Biological fitness of Trichoderma atroviride during long-term storage,after production in different culture conditions

Identification of the production and storage factors that affect conidium germination and bioactivity (fitness) will assist the success of biological control agents. Effects of culturing conditions on conidium fitness of Trichoderma atroviride LU132 were examined in different storage conditions over time. Abiotic factors (temperature, nutrients, water activity and pH) during production were studied. Conidia from the culturing regimes which resulted in greatest and least bioactivity against Rhizoctonia solani in dual culture were selected to assess effects of storage conditions on conidium fitness. Fitness of the test conidia was examined after storage at 30°C and at 0% or 50% relative humidity (RH) over 6 months. Fitness declined over time, and the decline was greater for 50% RH than 0% RH, probably through reduced metabolic activity of conidia during long-term storage. Stored conidia were probably affected by dehydration, temperature and other factors such as oxidation, before and during storage, and also by rehydration after storage. The greatest number of conidia and germination percentage resulted from production at 25°C, but greatest bioactivity resulted from those produced at 30°C. No significant effects on bioactivity were detected between the conidium production treatments C?:?N 5?:?1 and C?:?N 160?:?1, indicating that C?:?N ratio in culture medium is not important for conidium survival of T. atroviride.     

The importance of water potential range tolerance as a limiting factor on Trichoderma spp. biocontrol of Sclerotinia sclerotiorum

Biological control of fungal phytopathogens is often more variable in efficacy compared with disease suppression achieved by conventional pesticide use. Matching the environmental range of a potential biocontrol agent with that of the target phytopathogen is necessary if consistent disease suppression is to be achieved under field conditions. Strains of Trichoderma that could parasitise sclerotia of Sclerotinia sclerotiorum had their spore germination and mycelial growth (five strains) and ability to parasitise sclerotia (two strains) tested under a range of water potentials under laboratory conditions. Relative mycelial growth and germination of all strains decreased with decreasing osmotic and matric potentials, with matric potential having a greater impact on growth and germination over the range examined. Trichoderma harzianum LU698 mycelial growth was the least affected by decreasing osmotic potential than the other isolates, and Trichoderma atroviride LU141 growth least affected by decreasing matric potential. The germination of LU698 and LU144 was also generally less affected by decreasing osmotic potential, although generally decreasing matric potential had the greatest affect on the germination of LU698 along with T. atroviride LU132. Soil treatments of LU698 and Trichoderma asperellum LU697 reduced sclerotial viability in all but the lowest soil water potential tested, with LU698 being most effective at ?0.1 and ?0.3 MPa after 28 days and LU697 most effective at ?0.01 and ?1.5 MPa after 28 days. We conclude that differences in the tolerance of potential biocontrol agents to changing water potential is an important experimental factor to consider when assaying biocontrol or making predictions of biocontrol efficacy in the field.     

Trichoderma atroviride LU132 promotes plant growth but not induced systemic resistance to Plutella xylostella in oilseed rape

Several species of the fungus Trichoderma can promote plant health and are widely used as commercial biopesticides. Beneficial effects of this fungus are attributed to various mechanisms such as mycoparasitism, plant-growth promotion, increased stress tolerance and elicitation of induced systemic resistance against pathogens via jasmonic acid/ethylene-dependent pathways. Despite such well-established effects on pathogens, surprisingly little is known about the influence of Trichoderma on plant defences against herbivorous insects. This study investigated whether soil-supplementation of the established biocontrol agent Trichoderma atroviride LU132 affected the performance of oilseed rape (Brassica napus) and the development of Plutella xylostella caterpillars. Furthermore, induction and priming of defence-related phytohormones, genes and secondary metabolites by fungus and herbivore were assessed. Plants colonized by T. atroviride LU132 had significantly larger root and shoot biomass than controls. No effects of fungal inoculation were found on herbivore development. Leaf feeding of the herbivore induced higher jasmonic acid levels, but this was not influenced by fungal treatment. Similarly, the defence-related genes MYC2 and TPI were induced by herbivory but not primed or induced by T. atroviride. Expression of the gene PDF1.2 was repressed by herbivore feeding while no effects on the gene ACO and glucosinolates were observed. We conclude that T. atroviride LU132 has positive effects on the growth of oilseed but it does not enhance above-ground insect defences.     

