Application of Impedance Spectroscopy for Selecting Frost Hardy Varieties of English Ryegrass

The frost hardening and frost damage of 12 varieties of Englishryegrass (Lolium perenne) was studied by electrical impedancespectroscopy. For the measurement of the impedance spectrum(80 Hz to 1 MHz) a 10 mm length sample was cut from the stemabove the growing point, but the growing point was included.The impedance spectra were analysed by an asymmetric distributedcircuit model. The impedance spectra were measured at two phasesof hardiness and after freezing, i.e. (a) before hardening,(b) after hardening in controlled conditions, and (c) aftercontrolled frost exposure at -16 °C after hardening. Twomodel parameters, i.e. intra- and extracellular resistance,increased with hardening. The intracellular resistance and theskewness factor before hardening, and the ratio between thosetwo parameters before and after hardening, strongly correlatedwith hardening of different varieties of English ryegrass. Theextracellular resistance and the relaxation time decreased asa result of the frost exposure at -16 °C. Cold acclimation; electrical impedance; English ryegrass; frost hardiness; impedance spectroscopy; Lolium perenne     

Relationship between quantum efficiency of PSII and cold-induced plant resistance to fungal pathogens

The aim of the presented work was to study whether the efficiency of photosynthesis may influence resistance of hardened plants to disease. Seedlings of spring barley, meadow fescue and winter oilseed rape were chilled at 5 °C for 2, 4 or 6 weeks and at these deadlines the changes in cell membrane permeability (expressed as electrolyte leakage), chlorophyll fluorescence (initial fluorescence - F₀, maximal fluorescence - F_m, quantum yield of PSII - F_v/F_m) and net photosynthesis rate (F_N) were measured. Also, the influence of cold on the degree of plant resistance to economically important pathogens -Bipolaris sorokiniana or Phoma lingam was estimated. Two, four or six week-hardened plants were artificially infected: barley and fescue by B. sorokiniana, and oilseed rape by P. lingam. Hardening at 5 °C stimulated resistance of barley, fecue and rape to their specific pathogens. Six-week long acclimation was the most effective for plant resistance. Cold significantly changed cell membrane permeability and decreased chlorophyll fluorescence (F₀, F_m and F_v/F_m) of all studied plant species, while net photosynthesis rate was found to decrease only in barley. The results indicate that cold-induced resistance of plants to pathogens was correlated with a decrease in cell membrane permeability. In the case of fescue and barley a significant connection between the quantum yield of PSII and their resistance to B. sorokiniana was shown. Additionally, the resistance of barley to fungus was depended on net photosynthesis rate. In general this research shows that the efficiency of photosynthesis may be used as an indicator of plant resistance to disease.     

The intensity of respiration and heat emission from seedlings of Festuca pratensis (Hud.) and Hordeum vulgare L. during pathogenesis caused by Bipolaris sorokiniana (Sacc.) Shoem

The presented work was conducted on seedlings of spring barley and meadow fescue which differ in the degree of sensitivity to leaf spot pathogen Bipolaris sorokiniana (Sacc.) Shoem. The seedling reaction to inoculation with mycelium and conidia was examined in glasshouse conditions on the basis of respiration intensity and heat production. The leaf respiration was measured using Clark-type electrode, while heat emission was evaluated by means of isotermic microcalorimeter. The measurements were performed after 1, 3, 6, 10, 24, 48, 72, 168 and 240 hours since the inoculation moment. Leaves of meadow fescue were characterized by the most intense respiration at the 6^th hour, while barley leaves at the 24^th and 72^nd hour after inoculation. In the case of meadow fescue the greatest heat production was noted in the period between 24 and 168 hours after inoculation. Simultaneously, at the 48^th hour the smallest rate of respiration was observed. Barley leaves emitted the greatest amount of heat only in the first 3 hours of the pathogenesis. In these hours the smallest respiration rate was noted. The observed, opposing reaction of respiration intensity and heat emission in the infected seedlings of both species may illustrate a disorder in metabolic processes in plants during pathogenesis. The plants studied differed in the time of their reaction to pathogen attack: barley responded earlier in heat production, while fescue extended respiration rate in the first hours after inoculation. This is clearly observable, when coefficients of metabolic inefficiency (heat rates per mole O₂) are compared. In the case of barley the highest rates were noticed just after inoculation, whereas in fescue at the 48^th hour. In both species attack of pathogen caused high metabolic efficiency.     

