Anhydrobiosis in nematodes—I. The role of glycerol myo-inositol and trehalose during desiccation

• 1.1. The concentrations of free glycerol, inositol and trehalose in five species of nematodes were determined. Analyses of total inositol content were also made.
• 2.2. Significant differences in free and bound sugar levels were found between the two good anhydrobiotes Anguina tritici and Ditylenchus dipsaci and the three poor survivors Pangrellus redivivus, D. myceliophagous and Turbatrix aceti.
• 3.3. Highest trehalose contents were found in desiccated A. tritici and D. dipsaci, but glycerol levels were low.
• 4.4. P. redivivus and T. aceti contained high concentrations of free glycerol.
• 5.5. Desiccated A. tritici larvae contained more free and bound inositol than all other species studied, but desiccated D. dipsaci larvae had higher levels of bound inositol than P. redivivus, D. myceliophagous and T. aceti.
• 6.6. Dramatic reductions in inositol and trehalose contents were found in revived A. tritici larvae and freshly extracted D. dipsaci larvae. This was accompanied by an increase in glycerol content.
• 7.7. The results are discussed in relation to the possible biochemical adaptations employed by anhydrobiotes during desiccation.

     

Anhydrobiosis in Nematodes II: Carbohydrate and Lipid Analysis in Undesiccated and Desiccate Nematodes

Glycogen, trehalose, glucose, and total lipid contents of six nematode species were studied. Anhydrobiotic Anguina tritici and Ditylencbus dipsaci stored trehalose in preference to glycogen and only small amounts of glucose were detected. Glycogen content was also reduced in anhydrobiotic Aphelenchus avenae. Conversely, Panagrellus redivivus and Turbatrix aceti contained large amounts of glycogen, appreciable amounts of glucose, and minimal amounts of trehalose. Ditylenchus myceliophagous "curds" contained low amounts of glycogen and very little trehalose; total lipid was 60% of that in fresh samples. The lipid contents of fresh samples of P. redivivus, T. aceti, and A. avenae were high (23.1, 21.9, and 36.7% dry weight, respectively), but in anhydrobiotic A. avenae larvae the level was reduced by over 60%. In contrast, lipid levels remained high in anhydrobiotic A. tritici and D. dipsaci larvae (40.6 and 38.3%, respectively). Analysis of lipid composition in anhydrobiotic A. tritici and A. avenae did not indicate any specific metabolic adaptations to desiccation survival.     

DNA stability in the anabiotic fourth-stage juveniles of Ditylenchus dipsaci (Nematoda)

Water plays an important role in DNA structure and desiccation of microorganisms has been correlated with breaks in their DNA. The fourth-stage juveniles of the anabiotic nematode Ditylenchus dipsaci can lose up to 90% of their body water and survive in a dehydrated state for long periods of time; however, there was no increase in the frequency of breaks in DNA isolated from anabiotic as opposed to hydrated juveniles. Desiccation of the non-anabiotic nematode Panagrellus redivivus similarly did not lead to an increased frequency of DNA breaks, nor did the polyol glycerol significantly effect DNA stability. The stability of DNA during tissue desiccation, like the stability of proteins, seems to be a general feature of biological material and not a unique property of anabiotic organisms.     

A Comparative and Phylogenetic Study of the Ditylenchus dipsaci,Ditylenchus destructor and Ditylenchus gigas Populations Occurring in Poland