Optimising sporulation and virulence in Drechslera avenacea

Studies were conducted on agar media to optimise sporulation of Drechslera avenacea, a fungal pathogen being evaluated as a biological control agent for Avena species (wild oats). Conidium production was affected by nutrition, pH, temperature and light conditions. Of the agar media tested, Czapek Dox agar (CZA) and half-strength oatmeal agar (½OMA) were the only media where sporulation occurred at all temperatures tested under a 12-h light:12-h dark photoperiod (L/D). The optimum temperature for conidium production was 20°C on ½OMA, whereas there was no optimum temperature on CZA. Under a 12-h near-ultraviolet (NUV):12-h dark photoperiod (NUV/D), similar numbers of conidia were produced on CZA at 6.66, 14.56, and 22.78 W m^?2, whereas on ½OMA conidium production was the highest at 14.56 W m^?2. When NUV/D and L/D conditions were compared, similar numbers of conidia where produced on CZA, whereas ½OMA conidium production was superior under the NUV/D photoperiod. Considerable variation in sporulation and degree of virulence of D. avenacea was detected among isolates from different geographic areas. The most virulent conidia were obtained on ½OMA at 20°C incubated under continuous illumination NUV light. Therefore, the most suitable conditions for conidium production of D. avenacea were growth for 1 week on ½OMA at 20°C under continuous NUV at an intensity of 14.56 W m^?2. Under these conditions, 1.1×10⁵ conidia mL^?1 were produced which is the highest sporulation yet reported for any Drechslera spp., which are traditionally poor sporulators.     

Biological control of southern corn leaf blight by Trichoderma atroviride SG3403

Trichoderma atroviride SG3403 showed high biocontrol activity against southern corn leaf blight (SCLB; pathogen: Cochliobolus heterostrophus). T. atroviride SG3403 could cause death of C. heterostrophus race O hypha on plates. Spraying T. atroviride SG3403 conidia suspension over maize seedling leaves protected the corn from SCLB infection. Biocontrol effect lasted for 30 days in the field. Trichoderma strain was able to induce resistance response in corn leaves against pathogen infection. In corn leaves treated with T. atroviride SG3403, the enzyme activities of phenylalanine ammonia lyase (PAL) and superoxide dismutase (SOD) reached the highest at 24 h, enzyme activity of catalase (CAT) reached the highest at 36 h after inoculation of pathogen C. heterostrophus race O. RNA expression levels of Pal, Sod and Cat (which synthesis enzyme PAL, SOD and CAT) were also upregulated and corresponded to the enzyme activity at the same time point. Enzyme activities and corresponding genes expression induced by Trichoderma SG3403 was more obvious than that induced by pathogen only, which implies that T. atroviride SG3403 induced corn defense gene expression against pathogen infection. Thus, induced resistance mechanism was possibly involved in the biocontrol of SCLB by T. atroviride SG3403.     

Enhancing viability of two biocontrol yeasts in liquid formulation by applying sugar protectant combined with antioxidant

Survivals of Cryptococcus laurentii and Pichia membranaefaciens in liquid formulations with sugar protectants (trehalose and galactose) and L-ascorbic acid (Vc) were investigated during storage at 4°C and 25°C. When galactose or trehalose was used alone as protectant, C. laurentii maintained relatively high viability in potassium phosphate buffer. Addition of Vc to trehalose improved its protective effect. P. membranaefaciens maintained viability >60% after 90 days at 4°C when 5% galactose served as a protectant, and its combination with Vc was the most effective at maintaining viability. Moreover, liquid formulation kept higher viability of the two yeasts at 4°C than at 25°C. Biocontrol efficiency of the two yeasts was maintained after formulation and storage. The results indicate that trehalose is considered as a suitable protectant for liquid formulation of C. laurentii, while galactose is better for P. membranaefaciens. Combining Vc with the sugars improves the protective efficiency.     

