pinus elliotii

Phenylalanine ammonia-lyase (PAL) activity increased 8- to 12-fold in pine (Pinus elliotii Engelm.) callus tissue within 2 days after subculturing on fresh medium. Factors such as increasing the sucrose content of the media, imposing additional tissue in jury or subculturing more frequently did not cause additional stimulation of PAL activity. The rapid increase in PAL activity appeared to be due to enzyme activation, since cycloheximide did not appreciably reduce the stimulation of PAL activity. The subsequent loss of increased PAL activity with age was reduced by cycloheximide and a cool growth environment.     

Successive subculturing alters spore-bound Pr1 activity,germination and virulence of the entomopathogenic fungus,Beauveria bassiana

One of the hurdles in the development of entomopathogenic fungi such as Beauveria bassiana is loss of virulence when successively maintained in vitro. This may result in products of inferior quality in mass production programs. Also, there are many contradicting data and unclear points in this case. Three isolates of B. bassiana were subcultured successively 15 times. Spore-bound Pr1 activity, germination rate, and virulence of conidia against mealworm (Tenebrio molitor L.) larvae were studied. Results showed that isolates normally retained their virulence during 10 subculturings. However, they clearly offered decreased virulence (elevated LT₅₀ values and lower percent mortality). The activity of Pr1 bound to conidia declined as subculturing continued; the lowest spore-bound activity and germination potential of conidia was recorded for the 15th subculture. Virulence data were in agreement with Pr1 activity and germination rate as there was a positive correlation between germination rate and spore-bound Pr1 activity with fungal virulence. This explains that at least a part of attenuation in fungal virulence can be explored in enzymatic activity, especially in the important cuticle-degrading protease, Pr1.     

植物乳植杆菌P-8连续传代过程中遗传稳定性分析

【背景】植物乳植杆菌(Lactiplantibacillus plantarum) P-8是一株具有优良益生特性的乳酸菌,探究其短期连续传代过程中的遗传稳定性对评估其加工生产的稳定性有着重要的指导作用。【目的】研究植物乳植杆菌P-8在37℃恒温环境下、MRS培养基中连续传代100代过程中的遗传稳定性。【方法】在MRS培养基、37℃恒温环境下将植物乳植杆菌P-8连续传代100代,测定不同代菌株(第0、25、50、75和100代)的菌体形态和碳水化合物利用能力,并利用二代、三代测序相结合的技术完成不同代菌株全基因组测序,通过比较基因组学综合分析其在连续传代100代过程中的遗传稳定性。【结果】植物乳植杆菌P-8在连续培养100代过程中,其菌体形态和碳水化合物利用能力均无明显变化。以植物乳植杆菌P-8的原始菌株基因组作为参考,比较分析了不同代菌株的基因组稳定性,发现菌株均有单核苷酸多态性(single nucleotide polymorphism, SNP)位点存在,但数量较少(SNP位点<21个)。不同代菌株基因组共线性良好,具有极高的相似性,且不同代菌株碳水化合物活性酶注释结果无显...     

Freezing Resistance and Cryopreservation of Cell Strains of Rhaponticum carthamoides and Thalictrum minus

Effects of long-term (few months) culturing and short pregrowth (up to 7 days prior to deep freezing) in the presence of mannitol (5–6%), ABA (5.0–7.5 × 10^–5 M), or both substances on cryogenic resistance of leusea (Rhaponticum carthamoides, strains Rhs-2 and Rhs-8) and meadow rue (Thalictrum minus L., strain B-233) cell suspension cultures were studied. Cryoprotective capacities of 48 solutions were studied at slow (0.33°C/min) freezing to the temperature of liquid nitrogen with an automatic initiation of crystallization. Cells were stored in liquid nitrogen for several days or months. ABA had a cryoprotective effect, provided that subculturing intervals were 12–14 days. At more frequent subculturing (every 7 days), pregrowth on ABA-containing medium did not increase survival percentage compared to pregrowth with mannitol. Successful cryopreservation of these strains has been achieved due to strict standardization of 7-day subculturing regime, pregrowth in the presence of mannitol prior to freezing, and cryopreservation with dimethyl sulfoxide, sucrose, trehalose, and glycerol. The cell survival rates after thawing were 60% (Rhs-8), 80% (Rhs-2), and 70% (B-233). The cell growth resumed on the third to seventh day. The growth indices and protoberberine synthesizing activity in B-233 strain reached their control values at the ninth subculturing after a post-thaw recovery.     