The genus Ditylenchus contains more than 80 recognized nematode species with a very wide host range. The most serious species are Ditylenchus dipsaci and Ditylenchus destructor. Populations of D. dipsaci species complex were collected from Allium cepa, Cichorium endivia and Phlox paniculata in Poland. The Ditylenchus gigas population was collected from Vicia faba minor, and populations of D. destructor, from Solanum tuberosum spp. tuberosum. Analyses of the rDNA sequences spanning both ITS1 and ITS2 fragment regions were carried out on the collected populations. The obtained DNA sequences were compared with those DNA sequences deposited in GenBank of populations isolated in other countries. Phylogenetic analysis was performed using the data obtained from the DNA sequence comparisons. The results indicated that there is no clear distinction between European and non‐European populations within D. dipsaci. The results also showed no clear distinction between populations isolated from different host plant species, including populations found in Poland. The populations of D. destructor described here constitute a common group together with American and Chinese populations belonging to the haplotype C of the D. destructor species. On the other hand, the D. gigas population was localized separately from those populations that have been described up until now, from Europe and Africa. This is also the first report on the occurrence of D. gigas in Poland.     

The effect of myo-inositol on the ability of Ditylenchus dipsaci,D. myceliophagus and Anguina tritici to survive desiccation

• 1.1. The effect of myo-inositol on the ability of three species of nematodes to survive desiccation has been studied.
• 2.2. Survival rates obtained from worms treated with an inositol bathing medium were compared with survival rates of worms treated with distilled or tapwater media.
• 3.3. Highest survival rates were found in those nematodes that were placed in an inositol solution prior to desiccation.
• 4.4. Tapwater facilitated higher revival rates than did distilled water in both D. dipsaci and D. myceliophagous.
• 5.5. No such differences were found for A. tritici.
• 6.6. The results are discussed in relation to the possible mechanisms of protection afforded by the different bathing media.

     

Cold tolerance of an Antarctic nematode that survives intracellular freezing: comparisons with other nematode species

Panagrolaimus davidi is an Antarctic nematode with very high levels of cold tolerance. Its survival was compared with that of some other nematodes (P. rigidus, Rhabditophanes sp., Steinernema carpocapsae, Panagrellus redivivus and Ditylenchus dipsaci) in both unacclimated samples and those acclimated at 5°C. Levels of recrystallization inhibition in homogenates were also compared, using the splat-cooling assay. The survival of P. davidi after the freezing of samples was notably higher than that of the other species tested, suggesting that its survival ability is atypical compared to other nematodes. In general, acclimation improved survival. Levels of recrystallization inhibition were not associated with survival but such a relationship may exist for those species that are freezing tolerant.     

The Influence of Environmental Factors on the Respiration of Plant-Parasitic Nematodes

Respiration of selected nematode species was measured relative to CO₂ level, temperature, osmotic pressure, humidity, glucose utilization and high ionic concentrations of sodium and potassium.In general, respiration was stimulated most by the dominant environmental factors at levels near those expected in the nematode''s "natural" habitat. Soil-inhabiting nematodes utilized O₂, most rapidly with high (1-2%) CO₂ whereas a foliar nematode (Aphelenchoides ritzemabosi) did so with 0.03% CO₂, the concentration typically found in air. Temperature optima for respiration corresponded closely to those for other activities. Ditylenchus dipsaci and Pratylenchus penetrans adults and Anguina tritici and A. agrostis second-stage larvae respired within the range of osmotic pressures from 0 to 44.8 arm and respiration of their drought-resistant stages was stimulated by increasing osmotic pressure which accompanies the onset of drought. Rehydration of A. tritici and A. agrostis larvae with RH as low as 5% stimulated measurable respiration. Glucose utilization from liquid medium by A. tritici larvae or A. ritzembosi was not detectable. Supplemental Na⁺ stimulated respiration of Anguina tritici, K⁺ did not.     

Effect of Acclimation Temperature on Infection of Alfalfa by Ditylenchus dipsaci

Ditylenchus dipsaci showed an affinity, in relation to infection, for the temp at which it had been acclimated. The optimum infective temp was also correlated with field temp when collections were made during different seasons and from climatically different geographical areas. Nematode developmental stage did not influence infectivity.     