Changes in soluble carbohydrate level in Hordeum vulgare (L.) and Festuca pratensis (Huds.) calli treated with metabolites of Bipolaris sorokiniana (Sacc.) Shoem

Calli of spring barley (Hordeum vulgare L.) and meadow fescue (Festuca pratensis Huds.) were treated with metabolites of Bipolaris sorokiniana and then the level of soluble carbohydrates was estimated. Fructose and glucose occurred in the greatest amount in non-treated calli (control). Control tissue of both species responded to a change in culture conditions with fluctuation in the sugar level. Calli treated with fungal phytotoxins demonstrated rapid decrease in sugar content 1, 3 and 24 hrs after elicitation. Fescue calli, as less susceptible, showed moderate increase in carbohydrate level, yet it was still significantly lower than that in control. In barley very small amount of carbohydrates was observed as soon as 24 hrs after elicitation. In the elicited tissue of both species rapid increase in soluble carbohydrate level was noted in the 10^th hour. It is suggested that a defence response of barley and fescue takes place in two phases. The 1^st phase occurred between the 1^st and the 10^th hour after elicitation with phytotoxins and it seems to be an adaptation time to this stress factor. This stage is typical for both studied species. The 2^nd phase was observed after 10 hrs of pathogenesis. Its course may reflect a various sensitivity degree of both species to B. sorokiniana metabolites.     

Impedance Spectroscopy in Frost Hardiness Evaluation of Rhododendron Leaves

Impedance spectroscopy was used in studying frost hardinessof leaves of two diploid rhododendron cultivars, RhododendronL. ‘PJM’ and R. ‘Cunningham's White’,and their tetraploid derivatives, R. ‘Northern Starburst’(NSB) and CW4. After the growing season and initial hardeningin a greenhouse, plants were subjected to an acclimation regimein a phytotron: 3 consecutive weeks at +5, +1 and -2°C each.Hardiness was studied with controlled freezing tests beforeeach decrease in temperature and at the end of the experiment,based on data of extracellular resistance r_eand relaxation time of the frost-exposed leaves. The correlation of the two estimateswas 0.92. Generally, the diploid clones had better frost hardinessthan the tetraploid clones. At the end of the experiment, frosthardiness of the diploid ‘PJM’ was -28.7°C andthat of the tetraploid NSB -20.6°C. Leaves of the diploid‘Cunningham's White’ and of the tetraploid CW4 hardenedto -32.0°C and -20.9°C, respectively. Frost hardinessestimated by impedance spectroscopy correlated well with earlierresults based on visual scoring (r = 0.81–0.86) and electrolyteleakage tests (r = 0.84–0.90), but results from impedancespectroscopy indicated weaker hardiness than the other tests.The difference between the results from impedance spectroscopyand the other tests was smaller and more coherent within the‘Cunningham's White’ clones than within ‘PJM’and NSB. Changes in extracellular and intracellular resistanceof non-frozen leaves during the acclimation correlated withthe changes in frost hardiness of ‘Cunningham's White’clones, but not with those of ‘PJM’ and NSB, whichbelong to another subspecies.Copyright 2000 Annals of BotanyCompany Cold resistance, evergreen, frost hardiness, impedance spectroscopy, polyploid, Rhododendron, tetraploid     

Cold acclimation of forage grasses in relation to pink snow mould (Microdochium nivale) resistance

The aim of our work is to investigate the changes in phenolic level, PAL activity and heat production rate induced during pre-hardening at 12°C and cold acclimation at 2°C of the forage grasses Festulolium, meadow fescue, tall fescue and Italian ryegrass in relation to their resistance to snow mould caused by Microdochium nivale. Meadow fescue and tall fescue were most resistant to M. nivale infection, while Italian ryegrass demonstrated the least resistance to this fungus inoculation. Festulolium, meadow fescue and tall fescue responded similarly to low temperature, while Italian ryegrass demonstrated considerable disturbance of energy balance and lower phenolic concentration, which could explain a higher susceptibility of the latter species to infection by M. nivale. The enhanced level of phenolic compounds, probably utilised for cell wall lignification as well as equilibrium of the metabolic activity observed in meadow fescue and tall fescue, is very important for both cold and pathogen-resistance mechanisms. The studied Festulolium cultivar ‘Felopa’, a hybrid of the Lolium multiflorum and Festuca pratensis genomes, was characterised by changes in biochemical parameters similar to the resistant meadow fescue and tall fescue.     