Repeated in vitro subculturing alters spore surface properties and virulence of Metarhizium anisopliae

Repeated subculturing caused rapid changes in the spore surface properties and virulence of Metarhizium anisopliae. Of the two strains evaluated, M. anisopliae V245 attenuated more rapidly than V275. Electrophoretic mobility and Radial Flow Chamber assays were used for the first time to generate qualitative and quantitative information on the adhesive forces of M. anisopliae conidia. Independent of strain, adhesion, hydrophobicity and spore-bound Pr1 declined after the first subculture; however, spore surface charge decline was erratic. Adhesion and hydrophobicity stabilized after the third subculture, whereas spore-bound Pr1 continues to decline following repeated subculturing. Decline in spore bound Pr1 was directly correlated with decline in virulence, however, such correlation with adhesion, hydrophobicity or surface charge could not be established. Because spore-bound Pr1 activities were directly correlated with M. anisopliae virulence; it could be used as a quality-control marker to monitor changes in virulence.     

The influence of a single-spore isolate and repeated subculturing on the pathogenicity of conidia of the entomophagous fungus Beauveria bassiana

The potential of monospore isolations for detecting strains of the entomophagous fungus Beauveria bassiana with increased virulence was investigated as well as the effect of repeated subculturing of both multispore parent strains and 28 newly obtained single-spore isolates on virulence. The use of the singlespore method enabled the disclosure of six spontaneous mutants surpassing the mother strain in pathogenicity, i.e., in a quicker effect and a higher virulence. The use of an optimal medium enabled an enhancement of virulence of all types of isolates. Differences among spontaneous mutants were indicated by the slope of the probit line. Our newly formed positive mutants retained their high pathogenicity both in repeated subculturing and in materials subcultured after a 12-month storage.     

Effects of successive subculturing on stability, virulence, conidial yield, germination and shelf-life of entomopathogenic fungi

Aims: To determine the stability and conidial yield of two strains of the entomopathogenic fungus Metarhizium anisopliae and one strain of M. brunneum, being developed for the control of insect pests. Methods and Results: The conidial yields and the shelf‐life of the conidia of two commercially viable strains of M. anisopliae V275 (=F52) and ARSEF 4556 and one strain of M. brunneum (ARSEF 3297) were determined after harvesting conidia from in vitro subcultures on Sabouraud dextrose agar (SDA) and broken basmati rice. The strains were stable and showed no decline in virulence against Tenebrio molitor, even when subcultured successively 12 times on SDA. Conidia‐bound Pr1 protease activity decreased in conidia harvested from SDA and mycosed cadavers after the 1st subculture, but increased in conidia produced on rice. The C:N ratio of conidia from mycosed cadavers was lower than that of conidia from rice or SDA. Irrespective of the number of subcultures, strain ARSEF 4556 produced significantly higher conidial yields than ARSEF 3297 and V275. The 12th subculture of V275 and ARSEF 3297 produced the lowest conidial yield. Shelf‐life studies showed that conidia of strain ARSEF 4556 had a higher conidial viability than V275 and ARSEF 3297 after 4 months, stored at 4°C. Conclusions: The current study shows that determining strain stability and conidial yield through successive subculturing is an essential component for selecting the best strain for commercial purposes. Significance and Impact of the Study: This is the first study to compare quality control parameters in the production of conidia on rice, and it shows that the level of Pr1 is comparatively high for inoculum produced on rice.     

Comparative investigation of different methods of storage of lactic acid bacteria

The study was undertaken to elucidate how different methods of storage (immersing in mineral oil, lyophilization, and subculturing) of lactic acid bacteria belonging to the generaLactobacillus andLactococcus affect their viability, antibiotic activity, and ability to accumulate organic acids. Storage of the lactic acid bacteriumLactococcus lactis subsp.lactis by immersion in mineral oil proved to be ineffective. Lyophilization allowed the survival of a sufficiently large number of cells, although their antibiotic activity somewhat decreased. The resuscitation of lyophilized bacteria by subculturing them in rich nutrient media, such as skim milk, led to the restoration of their physiological activity, including the effective antimicrobial spectrum