Resistance and tolerance of potato varieties to potato rot nematode (Ditylenchus destructor) and stem nematode (Ditylenchus dipsaci)

Ditylenchus destructor and Ditylenchus dipsaci are economically important plant‐parasitic nematodes, affecting potato production mostly in temperate climates. Management through crop rotation is not feasible because of their wide host range. These nematodes are listed as quarantine pests in many countries. Limited information exists on the resistance and tolerance of currently cultivated potatoes to D. destructor and D. dipsaci. Two greenhouse experiments were conducted to screen 25 potato varieties for resistance to and tolerance for D. destructor and D. dipsaci infections. Reproduction factor (RF) and relative susceptibility (RS) were used to evaluate resistance, while potato tuber damage and tuber weight reduction was used to evaluate tolerance. Based on the RF, 16 varieties were evaluated as susceptible (S) while 5 varieties were evaluated as resistant (R) to D. destructor; varieties ‘Innovator’, ‘Aveka’ and ‘Spunta’ were resistant to D. dipsaci based on RF. ‘Désirée’ was observed to be highly susceptible to D. destructor and D. dipsaci in both experiments and was used as the standard susceptible control variety for the calculation of RS. A scale of 1–9 was used to classify RS of the potato varieties to D. destructor and D. dipsaci, where 9 indicated the highest level of resistance. All classes of resistance to D. destructor and D. dipsaci were observed in the potato varieties tested in the experiments. Six varieties had significantly lower RS to D. dipsaci than the standard susceptible control variety. Tolerant to highly sensitive potato varieties to both nematodes were also observed. RS and external potato tuber damage were identified as suitable methods for resistance and tolerance determination, respectively. This study provides essential information on the status of resistance and tolerance in potato varieties against D. destructor and D. dipsaci but needs to be confirmed under field conditions.     

Effect of desiccation on the photosynthesis of seaweeds from the intertidal zone in Honshu, Japan

Intertidal seaweeds are periodically exposed during low tide and thus experience extreme levels of desiccation. The physiological activity of seaweeds changes during this water loss process. This study examined how desiccation affects the photosynthesis and respiration of seaweeds from different intertidal levels, and whether the ability to retain photosynthesis and respiration rates during desiccation varies among these species. Photosynthesis and respiration rates of 12 species of seaweeds were measured under various levels of desiccation, using an infrared CO₂ gas analyzer. High levels of drought negatively affected photosynthesis, while most species showed initial rises in photosynthetic rates. The ability to retain photosynthesis and respiration activities under desiccation conditions varied among species. These physiological responses were not related to the intertidal level at which these species occur, but to their ability to prevent water loss. The species with lower rates of water loss had slower declines in the rate of photosynthesis and respiration.     

Control of Ditylenchus dipsaci in Infected Garlic Seed Cloves by Nonfumigant Nematicides

Different rates of granular formulations ofaldicarb, carbofuran, ethoprop, fensulfothion, and phenamiphos were applied directly onto garlic seed cloves in the seed furrow in sandy clay loam, clay loam, and loam soils at planting to assess efficacy for control of Ditylenchus dipsaci in infected seed cloves. All treatments were compared to hotwater-formalin clove dip disinfection treatment and to nontreated infected controls. Aldicarb and phenamiphos at 2.52 and 5.04 kg a.i./ ha, but not at lower rates, effectively suppressed infection by D. dipsaci and increased yields. Although both nematicides slightly slowed the rate of plant emergence, normal stands were established. Trace levels of infection occurred in all treatments, including the hotwater-formalin dip. Carbofuran at 5.04 kg a.i./ha controlled the nematode but was phytotoxic. Ethoprop was phytotoxic. Fensulfothion did not control D. dipsaci even at the highest application rate, 8.90 kg a.i./ha. Single and multiple applications of oxamyl at 1.12-8.96 kg a.i./ha, applied as a surface spray or in furrow irrigation water, slowed the early progression of disease symptoms but failed to provide season-long nematode control.     