Effect of temperature and water activity on spore germination and mycelial growth of three fungal biocontrol agents against water hyacinth (Eichhornia crassipes)

Aims: To determine the effect of water activity (a_w = 0·880–0·960) and temperature (15–35°C) on the percentage of viable conidia and mycelial growth of three biocontrol agents effective against water hyacinth in Mali: Alternaria sp. isolate Mlb684, Fusarium sacchari isolate Mln799 and Cadophora malorum isolate Mln715. Methods and Results: The fungi were grown in vitro on plates containing potato dextrose agar medium at different a_w values (glycerol being added to adjust the a_w). The percentage of viable conidia and radial growth rate decreased with decreasing water activity. Statistical analysis showed a significant effect of a_w, temperature and the a_w × temperature interaction on mycelial growth (P < 0·0001). Water activity emerged as the factor exerting the greatest influence. Differences were observed between the fungi tested, the C. malorum appearing more tolerant to low a_w and the F. sacchari more tolerant to high temperature (35°C). Growth models predicting the combined effect of a_w and temperature were developed and response surfaces generated, showing fairly good agreement with the experimental values. Conclusions: Our results confirm the previous finding that a_w has a greater influence than temperature on fungal growth. Under most conditions, variation of environmental factors has a detrimental influence on the percentage of viable conidia and mycelial growth rate of fungal isolates. Significance and Impact of the Study: The developed models may contribute to predicting the best environmental conditions for use of these fungi as effective biocontrol agents against water hyacinth.     

Physiological manipulation and formulation of the biocontrol yeast Pichia anomala for control of Penicillium verrucosum and ochratoxin A contamination of moist grain

The major hurdle in the production of commercial biocontrol agents (BCAs) has been the lack of production of appropriate formulations. Of particular importance is the conservation of viability and ecological competence after application. With this in mind studies were conducted to develop formulations of P. anomala which would have these attributes. Cells were grown in molasses-based medium modified with proline to different water availability levels (0.98 and 0.96) which significantly increased (up to 50%) the content of trehalose and arabitol in the yeast cells during liquid broth fermentation. The use of isotonic solutions for harvesting the yeast cells further increased the endogenous content of these compatible solutes as well as glycerol. Fluidised bed drying of cells at 30–80°C was carried out for 10 and 20 min and showed that viability was significantly decreased at 70–80°C. A temperature of 50°C for 20 min was found to be best for viability (70%) and moisture content of <10%. Several additives for conservation of viability showed that cotton seed flour+skimmed milk was the best treatment when dried at 50°C. The biocontrol efficacy of formulated P. anomala cells was tested in laboratory scale studies and this showed that they inhibited growth of Penicillium verrucosum and reduce ochratoxin A production in moist wheat grain under some combinations of water availability. Physiologically modified formulated yeast cells with increased levels of trehalose and arabitol gave similar efficacy as fresh cells. This suggests that ecophysiological manipulation of such BCAs can result in improved ecological competence of such formulations and effective biocontrol.     

Comparison of cold hardiness and sugar content between diapausing and nondiapausing pupae of the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