Freezing Tolerance and Solute Changes in Contrasting Genotypes of Lolium perenne L. Acclimated to Cold and Drought

Clonal ramets of 12 contrasting genotypes of Lolium perenneL. were grown in sand or soil-based compost and maintained underwell-watered conditions at 20/15°C or acclimated to lowtemperature (2°C) or to a restricted water supply. Freezingtolerance was measured as LT₅₀ following exposure to sub-zerotemperatures in a freezing tank. Measurements were also madeof osmotic potential, water-soluble carbohydrates, free proline,free amino acids, and minerals in entire tillers. Acclimationto both drought and cold lowered LT₅₀, induced osmotic adjustment,and increased concentrations of proline and amino acids, Rootingmedium had little effect on LT₅₀, but caused large differencesin osmotic potential and in proline and amino-acid concentrations.There was considerable genetic diversity for all charactersmeasured, except for mineral contents. There was, however, norelationship between LT₅₀ and osmotic potential or solute contentthat was consistent across the three sources of variation (growingmedium, acclimation, genotype). Furthermore, the diverse genotypicvalues of cold-induced freezing tolerance were not correlatedwith those of drought-induced tolerance. It is concluded thatmore precise measurements are needed of the partitioning ofsolutes during acclimation and of the sensitivity of differentorgans and tissues to freezing.Copyright 1993, 1999 AcademicPress Perennial ryegrass, hardening, acclimation, osmotic potential, solute potential, carbohydrates, proline     

Regulation of frost resistance during cold de-acclimation and re-acclimation in oilseed rape. A possible role of PSII redox state

A possible role of photosynthetic apparatus during cold de-acclimation was studied in oilseed rape ( Brassica napus var. oleifera ). Plants of spring (Star) and winter (Górczañski) cultivars were cold acclimated at + 5°C, and de-acclimated during 4 weeks at combinations of + 12 and + 20°C operating in the light or/and dark, with a 12-h photoperiod. Evidence is presented that the photosynthetic apparatus may be involved in temperature perception during de-acclimation. De-acclimation was faster under a 20/12°C (day/night) treatment than under the reverse 12/20°C (day/night). De-acclimation rate was constant when the day temperature was constant, irrespective of the night temperature both under cold day temperature regimes (12/20, 12/12°C (day/night) and warm-day treatments (20/12, 20/20°C (day/night). The fast decrease in frost resistance observed under warm-day de-acclimation was always accompanied by an acceleration of elongation growth. In the spring cultivar, elongation growth increased starting from the second week of de-acclimation, regardless of temperature conditions. Once elongation growth had commenced during de-acclimation, it continued throughout the period necessary for re-acclimation to low temperature. Re-acclimation to the initial freezing tolerance level was only possible when plant elongation was reduced. In addition re-acclimation of the photosynthetic apparatus to low temperature was impossible in fast growing plants. A possible relationship between PSII, growth rate and frost resistance during cold acclimation and de-acclimation is discussed.     

Effects of Hormones on Morphogenesis and Cold Resistance in Berseem Clover (Trifolium alexandrinum L.)

Plants of berseem clover (Trifolium alexandrinum L.) cv. Taborwere raised under conditions inhibiting the acquisition of coldhardiness (non-hardened) or inducing cold hardiness (hardened).All non-hardened plants developed an elongated shoot and exhibitedconsiderable frost sensitivity, as measured by the extent ofthe reduction in yield of variable chlorophyll fluorescenceafter exposure to sub-zero temperature. Hardened plants developeda shorter shoot, with fewer leaves and a greater percentageof dry matter in the root system. These parameters were associatedwith a marked increase in frost resistance. Exogenous applicationof ABA to plants effected similar morphological modificationsin both hardening and non-hardening temperature regimes; plantsdeveloped a shorter primary shoot axis and leaves exhibiteda marked increase in frost hardiness. In berseem clover ABAcan thus substitute, at least partially, for the low temperaturetreatment required to induce cold hardiness. Spraying plantsraised under hardening conditions with gibberellic acid reversedthe effects of the hardening treatment, since they developedan elongated shoot and exhibited frost sensitivity comparableto non-treated plants grown under non-hardening conditions.It is concluded that these endogenous hormones are directlyinvolved in triggering changes in morphogenesis which accompanyphysiological and metabolic events associated with the inductionof plant cold hardiness. Key words: Frost resistance, morphogenesis, abscisic acid, giberellic acid, Trifolium alexandrinum     

