Hybridisation and genetic selection for improving desiccation tolerance of the entomopathogenic nematode Steinernema feltiae

Shelf life of biological control products based on the entomopathogenic nematode Steinernema feltiae is rather limited. In order to prolong shelf life, the metabolism of nematodes during storage must be reduced, either by low temperature or by means of desiccation of the third stage dauer juveniles (DJs). Tolerance to desiccation is limited in S. feltiae. Their tolerance can be increased by an adaptation to moderate desiccation conditions. The objective of this study was to screen for tolerant strains among wild type populations of S. feltiae, hybridise most tolerant strains and further improve desiccation tolerance by subjection of the hybrid strain to genetic selection. Dehydrating conditions, measured as water activity (a _w-values), were produced by treating DJs with different concentrations of the polymer polyethylene glycol 600. Significant variation was recorded among 24 S. feltiae strains. The mean tolerated water activity survived by 50% of the population (WA₅₀) ranged from 0.78 to 0.93 when nematodes were not adapted to desiccation stress and from 0.66 to 0.88 when preadapted to desiccation stress. The six most desiccation tolerant strains of non-adapted and adapted nematode populations were crossed. Preadapted tolerance of hybrids was superior to the tolerance of parental strains, whereas non-adapted tolerance was not increased. The most tolerant hybrid had a WA₅₀ when preadapted of 0.67 and 0.86 when not adapted. The tolerance was lost after few reproductive cycles in the insect Galleria mellonella but was recovered again after six selection cycles with exposure to increasing stress conditions. Virulence and reproduction potential was not negatively affected by the selection. Stabilisation of the selection progress will be a major challenge to enable commercial exploitation of the genetic improvement.     

Improvement of heat and desiccation tolerance in <Emphasis Type="Italic">Heterorhabditis bacteriophora</Emphasis> through cross-breeding of tolerant strains and successive genetic selection

Genetic selection can be a powerful tool to increase beneficial traits in biological control agents. In this study the heat and desiccation tolerance of the entomopathogenic nematode Heterorhabditis bacteriophora Poinar (Rhabditidomorpha: Strongyloidea) were significantly increased by cross breeding tolerant parental strains and successive genetic selection. These strains originated from a prior screening among 60 strains for increased stress tolerance. During genetic selection, the selection pressure was constantly increased and only the most tolerant 10% of the nematode populations were propagated for further selection steps. Assessment of tolerance and selection for both traits was performed with and without prior adaptation to the stress conditions. Eleven selection steps were performed to increase heat tolerance. A final overall increase in mean heat tolerance of 5.5°C was achieved when nematodes had been adapted to heat stress. For non-adapted tolerance an increase of 3.0°C from 40.1°C to 43.1°C was recorded. For comparison, a commercial strain had a mean tolerated temperature after adaptation of 38.2°C and of 36.5°C without adaptation. For assessment of the desiccation tolerance the mean tolerated water activity (a_w-value) of a population was measured. Cross-breeding most tolerant strains reduced the a_w-value from 0.67 to 0.65 after adaptation and from 0.9 to 0.7 without prior adaptation. The following six selection steps could not increase the tolerance whether nematodes had been adapted to stress or not. In comparison, the commercial strain tolerated a mean a_w-value of 0.985 after adaptation and 0.951 without adaptation. Further investigation will have to assess trait stability and possible trade-off effects. This study is a first important step on the road towards domestication of the entomopathogenic nematode H. bacteriophora.     

Tolerance of Bradyrhizobium sp. (Lupini) strains to salinity, pH, CaCO3 and antibiotics

AIMS: Ten rhizobial isolates obtained from different locations in Egypt were examined for their ability to survive under stress conditions and their growth response to increasing levels of NaCl (1-8% w/v), pH (4-10), CaCO3 (1-10% w/v) and 12 antibiotics. METHODS AND RESULTS: All the rhizobial isolates tolerated a NaCl concentration up to 5% and were divided into two groups with respect to NaCl tolerance. The rhizobial isolates from group two showed significantly (P < 0.05) better survival under high NaCl concentration. All the tested isolates survived acidic (pH 4-5) and alkaline conditions (pH 9-10) and CaCO3 (up to 10% w/v) in liqued YEM medium. CONCLUSION: Antibiotic resistance patterns did not correlate to NaCl, pH or CaCO3 tolerance. Variations among different strains showed that there is potential to improve strain performance under stress conditions. Significance and Impact of the Study: The results suggest that selection of adapted strains under stress conditions is possible and can be used as inoculants for successful lupin growth.     