Abstract. To understand overwintering of the cotton boll worm Helicoverpa armigera, cold hardiness and sugar content are compared between diapausing and nondiapausing pupae. Diapausing and nondiapausing pupae reared at 20 °C under short and long photoperiods are acclimatized with a reduction of 5 °C per 5 days to 0 °C. When the acclimation temperature reaches 0 °C, the survival of diapausing pupae is assessed. The survival gradually decreases as the period of treatment progresses and approximately half survive for 112 days. However, nondiapausing pupae survive only 14 days after exposure to 0 °C. The surpercooling points of nondiapausing, diapausing and acclimatized pupae are approximately −17 °C. The major sugars contained in pupae are trehalose and glucose. Even though trehalose contents in diapausing pupae (initial level: 0.6 mg 100 mg⁻¹ fresh weight) increase significantly during cold acclimation and continue increasing until 58 days after exposure to 0 °C (maximum level: 1.8 mg 100 mg⁻¹), glucose is maintained at low levels (0.02 mg 100 mg⁻¹) for 56 days at 0 °C. However, glucose contents increase (maximum level: 0.8 mg 100 mg⁻¹) with decreasing contents of trehalose 84 days after exposure to 0 °C. Glycogen content gradually decreases during cold acclimation. When nondiapausing pupae are acclimatized with a reduction of 5 °C per 5 days to 5 °C from the beginning of pupation until the eyespots move, trehalose content increases (maximum level: 1.0 mg 100 mg⁻¹). Glucose contents in nondiapausing pupae increase before eclosion (0.09 mg 100 mg⁻¹). From these results, diapausing pupae of H. armigera can overwinter in regions where average winter temperatures are higher than 0 °C, but nondiapausing pupae cannot.     

Isolation and partial characterization of esters of trehalose from Corynebacterium ovis (C. pseudotuberculosis)

A glycolipid fraction was isolated from Corynebacterium ovis (C. pseudotuberculosis). It had [α]_D²⁵ = + 63.2° (C = 0.5, CHCl₃) and m.p. 43–46°C; the sugar content was 26%, determined as trehalose. Alkaline hydrolysis of the isolated fraction found trehalose as the sole water-soluble component, while glucose was found only after acid hydrolysis of the aqueous phase. Saturated and unsaturated short-chain mycolic acids with carbon atoms ranging from C₃₀ to C₃₆ were the constituents of the fatty acid moiety. The glycolipid fraction of C. ovis is therefore assumed to be a mixture of trehalose esters in which the trehalose molecule is esterified by saturated and unsaturated short-chain (C₃₀–C₃₆) mycolic acids.     

Competition is the mechanism of biocontrol of brown rot in stone fruit by <Emphasis Type="Italic">Penicillium frequentans</Emphasis>

The mechanism of biocontrol of brown rot in stone fruit by Penicillium frequentans Westling (Pf909) was investigated using in vitro and in vivo growth assays and a benomyl-resistant strain of Monilinia fructicola (G Winter) Honey (Mf3C). For the in vitro assays, Pf909 and Mf3C conidia were suspended in Czapek-Dox broth, which was amended or not amended with a skin extract of mature peaches. The growth and germination of Pf909 and Mf3C conidia were determined by counting the number of colony-forming units on potato dextrose agar plates, which were amended or not amended with 0.5 g ml^?1 benomyl. In some of the assays, germinated Pf909 conidia were used before their exposure to Mf3C conidia. For the in vivo assays, healthy cherries were inoculated with Mf3C conidia before and after applying Pf909 conidia on the cherry surface and the incidence of brown rot was recorded for seven days. Since we found that Pf909 conidia compete with Mf3C conidia for space and nutrients in the different assays, we concluded that competition is the probable primary mechanism of biocontrol of Pf909.     

A new technique for monoconidial culture of the most aggressive isolate in a given population of Bipolaris sorokiniana, cause of foliar spot blotch in wheat and barley

We developed a new technique for monoconidial culture of the most aggressive isolate in a given population of Bipolaris sorokiniana, to facilitate the evaluation of spot blotch resistance in wheat and barley. Blotched portions of infected barley leaves were placed on a glass slide in a moist chamber for production of conidia by associated fungal hyphae. Conidia were collected separately and grown on water agar discs. Individual water agar discs having conidium growth were inoculated on barley leaves. The conidium producing the earliest symptom with the largest lesion was considered most aggressive. This lesion was incubated in a moist chamber and the conidial offspring were tested for pathogenicity. When a uniform infection was observed, a small piece of the lesion was cut using a sterilized scalpel, surface sterilized with NaOCl, and inoculated in the centre of Petri dishes containing potato dextrose agar medium. The inoculated Petri dishes were incubated at 25 ± 1 °C to yield monoconidial cultures of the most aggressive isolate. Variability in symptom expression caused by the most aggressive isolate of a given population was much less than variability in symptom expression caused by all isolates collectively. The techniques will be useful for plant pathologists and breeders in screening for spot blotch resistance in wheat and barley.     