Increased Levels of Cytokinins in Barley Leaves Having the Systemic Acquired Resistance to Bipolaris sorokiniana (Sacc.) Shoemaker

The role of endogenously induced higher level of cytokinins and exogenously applied kinetin in relation to the development of barley leaf spot caused by Bipolaris sorokiniana (Sacc.) Shoemaker (syn. Helminthosporium sativum Pammel, King and Bakke) was studied. Spraying barley leaves with kinetin suppressed the number and the size of necrotic spots caused by the fungus. Inoculation of the lower leaves of barley by a spore suspension of the fungus B. sorokiniana induced resistance on the upper leaves against a subsequent challenge inoculation by the same pathogen 10 days later. An increase in the level of cytokinins was observed in these resistant leaves. Elevated levels of cytokinins may cause a type of juvenility in leaf tissues. The juvenile state could be in a causal relationship with the suppression of necrotic spots caused by the fungus.     

Leaf cold acclimation and freezing injury in C3 and C4 grasses of the Mongolian Plateau

The scarcity of C4 plants in cool climates is usually attributed to their lower photosynthetic efficiency than C3 species at low temperatures. However, a lower freezing resistance may also decrease the competitive advantage of C4 plants by reducing canopy duration, especially in continental steppe grasslands, where a short, hot growing season is bracketed by frost events. This paper reports an experimental test of the hypothesis that cold acclimation is negligible in C4 grasses, leading to greater frost damage than in C3 species. The experiments exposed six C3 and three C4 Mongolian steppe grasses to 20 d chilling or control pre-treatments, followed by a high-light freezing event. Leaf resistance to freezing injury was independent of photosynthetic type. Three C3 species showed constitutive freezing resistance characterized by <20% leaf mortality, associated with high photosynthetic carbon fixation and electron transport rates and low leaf osmotic potential. One freezing-sensitive C4 species showed the expected pattern of chilling-induced damage to photosynthesis and >95% leaf mortality after the freezing event. However, three C3 and two C4 species displayed a cold acclimation response, showing significant decreases in osmotic potential and photosynthesis after exposure to chilling, and a 30-72% reduction of leaf freezing injury. This result suggested that down-regulation of osmotic potential may be involved in the cold acclimation process, and demonstrated that there is no inherent barrier to the development of cold acclimation in C4 species from this ecosystem. Cold acclimation via osmoregulation represents a previously undescribed mechanism to explain the persistence of C4 plants in cool climates.     

Phenylalanine Ammonia-lyase Activity in Barley After Infection with Bipolaris sorokiniana or Treatment with its Purified Xylanase

Phenylalanine ammonia-lyase (PAL) activity was determined from leaves and roots of two barley (Hordeum vulgare L.) cultivars after infection with a necrotrophic pathogen, Bipolaris sorokiniana (Sacc.) Shoem., and treatment with its purified xylanase. PAL activity increased in leaves of both cultivars 16 h after fungal inoculation but two phases, with activity peaks at 24–32 h and 40 h, were recorded only for the more resistant cultivar, Agneta. Attempts to use a PAL inhibitor, χ-amin, ooxyacetic acid, to increase susceptibility to B. sorokiniana in barley leaves were unsuccessful. Treatments of leaves with purified xylanase resulted in more rapid (4–12 h after injection), although reduced, induction of PAL compared with fungal injection. The higher the concentration of xylanase applied the earlier the activity peaks were detected. Fungal inoculation only slightly increased PAL activity in barley roots while xylanase treatment had no effect. The basal level of PAL was however much higher in roots than in leaves. In wheat, Triticum aestivum L. resistant to B. sorokiniana, the time-course of PAL induction after fungal infection and xylanase treatment resembled that for cv. Agneta, while in oats, Avena sativa L. (non-host) PAL activity did not change after the treatments. The results suggest that the second phase of PAL induction, associated only with responses of barley cv. Agneta and wheat, is linked with their resistance to B. sorokiniana infection. The possible role of xylanase as an elicitor of PAL is discussed.     