Dehydration-induced cross tolerance of Belgica antarctica larvae to cold and heat is facilitated by trehalose accumulation

Larvae of the Antarctic midge, Belgica antarctica (Diptera: Chironomidae), are frequently exposed to dehydrating conditions on the Antarctic Peninsula. In this study, we examined how rates and levels of dehydration alter heat and cold tolerance and how these relate to levels of trehalose within the insect. When dehydrated, larvae tolerated cold and heat stress more effectively, although resistance to cold was more pronounced than heat resistance. Slow dehydration was more effective than rapid dehydration in increasing temperature tolerance. Severe dehydration (50% reduction in water content) caused a much greater increase in temperature tolerance than did mild dehydration (e.g. 10% water loss). Larvae severely dehydrated at a slow rate (98% RH) were more temperature tolerant than those dehydrated quickly (0 or 75% RH). These results indicate that the slower dehydration rate allows the larvae to more effectively respond to reduced water levels and that physiological adjustments to desiccation provide cross tolerance to cold and heat. Levels of trehalose increased during dehydration and are likely a major factor increasing subsequent cold and heat resistance. This hypothesis was also supported by experimental results showing that injection of trehalose enhanced resistance to temperature stress and dehydration. We conclude that changes in temperature tolerance in B. antarctica are linked to the rate and severity of dehydration and that trehalose elevation is a probable mechanism enhancing this form of cross tolerance.     

Desiccation tolerant plants as model systems to study redox regulation of protein thiols

While the majority of plants and animals succumb to water loss, desiccation tolerant organisms can lose almost all of their intracellular water and revive upon rehydration. Only about 300 ‘resurrection’ angiosperms and very few animals are desiccation tolerant. By contrast, many bryophytes and most lichens are desiccation tolerant and so are the seeds and pollen grains of most flowering plants. The current literature reveals that the extreme fluctuations in water content experienced by desiccation tolerant organisms are accompanied by equally extreme changes in cellular redox state. Strongly oxidizing conditions upon desiccation can cause irreversible oxidation of free cysteine residues of proteins, which can change protein structure and function, and contribute to protein denaturation. It appears likely that reversible formation of disulphide bonds, in particular through protein glutathionylation, contributes to the set of protection mechanisms that confer desiccation tolerance. Upon rehydration, de-glutathionylation can be catalyzed by glutaredoxins (GRXs) and protein disulphide bonds can be reduced through NADPH-dependent thioredoxins (TRXs). Due to their ability to survive severe oxidative stress, desiccation tolerant plants and seeds are excellent models to study protein redox regulation, which may provide tools for enhancing tolerance to drought and more generally, to oxidative stress, in crops.     

Survival of rhizobia on arrowleaf clover seeds under stresses of seed-coat toxins,heat and desiccation

Eighteen strains of Rhizobium leguminosarum bv. trifolii that had been found to be heat- and desiccation-stress tolerant in soil were tested for their resistance to Trifolium vesiculosum, Savi (arrowleaf clover) coat toxins using an agar plate technique and for survival on seed. They were further tested for their tolerance to seed coat toxins in combination with heat and desiccation stresses. The zone of inhibition in agar ranged from no inhibition to 21 mm diameter of growth inhibition around seed. Inoculation on seed under conditions of 28 °C and 100% relative humidity for 7 days resulted in a range of survival from nearly 100% to less than 1%. The correlation between zone of growth inhibition and survival on seed was not statistically significant. Strains that were desiccation or heat tolerant in soil were not necessarily desiccation or heat tolerant on seed but strains that were heat tolerant in soil were better able to survive on glass beads at 37 °C than strains that were not heat tolerant in soil. The zone of rhizobial inhibition on an agar medium was not a valid method to screen for isolates better able to survive on seed and the ability of rhizobia to withstand heat or drought stress on seed was not related to their ability to survive in soil. This revised version was published online in August 2006 with corrections to the Cover Date.     

Dehydration-induced redistribution of amphiphilic molecules between cytoplasm and lipids is associated with desiccation tolerance in seeds