The survival ofZoophthora radicans (Zygomycetes: entomophthorales) isolates as hyphal bodies in mummified larvae ofPlutella xylostella (Lep.: Yponomeutidae)

The survival of three isolates ofZoophthora radicans (NW 250, NW 253 & NW 182) as hyphal bodies in dried larvae ofPlutella xylostella stored at 4, 10 and 20°C and 20% R.H was determined. After storage at 20°C, the production of conidia by all isolates was unaffected after 2 weeks but diminished increasingly after 4 and 8 weeks and was entirely lost after 16 weeks. By comparison conidium production at 10°C was unaffected after 16 weeks (isolates NW 250 and NW 182) and, 24 weeks (NW 253) of storage though it declined rapidly in all isolates thereafter. At 4°C many conidia were produced by all isolates even after 34 weeks of storage. These results are consistent with work on other entomophthoralean fungi in dried cadavers suggesting that this may be a common survival strategy in these fungi. NW 250, 253 and 182 were isolated fromP. xylostella in Malaysia and Taiwan, where conditions allow the host to remain active throughout the year. None produced resting sporesin vivo orin vitro but as hosts are always available the ability to survive short dry periods is probably more important than long-term survival for which resting spores are most adapted.      

Cloning and expression of Burkholderia polyyne biosynthetic gene clusters in Paraburkholderia hosts provides a strategy for biopesticide development

Burkholderia have potential as biocontrol agents because they encode diverse biosynthetic gene clusters (BGCs) for a range of antimicrobial metabolites. Given the opportunistic pathogenicity associated with Burkholderia species, heterologous BGC expression within non-pathogenic hosts is a strategy to construct safe biocontrol strains. We constructed a yeast-adapted Burkholderia-Escherichia shuttle vector (pMLBAD_yeast) with a yeast replication origin 2 μ and URA3 selection marker and optimised it for cloning BGCs using the in vivo recombination ability of Saccharomyces cerevisiae. Two Burkholderia polyyne BGCs, cepacin (13 kb) and caryoynencin (11 kb), were PCR-amplified as three overlapping fragments, cloned downstream of the pBAD arabinose promoter in pMLBAD_yeast and mobilised into Burkholderia and Paraburkholderia heterologous hosts. Paraburkholderia phytofirmans carrying the heterologous polyyne constructs displayed in vitro bioactivity against a variety of fungal and bacterial plant pathogens similar to the native polyyne producers. Thirteen Paraburkholderia strains with preferential growth at 30°C compared with 37°C were also identified, and four of these were amenable to genetic manipulation and heterologous expression of the caryoynencin construct. The cloning and successful heterologous expression of Burkholderia biosynthetic gene clusters within Paraburkholderia with restricted growth at 37°C opens avenues for engineering non-pathogenic biocontrol strains.     

Enhancing the adhesion of Epicoccum nigrum conidia to peach surfaces and its relationship to the biocontrol of brown rot caused by Monilinia laxa