Short-day and Low-temperature Control of Floral Induction in Festuca

The conditions necessary for floral induction to occur in tallfescue (Festuca arundinacea), meadow fescue (Festuca pratensis),and red fescue (Festuca rubra), have been investigated. Onlya Tunisian ecotype of tall fescue produced inflorescences undershort-day conditions when air temperatures were above 8 °C.Under short days with low temperatures nearly all plants ofS. 170 tall fescue and S. 215 meadow fescue produced inflorescencesafter 15 weeks' exposure, but S. 59 red fescue showed only asmall response. Evidence was obtained for the existence in bothtall fescue and meadow fescue of a juvenile stage during whichplants showed a reduced response to inductive conditions. Avariation of 35 days in the required length of exposure to inductiveconditions was demonstrated between families within the S. 170variety of tall fescue, indicating the possibility of selectingfor larger or smaller inductive requirements. A second generationof seed was produced within a 12-month period from inflorescenceswhich had developed in a heated glasshouse during the wintermonths.     

Relationship between soluble carbohydrate level and tolerance of meadow fescue callus to Bipolaris sorokiniana (Sacc.) Shoem. and Drechslera dictyoides (Drechsl.) Shoem. metabolites

The aim of the presented work was the search for the relationship between the level of soluble carbohydrates in callus tissues of eight meadow fescue (Festuca pratensis Huds.) cultivars and their growth ability on media containing Bipolaris sorokiniana and Drechslera dictyoides metabolites. Calli were induced from mature grains using the method previously described (Płażek 1994). Callus obtained from single caryopsis was cut into three pieces which were weighted and put on the media with or without pathogen metabolites. Tissue selection was performed by means of “double-layer culture” technique (Lepoivre et al. 1986). After two-week culture in the darkness at temp. of 25°C the calli were weighted again. The sugar level in tissue was measured by means of colorimetric method of Klein & Weissman according to Snell (1961). Fresh mass decrease of calli developing on the media with fungus metabolites was observed by all studied object. The tolerance of calli of the tested cultivars to metabolites of both pathogens was significantly different. However, significant similarity between the tolerance of calli of particular varietes to both fungi was noted. The soluble carbohydrate contents in control tissue of all studied cultivars were similar and their values ranged between 2.4 and 3 % of fresh mass. B. sorokiniana metabolites caused a significant decrease of the sugar content in calli, while D. dictyoides metabolites did not decrease the sugar level.     

Influence of Seed‐borne Cochliobolus sativus (Anamorph Bipolaris sorokiniana) on Crown Rot and Root Rot of Barley and Wheat

The effect of seed‐borne pathogens of wheat and barley on crown and root rot diseases of seven barley cultivars (Jimah‐6, Jimah‐51, Jimah‐54, Jimah‐58, Omani, Beecher and Duraqi) and three wheat cultivars (Cooley, Maissani and Shawarir) was investigated. Bipolaris sorokiniana and Alternaria alternata were detected in seeds of at least eight cultivars, but Fusarium species in seeds of only two barley cultivars (Jimah‐54 and Jimah‐58). Crown rot and root rot symptoms developed on barley and wheat cultivars following germination of infected seeds in sterilized growing media. Bipolaris sorokiniana was the only pathogen consistently isolated from crowns and roots of the emerging seedlings. In addition, crown rot and root rot diseases of non‐inoculated barley cultivars correlated significantly with B. sorokiniana inoculum in seeds (P = 0.0019), but not with Fusarium or Alternaria (P > 0.05). These results indicate the role of seed‐borne inoculum of B. sorokiniana in development of crown rot and root rot diseases. Pathogenicity tests of B. sorokiniana isolates confirmed its role in inducing crown rot and root rot, with two wheat cultivars being more resistant to crown and root rots than most barley cultivars (P < 0.05). Barley cultivars also exhibited significant differences in resistance to crown rot (P < 0.05). In addition, black point disease symptoms were observed on seeds of three barley cultivars and were found to significantly affect seed germination and growth of some of these cultivars. This study confirms the role of seed‐borne inoculum of B. sorokiniana in crown and root rots of wheat and barley and is the first report in Oman of the association of B. sorokiniana with black point disease of barley.     