Multiyear Population Dynamics of Ditylenchus dipsaci Associated with Phlox subulata

Field population densities of Ditylenchus dipsaci associated with shoot tissue of Phlox subulata were monitored during two consecutive growing seasons and intervening periods of overwintering and plant storage. The population density increased significantly through four peaks during the first growing season, and decreased significantly during storage at 5-7 C or overwintering in the field. During the second growing season, there was only a single increase to a moderate population density, followed by a severe population decline associated with the poor physiological condition of the host. A simple model is proposed to explain the population dynamics of D. dipsaci during the first growing season.     

Fine Structure Analyses of Stem Nematode-Induced White Flagging in Medicago sativa

White flagging of alfalfa, Medicago sativa ''Ranger, found associated with Ditylenchus dipsaci in the Columbia River Basin was observed in northern Utah during 1971. This is a report on chloroplast changes, induced by D. dipsaci in alfalfa leaves, as observed with an electron microscope. Leaves from alfalfa plants infected with D. dipsaci were either devoid of normal pigmentation or displayed various shades of yellow-green. Cells of leaf tissue from noninfected plants exhibited normal chloroplast structure. By contrast, the chloroplast structure in cells of leaf tissue from infected plants showed progressive degradation as normal pigmentation decreased.     

Freezing and Storing Ditylenchus dipsaci in Liquid Nitrogen

After 18 months of storage at -150 C, some larvae of Ditylenchus dipsaci, which had been treated in a 7.5% solution of dimethyl sulphoxide and cooled to -25 C before storage, were still viable on thawing. Some survivors penetrated and developed normally in stems of alfalfa seedlings. Tests showed that active larvae could be frozen directly, thus eliminating the need to use the quiescent stage of this nematode previously thought necessary for successful storage at cryogenic temperatures. The method described is suitable for long-term storage of D. dipsaci and may, with slight modifications, be used to preserve other plant-parasitic nematodes.     

Water balance and resistance to desiccation in rock-dwelling snails

We have examined the resistance to desiccation among rock-dwelling land snails of various phylogenetic groups:Cristataria genezarethana (Clausiliidae),Rupestrella rhodia (Chondrinidae) andLevantina caesareana (Helicidae), all from the same location in Israel.L. caesareana was the most resistant andR. rhodia the least resistant to desiccation andC. genezarethana was of intermediate resistance. Differences in the rates of water loss during desiccation were determined mainly by rate of water loss during the first 2 days of desiccation. The high rates of water loss in rock-dwelling species exceed those of other snails in the Mediterranean habitat of Israel. However, snails collected in the field at the end of aestivation were in only a mild state of dehydration, suggesting that the rocky habitat protects its occupants against desiccation. We also suggest that among the rock-dwelling species, the protective role of the rock is more important in the more evolutionarily primitive genera (the chondrinidRupestrella and the clausiliidCristataria) and that physiological capacities are more effective in the more highly evolved helicidLevantina.     

Ultrastructural changes during desiccation of the anhydrobiotic nematode Ditylenchus dipsaci

Ultrastructural changes during desiccation of the anhydrobiotic nematode Ditylenchus dipsaci were followed and quantified after preparation of material at different levels of hydration using freeze substitution techniques. Some shrinkage was caused by processing in the more hydrated specimens but the changes observed correspond to those observed in live nematodes by light microscopy, indicating that the technique is useful for following changes during desiccation. The overall pattern of changes was a rapid decrease in the magnitude of the measured parameter during the first 5 min of desiccation, followed by a slower rate of decrease upon further desiccation. This was observed in the cuticle, the lateral hypodermal cords and the muscle cells and is consistent with the pattern of water loss of the nematode. The contractile region of the muscle cells, however, proved an exception and the muscle fibres appear to resist shrinkage and packing until water loss becomes severe. The mitochondria swell and then shrink during desiccation, which may indicate disruption of the permeability of the mitochondrial membrane. A decrease in the thickness of the cortical zone was the most prominent change in the cuticle and this may be related to the permeability slump which occurs during the first 5 min of desiccation.     