This study establishes a relationship between desiccation tolerance and the transfer of amphiphilic molecules from the cytoplasm into lipids during drying, using electron paramagnetic resonance spectroscopy of amphiphilic spin probes introduced into imbibed radicles of pea (Pisum sativum) and cucumber (Cucumis sativa) seeds. Survival following drying and a membrane integrity assay indicated that desiccation tolerance was present during early imbibition and lost in germinated radicles. In germinated cucumber radicles, desiccation tolerance could be re-induced by an incubation in polyethylene glycol (PEG) before drying. In desiccation-intolerant radicles, partitioning of spin probes into lipids during dehydration occurred at higher water contents compared with tolerant and PEG-induced tolerant radicles. The difference in partitioning behavior between desiccation-tolerant and -intolerant tissues could not be explained by the loss of water. Consequently, using a two-phase model system composed of sunflower or cucumber oil and water, physical properties of the aqueous solvent that may affect the partitioning of amphiphilic spin probes were investigated. A significant relationship was found between the partitioning of spin probes and the viscosity of the aqueous solvent. Moreover, in desiccation-sensitive radicles, the rise in cellular microviscosity during drying commenced at higher water contents compared with tolerant or PEG-induced tolerant radicles, suggesting that the microviscosity of the cytoplasm may control the partitioning behavior in dehydrating seeds.     

Proline biosynthesizing enzymes (glutamate 5-kinase and pyrroline-5-carboxylate reductase) from a model cyanobacterium for desiccation tolerance

Drought is the most important abiotic stress, challenging sustainable agriculture globally. For desiccation being the multigenic trait, a combination of identified genes from the appropriate organism may render crop tolerant to the water stress. Among the compatible solutes, proline plays multifaceted role in counteracting such stress. The genes encoding proline biosynthesizing enzymes, glutamate 5-kinase (G5K), and pyrroline-5-carboxylate reductase (P5CR) from the low-desiccation-tolerant cyanobacterium Anabaena sp. PCC 7120, were cloned and overexpressed in Escherichia coli BL21(DE3) individually. The recombinant E. coli cells harboring G5K, failed to exhibit enhanced desiccation tolerance relative to those with P5CR that showed increased growth/survival over the wild type. This may be ascribed to the overexpression of the reductase gene. Multiple sequence alignment showed P5CR to be conserved in all the organisms. We hypothesize that P5CR gene from high-desiccation-tolerant cyanobacteria may be adopted as the candidate for making transgenic N₂-fixing cyanobacterium for paddy fields and/or crop development in future.     

The function of melanin in the ectomycorrhizal fungus Cenococcum geophilum under water stress

Despite the ubiquity and importance of ectomycorrhizal fungi to ecosystem function, our understanding of their functional ecology remains poor. The highly melanized and common ectomycorrhizal fungus, Cenococcum geophilum, is drought tolerant and abundant in water-stressed habitats, yet the responsible functional traits have not been identified. The production of melanin, a class of complex dark polymers found in fungal cell walls, may be a key functional trait to water stress tolerance. To test this hypothesis, we devised a series of experiments determining the effect of the melanin biosynthesis inhibitor, tricyclazole, on response to osmotic and desiccation stresses. Melanin inhibition only had negative effects on growth when C. geophilum isolates were subjected to osmotic and desiccation stress (?1.7 MPa and desiccated) but not under control conditions (?0.01 MPa and non-desiccated). This suggests that melanin production is an important functional trait that contributes to water stress tolerance of this cosmopolitan ectomycorrhizal fungus.     

Crucial role of extracellular polysaccharides in desiccation and freezing tolerance in the terrestrial cyanobacterium Nostoc commune

The cyanobacterium Nostoc commune is adapted to the terrestrial environment and has a cosmopolitan distribution. In this study, the role of extracellular polysaccharides (EPS) in the desiccation tolerance of photosynthesis in N. commune was examined. Although photosynthetic O2 evolution was not detected in desiccated colonies, the ability of the cells to evolve O2 rapidly recovered after rehydration. The air-dried colonies contained approximately 10% (wt/wt) water, and field-isolated, natural colonies with EPS were highly water absorbent and were rapidly hydrated by atmospheric moisture. The cells embedded in EPS in Nostoc colonies were highly desiccation tolerant, and O2 evolution was not damaged by air drying. Although N. commune was determined to be a mesophilic cyanobacterium, the cells with EPS were heat tolerant in a desiccated state. EPS could be removed from cells by homogenizing colonies with a blender and filtering with coarse filter paper. This treatment to remove EPS did not damage Nostoc cells or their ability to evolve O2, but O2 evolution was significantly damaged by desiccation treatment of the EPS-depleted cells. Similar to the EPS-depleted cells, the laboratory culture strain KU002 had only small amount of EPS and was highly sensitive to desiccation. In the EPS-depleted cells, O2 evolution was also sensitive to freeze-thaw treatment. These results strongly suggest that EPS of N. commune is crucial for the stress tolerance of photosynthesis during desiccation and during freezing and thawing.     