Metabolic changes associated with cold-acclimation in contrasting cultivars of barley

Cereal plants become more resistant to freezing when first exposed to a period of cold-acclimation. Many physiological and molecular changes have been shown to occur at low temperatures, but the role and the contribution of each to frost resistance is still poorly understood. Two cultivars of barley ( Hordeum vulgare L.), the winter barley Onice and the spring barley Gitane, were acclimated under controlled conditions under an 8-h photoperiod at 4°C (light) and 2°C (dark) for 21 days. Changes in free proline, ABA, water-soluble carbohydrates and free fatty acids were measured to assess their involvement in cold-acclimation and to explain the different frost-resistant capacities of the two cultivars. Exposure of barley plants to low temperature resulted in an equal increase in proline in both cultivars. During the first days of cold acclimation, ABA levels showed a peak in the frost-resistant cultivar, lasting about 24 h, followed by a decrease. The water soluble carbohydrates reached their highest content after 3 days of hardening, although after 14 to 21 days of acclimation the carbohydrate content was similar to that of unhardened plants. The frost-resistant Onice had a much higher free fatty acid content than the frost-sensitive Gitane. Furthermore in Onice 86% of free farty acids was represented by unsaturated molecular species. Inolenic acid alone being 71%. In contrast, in the frost-sensitive cultivar only 31% of free fatty acids was unsaturated and a large amount of 9-oxo-nonanoic acid, a product present in the linolenic acid cascade, was also detected.
The ABA content after 2 days of hardening and the free fatty acid composition were clearly different between the two cultivars and may explain, at least in part, the different frost-resistant capacities of Onice and Gitane.     

A Second-order Dynamic Model for the Frost Hardiness of Trees

The development of frost hardiness in forest trees is describedby a dynamic model in which the input variables are the prevailingenvironmental conditions and the developmental stage of trees.The assumption of the model is that for each temperature andphotoperiod there is a discrete stationary level of frost hardiness,which is attained if these environmental factors remain constant.The dependence of the stationary level on temperature and photoperiodis assumed to be piece-wise linear and additive. The rate ofacclimation, i.e. frost hardening or dehardening, is describedas a second-order dynamic process with two time constants, thesecond of which changes depending on the stage of the annualdevelopment of the trees. The frost hardiness model was calibratedand tested using experimental data from Douglas fir [Pseudotsugamenziesii var. glauca (Beissn.) Franco] seedlings. The resultssuggest that the second-order model describes the changes infrost hardiness better than the first-order model with onlyone time constant.Copyright 1995, 1999 Academic Press Acclimation, developmental stage, Douglas fir, dynamic model, frost hardiness, photoperiod, Pseudotsuga menziesii, temperature     

Osmotic Adjustment and the Development of Freezing Resistance in Fragaria virginiana

Cold temperature acclimation in strawberry (Fragaria virginiana) leaves apparently involves the alteration of cellular osmotic properties. Alterations in leaf osmotic potential were closely correlated with alterations in soluble carbohydrate content of the leaf tissue and changing temperatures. Leaf starch content was inversely related to soluble carbohydrate levels, suggesting that starch is a partial source of osmoticum during osmotic adjustment associated with cold temperature stress. Free amino acid changes were more closely linked to senescence and growth processes while changes in ion content suggested a rapid mobilization of solutes at the onset of freezing temperatures. This was supported by changes in whole plant gradients in leaf osmotic potential before and after exposure to freezing temperatures. In terms of freezing resistance and the role of osmotic adjustment in the development of resistance, it was found that of all leaves undergoing osmotic adjustment only the younger leaves survived, suggesting an age-dependent component to freezing resistance in leaves. Freezing resistance appears to involve alterations in several cellular properties that act in concert to confer a hardy state of the tissue. Although osmotic adjustment may be an important component of the final combination of cellular properties, this study indicates that solute accumulation does not function alone to confer freezing resistance.