Separation of Three Species of Ditylenchus and Some Host Races of D. dipsaci by Restriction Fragment Length Polymorphism

This study examined the ribosomal cistron of Ditylenchus destructor, D. myceliophagus and seven host races of D. dipsaci from different geographic locations. The three species showed restriction fragment length polymorphisms (RFLPs) in the ribosomal cistron, the 18S rDNA gene, and the ribosomal internal transcribed spacer (ITS). Southern blot analysis with a 7.5-kb ribosomal cistron probe differentiated the five host races of D. dipsaci examined. Polymerase chain reaction (PCR) amplification of the ITS, followed by digestion with some restriction endonucleases (but not others), produced restriction fragments diagnostic of the giant race. Because the PCR product from D. myceliophagus and the host races of D. dipsaci was about 900 base pairs and the ITS size in D. destructor populations was 1,200 base pairs, mixtures of populations could be detected by PCR amplification. ITS fragments differentiated between D. dipsaci and Aphelenchoides rhyntium in mixed populations. This study establishes the feasibility of differentiation of the host races of D. dipsaci by probing Southern blots with the whole ribosomal cistron.     

A Comparative Study of the Effects of Five Chemicals on the Survival of Chlamydospores of Fusarium oxysporum f. sp. narcissi

Formaldehyde is routinely used in hot water treatment for the control of narcissus basal rot (caused by Fusarium oxysporum f. sp. narcissi) and stem and bulb eelworm (Ditylenchus dipsaci). It does not kill all the thick walled chlamydospores of F. oxysporum. Four chemicals were evaluated for their efficacy against F. oxysporum by comparing them with the standard formaldehyde rate. At rates of 0.25% a.i. or more, a glutaraldehyde formulation (Cidex) killed 100% of spores within 160 min. Peratol, a hydrogen peroxide and peracetic acid containing formulation, gave 100% kill after 80 min at a concentration of 0.5%. A thiabendazole formulation (Storite Clear Liquid) killed all the chlamydospores. Decon 90 failed to control the fungus adequately. Formaldehyde (as 0.5% commercial formalin) gave good, but not total, control of the fungus but, at higher rates (2.5%), caused flower malformation and corkiness of bulb base plates. The results suggest that Cidex and Peratol, as they are more effective than formaldehyde in controlling the fungus and less unpleasant to use, may be suitable replacements.     

Divergent mechanisms for water conservation in Drosophila species

The role of melanization and cuticular lipids in water conservation has been studied in many Drosophila species (Diptera: Drosophilidae). Nevertheless, a comparative approach to larval and adult stages of ecologically diverse, wild Drosophila species is still required. Based upon abdominal cuticular melanization patterns, wild‐caught Drosophila species were categorized as (1) melanic, (2) fixed‐melanic, or (3) non‐melanic. At the interspecific level, the ecological significance of melanization and cuticular lipids was determined by the inverse association of melanization and cuticular water loss in melanic species, and of cuticular lipids and cuticular water loss in fixed‐melanic and non‐melanic species. Interestingly, higher amounts of cuticular lipids were also evident in fixed as well as non‐melanic species, as compared to melanic species at larval stages, which is consistent with their differences in reduced water loss rates. Moreover, fixed‐melanic and non‐melanic species exhibited comparatively higher (ca. 1.8–2.0 fold) desiccation resistance. Thus, cuticular lipids provide a better waterproofing mechanism than melanization. Furthermore, acclimation to dehydration stress in adults improved desiccation resistance in melanic species, whereas such effects were lacking in fixed‐melanic and non‐melanic species. However, there were no changes in cuticular components as a consequence of desiccation acclimation. Thus, our results indicate that melanic, fixed‐melanic, and non‐melanic Drosophila species differ in the evolved physiological mechanisms of water conservation to adapt to dry conditions.