Aims: To find a formulation of Epicoccum nigrum conidia that enhances its adhesion to peach surfaces and improves its biocontrol efficacy against brown rot caused by Monilinia laxa. Methods and Results: The stickers, glycerol, sodium alginate and methylcellulose; the desiccants, silica powder and talc; and a commercial adhesive (NU FILM 17^®) were added at two different points during the production of an E. nigrum conidial formulation to improve conidial adhesion to peach surfaces. Conidial adhesion levels were determined from the number of E. nigrum conidia that adhered to glass slides or peach surfaces and conidial viability of adherent E. nigrum conidia was determined from the number of colony‐forming units of glass or peach‐adherent E. nigrum that grew on Petri dishes that contained potato dextrose agar. Compared to dried E. nigrum conidia without additives, the adhesion and viability of adherent E. nigrum conidia to peach surfaces were enhanced when either 1·25% sodium alginate or 2·5% methylcellulose was added to the conidial mass after fluid‐bed drying, and when 2·5% methylcellulose was added to the conidial mass after its production and before fluid‐bed drying. Epicoccum nigrum conidial formulations with 2·5% methylcellulose were more effective than dried E. nigrum conidia without additives in reducing the incidence of brown rot in peaches caused by M. laxa. Conclusions: When 2·5% methylcellulose is incorporated into an E. nigrum conidial formulation, the adhesion of E. nigrum conidia to peach surfaces improves and results in efficacious biocontrol of brown rot. Significance and Impact of the Study: A new improved formulation of a biocontrol agent has been developed to improve the control of M. laxa on peaches.     

Industrial wastewaters and dewatered sludge: rich nutrient source for production and formulation of biocontrol agent, <Emphasis Type="Italic">Trichoderma viride</Emphasis>

Axenic cultivation of biocontrol fungus Trichoderma viride was conducted on a synthetic medium and different wastewaters and wastewater sludges in shake flasks to search for a suitable raw material resulting in higher biocontrol activity. Soluble starch based synthetic medium, dewatered municipal sludge, cheese industry wastewater sludge, pre-treated and untreated pulp and paper industry wastewater and slaughter house wastewater (SHW) were tested for T. viride conidia and protease enzyme production. The maximum conidia production followed the order, soluble starch medium (>10⁹ c.f.u./mL), untreated pulp and paper industry wastewater (4.9 × 10⁷ c.f.u./mL) > cheese industry wastewater (1.88 × 10⁷ c.f.u./mL) ≈ SHW (1.63 × 10⁷ c.f.u./mL) > dewatered municipal sludge (3.5 × 10⁶ c.f.u./mL) > pre-treated pulp and paper industry wastewater (1.55 × 10⁶ c.f.u./mL). The protease activity of T. viride was particularly higher in slaughterhouse wastewater (2.14 IU/mL) and dewatered municipal sludge (1.94 IU/mL). The entomotoxicity of soluble starch based synthetic medium was lower (≈6090 SBU/μL) in contrast to other raw materials. The entomotoxicity inversely decreased with carbon to nitrogen ratio in the growth medium and the conidia concentration and protease activity also contributed to the entomotoxicity. The residual c.f.u./g formulation of T. viride conidia were up to approximately, 90% after 1 month at 4 ± 1 °C and about 70% after 6 months at 25 ± 1 °C. Thus, production of T. viride conidia would help in marketability of low cost biopesticide from the sludge and safe reduction of pollution load.     

Regulation of the synthesis of unsaturated fatty acids by growth temperature in Bacillus subtilis

Bacillus subtilis synthesizes, almost exclusively, saturated fatty acids, when grown at 37° C. When cultures were transferred from 37° C to 20° C, a chloramphenicol- and rifampicin-sensitive synthesis of a C-16 unsaturated fatty acid was observed. Synthesis of this compound reached a plateau after 5 h at 20° C, reaching levels of 20% of the total fatty acid content. [¹⁴C]-labelled fatty acids attached as thioesters to acyl-carriers compounds, such as coenzyme A (CoA) or acyl-carrier protein (ACP) synthesized de novo by glycerol-requiring auxotrophs deprived of glycerol to arrest phospholipid synthesis, could not be desaturated at 20° C. Desaturation of these fatty acids was readily observed when glycerol was restored to the cultures allowing resumption of transfer of acyl-moieties from acyl-thioesters to phospholipid. It was also observed that depletion of the pools of CoA and ACP by starvation of pantothenate auxotrophs had no effect on the observed synthesis of unsaturated fatty acid at 20° C. The overall results indicate that synthesis of unsaturated fatty acids in B. subtilis is a cold-inducible process and that phospholipids are obligate intermediates in this fatty acid desaturation pathway.     