Metabolic dysfunction and unabated respiration precede the loss of membrane integrity during dehydration of germinating radicles

This study shows that dehydration induces imbalanced metabolism before loss of membrane integrity in desiccation-sensitive germinated radicles. Using a photoacoustic detection system, responses of CO(2) emission and fermentation to drying were analyzed non-invasively in desiccation-tolerant and -intolerant radicles of cucumber (Cucumis sativa) and pea (Pisum sativum). Survival after drying and a membrane integrity assay showed that desiccation tolerance was present during early imbibition and lost in germinated radicles. However, tolerance could be re-induced in germinated cucumber radicles by incubation in polyethylene glycol before drying. Tolerant and polyethylene glycol (PEG)-induced tolerant radicles exhibited a much-reduced CO(2) production before dehydration compared with desiccation-sensitive radicles. This difference was maintained during dehydration. In desiccation-sensitive tissues, dehydration induced an increase in the emission of acetaldehyde and ethanol that peaked well before the loss of membrane integrity. Acetaldehyde emission from sensitive radicles was significantly reduced when dehydration occurred in 50% O(2) instead of air. Acetaldehyde/ethanol were not detected in dehydrating tolerant radicles of either species or in polyethylene glycol-induced tolerant cucumber radicles. Thus, a balance between down-regulation of metabolism during drying and O(2) availability appears to be associated with desiccation tolerance. Using Fourier transform infrared spectroscopy, acetaldehyde was found to disturb the phase behavior of phospholipid vesicles, suggesting that the products resulting from imbalanced metabolism in seeds may aggravate membrane damage induced by dehydration.     

Adaptations to environmental stress in altitudinal populations of two Drosophila species

Abstract.  Opposite clinal variation for desiccation and starvation tolerance are observed in four altitudinal populations (219–2202 m), each of two sympatric and cold adapted species: Drosophila takahashii and Drosophila nepalensis from northern India. The high-altitude populations are more tolerant to desiccation than those from lower altitudes, whereas the reverse trend occurs for starvation tolerance. The magnitude of tolerances are significantly high in D. nepalensis, which is better adapted to cold conditions. During winter months (November to February), there are significant decreases in T _max, T _min and relative humidity along the altitudinal transect. Higher desiccation resistance can develop under cold conditions over short-range, altitudinally varying, geographical areas (250 km) compared with our previously reported long-range (>2000 km), latitudinal variations under tropical climatic conditions. However, significant starvation tolerances are favoured by small body size, higher dispersal rate and higher ambient temperature of the site of origin of populations. Significant correlations of two climatic factors (the mean monthly coefficients of variation of temperature and relative humidity) with these two physiological traits can best explain the observed altitudinal clinal variations under natural conditions.     

Nitrogen Limitation and Slow Drying Induce Desiccation Tolerance in Conjugating Green Algae (Zygnematophyceae,Streptophyta) from Polar Habitats

Background

Filamentous Zygnematophyceae are typical components of algal mats in the polar hydro-terrestrial environment. Under field conditions, they form senescent vegetative cells, designated as pre-akinetes, which are tolerant to desiccation and osmotic stress.

Key Findings

Pre-akinete formation and desiccation tolerance was investigated experimentally under monitored laboratory conditions in four strains of Arctic and Antarctic isolates with vegetative Zygnema sp. morphology. Phylogenetic analyses of rbcL sequences revealed one Arctic strain as genus Zygnemopsis, phylogenetically distant from the closely related Zygnema strains. Algae were cultivated in liquid or on solidified medium (9 weeks), supplemented with or lacking nitrogen. Nitrogen-free cultures (liquid as well as solidified) consisted of well-developed pre-akinetes after this period. Desiccation experiments were performed at three different drying rates (rapid: 10% relative humidity, slow: 86% rh and very slow); viability, effective quantum yield of PS II, visual and ultrastructural changes were monitored. Recovery and viability of pre-akinetes were clearly dependent on the drying rate: slower desiccation led to higher levels of survival. Pre-akinetes survived rapid drying after acclimation by very slow desiccation.

Conclusions

The formation of pre-akinetes in polar Zygnema spp. and Zygnemopsis sp. is induced by nitrogen limitation. Pre-akinetes, modified vegetative cells, rather than specialized stages of the life cycle, can be hardened by mild desiccation stress to survive rapid drying. Naturally hardened pre-akinetes play a key role in stress tolerance and dispersal under the extreme conditions of polar regions, where sexual reproduction and production of dormant stages is largely suppressed.     

Bayesian reconstruction of ancestral expression of the LEA gene families reveals propagule-derived desiccation tolerance in resurrection plants

Desiccation tolerance is a complex trait that is broadly but infrequently present throughout the evolutionary tree of life. Desiccation tolerance has played a significant role in land plant evolution, in both the vegetative and reproductive life history stages. In the land plants, the late embryogenesis abundant (LEA) gene families are involved in both abiotic stress tolerance and the development of reproductive propagules. They are also a major component of vegetative desiccation tolerance. Phylogenies were estimated for four families of LEA genes from Arabidopsis, Physcomitrella, and the desiccation tolerant plants Tortula ruralis, Craterostigma plantagineum, and Xerophyta humilis. Microarray expression data from Arabidopsis and a subset of the Physcomitrella LEAs were used to estimate ancestral expression patterns in the LEA families and to evaluate alternative hypotheses for the origins of vegetative desiccation tolerance in the flowering plants. The results contradict the idea that vegetative desiccation tolerance in the resurrection angiosperms Craterostigma and Xerophyta arose through the co-option of genes exclusively related to stress tolerance, and support the propagule-derived origin of vegetative desiccation tolerance in the resurrection plants.     

The isolation of genes from the resurrection grass Sporobolus stapfianus which are induced during severe drought stress

A modification of the ‘cold plaque’ screening technique (Hodge et al., Plant Journal1992, 2, 257–260) was used to screen a cDNA library constructed from drought‐stressed leaf tissue of the desiccation tolerant (‘resurrection’) grass Sporobolus stapfianus. This technique allowed a large number of clones representing genes expressed at low abundance to be isolated. An examination of expression profiles revealed that several of these genes are induced in desiccation‐tolerant tissue experiencing severe drought stress. Further characterization indicated that the gene products encoded include an eIF1 protein translation initiation factor and a glycine‐ and proline‐rich protein which have not previously been associated with drought stress. In addition, genes encoding a serine/threonine phosphatase type 2C, a tonoplast‐intrinsic protein (TIP) and an early light‐inducible protein (ELIP) were isolated. A number of these genes are expressed differentially in desiccation‐tolerant and desiccation‐sensitive tissues, suggesting that they may be associated with the desiccation tolerance response of S. stapfianus. The results indicate that there may be unique gene regulation processes occurring during induction of desiccation tolerance in resurrection plants which allow different drought‐responsive genes to be selectively expressed at successive levels of water loss.     

Novel clues on abiotic stress tolerance emerge from embryo proteome analyses of rice varieties with contrasting stress adaptation

Cereal embryos sustain severe water deficit at the final stage of seed maturation. The molecular mechanisms underlying the acquisition of desiccation tolerance in seed embryos are similar to those displayed during water deficit in vegetative tissues. The genetic variation among six rice genotypes adapted to diverse environmental conditions was analysed at the proteome level to get further clues on the mechanisms leading to water-stress tolerance. MS analysis allowed the identification of 28 proteins involved in stress tolerance (late embryogenesis abundant proteins), nutrient reservoir activity, among other proteins implicated in diverse cellular processes potentially related to the stress response (e.g., mitochondrial import translocase). Hierarchical clustering and multidimensional scaling analyses revealed a close relationship between the stress-sensitive genotypes, whereas the stress-tolerant varieties were more distantly related. Besides qualitative and significant quantitative changes in embryo proteins across the distinct varieties, we also found differences at post-translational level. The results indicated that late embryogenesis abundant Rab21 was more strongly phosphorylated in the embryos of the sensitive varieties than in the embryos of the tolerant ones. We propose that the differences found in the phosphorylation status of Rab21 are related to stress tolerance.     

Identification of two hydrophilins that contribute to the desiccation and freezing tolerance of yeast (Saccharomyces cerevisiae) cells

Hydrophilins are a group of proteins that are present in all organisms and that have been defined as being highly hydrophilic and rich in glycine. They are assumed to play important roles in cellular dehydration tolerance. There are 12 genes in the yeast Saccharomyces cerevisiae that encode hydrophilins and most of these genes are stress responsive. However, the functional role of yeast hydrophilins, especially in desiccation and freezing tolerance, is largely unknown. Here, we selected six candidate hydrophilins for further analysis. All six proteins were predicted to be intrinsically disordered, i.e. to have no stable structure in solution. The contribution of these proteins to the desiccation and freezing tolerance of yeast was investigated in the respective knock-out strains. Only the disruption of the genes YJL144W and YMR175W (SIP18) resulted in significantly reduced desiccation tolerance, while none of the strains was affected in its freezing tolerance under our experimental conditions. Complementation experiments showed that yeast cells overexpressing these two genes were both more desiccation and freezing tolerant, confirming the role of these two hydrophilins in yeast dehydration stress tolerance.