The orlA gene from Aspergillus nidulans encodes a trehalose-6-phosphate phosphatase necessary for normal growth and chitin synthesis at elevated temperatures

A cosmid carrying the orIA gene from Aspergillus nidulans was identified by complementation of an orlA1 mutant strain with DNA from the pKBY2 cosmid library. An orlA1 complementing fragment from the cosmid was sequenced. orlA encodes a predicted polypeptide of 227 amino acids (26 360 Da) that is homologous to a 211-amino-acid domain from the polypeptide encoded by the Saccharomyces cerevisiae TPS2 gene and to almost the entire Escherichia coli of otsB-encoded polypeptide. TPS2 and otsB each specify a trehalose-6-phosphate phosphatase, an enzyme that is necessary for trehalose synthesis. orlA disruptants accumulate trehalose-6-phosphate and have reduced trehalose-6-phosphatate phosphatase levels, indicating that the gene encodes a tre-halose-6-phosphatate phosphatase. Disruptants have a nearly-wild-type morphology at 32°C. When germinated at 42°C, the conidia and hyphae from disruptants are chitin deficient, swell excessively, and lyse. The lysis is almost completely remedied by osmotic stabilizers and is partially remedied by N-acetylglucosamine (GlcNAc). The activity of glutamine:fructose-6-phosphate amido-transferase (GFAT), the first enzyme unique to aminosugar synthesis, is reduced and is labile in orIA disruption strains. The findings are consistent with the hypothesis that trehalose-6-phosphate reduces the temperature stability of GFAT and other enzymes of chitin metabolism at elevated temperatures. The results extend to filamentous organisms the observation that mutations in fungal trehalose synthesis are highly pleiotropic and affect aspects of carbohydrate metabolism that are not directly related to trehalose synthesis.     

Diversity of Yeasts from Puddles in the Vicinity of Midre Lovénbreen Glacier, Arctic and Bioprospecting for Enzymes and Fatty Acids

A total of 132 yeast strains were characterised from 4 sediment samples collected from small puddles in the vicinity of Midre Lovénbreen glacier, Arctic. Based on the D1/D2 domain sequence similarity, the isolates could be categorised into 6 groups. The nearest phylogenetic neighbour of groups I to VI were identified as Cryptococcus gastricus, Cryptococcus terricolus, Rhodotorula muscorum, Mrakia psychrophila, Mrakia gelida and Rhodotorula glacialis, respectively. Strains representative of the six groups were psychrophilic and salt tolerant but varied in their ability to produce cold-active extracellular enzymes such as lipase, protease, pectinase, cellulase and amylase. C_{18:1 (w9C)} and C_{18:2 (w9,12C)} were the only two fatty acids common to all the yeasts and branched and (or) unsaturated fatty acids increased in yeasts growing at 8°C compared to 22°C, probably as an adaptation to low temperature. The present study establishes that psychrophilic yeasts are predominant in Arctic and could be used as work horses to produce cold-active enzymes and poly unsaturated fatty acids which have been implicated in low temperature adaptation and also for their use in biotechnology.     

DNA supercoiling and thermal regulation of unsaturated fatty acid synthesis in Bacillus subtilis

Bacillus subtilis growing at 37° C synthesizes, almost exclusively, saturated fatty acids. However, when a culture growing at 37°C is transferred to 20°C, the synthesis of unsaturated fatty acids is induced. The addition of the DNA gyrase inhibitor novobiocin specifically prevented the induction of unsaturated fatty acid synthesis at 20° C. Furthermore, it was determined that plasmid DNA isolated from cells growing at 20°C was significantly more negatively supercoiled than the equivalent DNA isolated from cells growing at 37°C. The overall results agree with the hypothesis that an increase in DNA supercoiling associated with a temperature downshift could regulate the unsaturated fatty acids synthesis in B. subtilis.