Cold Hardening of Meloidogyne hapla Second-stage Juveniles

The effect of previous exposure to low temperatures on freezing tolerance was determined for second-stage juveniles of Meloidogyne hapla. Juveniles in 5% polyethylene glycol 20,000 were exposed to 0-24 C for 12-96 hours, and then freezing tolerance was assessed by freezing samples at -4 C for 24 hours, thawing, and determining survival. Freezing tolerance was inversely related to prefreeze temperatures of 4-24 C. Prefreeze exposure to 4 C resulted in fourfold greater freezing tolerance than did exposure to 24 C. Mortality occurred during prefreeze exposure to 0 C. Most of the increase in freezing tolerance at 4 C occurred during the first 12 hours. In soil, prefreeze exposure to 4 C resulted in greater freezing tolerance than did prefreeze exposure to 24 C.     

Overwintering and Cold Tolerance of the Mermithid Filipjevimermis leipsandra

Overwintering survival of Filipjevimermis leipsandra postparasitic adults ranged from 12 to 15% over 2 years. Nematodes recovered from biweekly samples oviposited within a week when exposed to warm temperatures. More nematodes were recovered from the lower portions of soil cores than from the upper. In laboratory tests of cold tolerance, the LD₅₀ at -5 C was 5.5 hours, whereas exposure to -2 C for as long as 96 hours resulted in no mortality.     

The Potential of Thiarubrine C as a Nematicidal Agent against Plant- parasitic Nematodes

Thiarubrine C, a polyacetylenic 1,2-dithiin isolated from the roots of Rudbeckia hirta (Asteraceae), exhibited strong nematicidal activity in in vitro and growth chamber assays. Thiarubrine C was toxic, in the absence of light, to the plant-parasitic nematodes Meloidogyne incognita and Pratylenchus penetrans at LC₅₀s of 12.4 ppm and 23.5 ppm, respectively. A minimum exposure time between 12 and 24 hours was the critical period for nematode mortality due to thiarubrine C. Although thiarubrine C was not totally dependent on light for toxicity, activity was enhanced in the presence of light, especially with the microbivorous nematode, Teratorhabditis dentifera. Upon exposure of M. incognita juveniles to 20 ppm thiarubrine C for 1 hour, infection of tomato plants was greatly reduced compared to untreated checks. Thiarubrine C was also effective in reducing plant infection when mixed with soil 24 hours prior to or at planting, unlike other related compounds such as δ-terthienyl.     

Cryopreservation of the Pinewood Nematode,Bursaphelenchus spp.

Populations of three isolates of Bursaphelenchus xylophilus, the pinewood nematode, and one of B. mucronatus were treated with three cryoprotectants at -70 C for 24 hours followed by deep freezing at -180 C in liquid nitrogen for different periods of time. A solution of 15% glycerol, 35% buffer S, and 50% M9, or 1% aqueous solution of dimethylsulfoxide (DMSO), or a mixture of 60% M9 and 40% S buffer were used as cryoprotectants. A significantly larger number of juveniles than adults survived deep freezing. Significantly more nematodes were motile after cryopreservation in the 15% glycerol-S-M9 soludon than in the M9-S buffer solution or the DMSO aqueous solution. When cryopreserved nematodes that had been treated with glycerol solution were plated onto Botrytis cinerea, they reproduced rapidly over several generations. Cryopreserved nematodes were as pathogenic as untreated nematodes to Scots pines.     

Effect of an Ice-Nucleating Activity Agent on Subzero Survival of Nematode Juveniles

Juveniles of five species of nematodes, Caenorhabditis elegans, Panagrellus redivivus, Pratylenchus agilis, Pristionchus pacificus, and Distolabrellus veechi, were added to solutions with (treatment) and without (control) a commercial ice-nucleating activity (INA) agent. Ten-microliter droplets of the solutions containing the juveniles were placed on glass microscope slides and transferred to a temperaturecontrolled freeze plate where the temperature was reduced to -6 to -8 °C. At this temperature, the droplets containing the INA agent froze while those without the agent remained liquid. After 2 minutes, the temperature of the plate was raised to 24 °C, and the slides were examined with a light microscope to determine the viability of the juveniles. The results showed that usually most juveniles (43% to 88%, depending on species) in solutions that did not contain the INA agent (controls) were active, indicating that the juveniles were capable of supercooling and were thereby protected from the subzero temperatures. Alternatively, less than 10% of the juveniles that had frozen for 2 minutes in solutions containing the INA agent remained viable, indicating that inoculative freezing of the solution was lethal to the supercooled juveniles. Our results suggest that, in geographical areas where winter temperatures may not be sufficiently low or sustained to freeze soil, the addition of an INA agent may help induce ice nucleation and thereby reduce the populations of nematode species that are unable to survive when the soil solution is frozen.     

Infectivity,Development, and Reproduction of Heterodera cajani on Pigeonpea: Influence of Soil Moisture and Temperature

The effect of soil moisture on penetration, development, and reproduction of Heterodera cajani on pigeonpea (cv. ICPL 87) was investigated in growth chambers held at 20 and 25 C, and in a greenhouse where temperature fluctuated between 25 and 32 C. Averaged across temperatures, the percentage of juveniles that penetrated roots was 34.3, 31.8, 8.8, and 3.7% at 24, 32, 16, and 40% soil moisture levels, respectively. Numbers of females per root system 4 weeks after infesting soil with second-stage juveniles was 79.6 at 24%, 65.3 at 32%, 26.1 at 16%, and 2.9 at 40% soil moisture. Nematode reproduction was greatest (P = 0.001) at 24% soil moisture and 25 C. Reproductive factor was 19.4 at 24%, 15.2 at 32%, 5.7 at 16%, and 0.5 at 40% soil moisture level. Nematode penetration, development, and reproduction at different moisture levels were greater (P = 0.01) at 25 and 25-32 C than at 20 C. Plant growth was retarded at 40% soil moisture and 20 C in comparison to that at 24 and 32% moisture levels and 25 C. This information on influence of temperature and soil moisture will be helpful in developing models for predicting changes in H. cajani densities in pigeonpea fields during rainy and postrainy dry seasons in the semi-arid tropics.     

Freezing and Desiccation Tolerance in Entomopathogenic Nematodes: Diversity and Correlation of Traits

The ability of entomopathogenic nematodes to tolerate environmental stress such as desiccating or freezing conditions, can contribute significantly to biocontrol efficacy. Thus, in selecting which nematode to use in a particular biocontrol program, it is important to be able to predict which strain or species to use in target areas where environmental stress is expected. Our objectives were to (i) compare inter- and intraspecific variation in freeze and desiccation tolerance among a broad array of entomopathogenic nematodes, and (ii) determine if freeze and desiccation tolerance are correlated. In laboratory studies we compared nematodes at two levels of relative humidity (RH) (97% and 85%) and exposure periods (24 and 48 h), and nematodes were exposed to freezing temperatures (-2°C) for 6 or 24 h. To assess interspecific variation, we compared ten species including seven that are of current or recent commercial interest: Heterorhabditis bacteriophora (VS), H. floridensis, H. georgiana, (Kesha), H. indica (HOM1), H. megidis (UK211), Steinernema carpocapsae (All), S. feltiae (SN), S. glaseri (VS), S. rarum (17C&E), and S. riobrave (355). To assess intraspecific variation we compared five strains of H. bacteriophora (Baine, Fl1-1, Hb, Oswego, and VS) and four strains of S. carpocapsae (All, Cxrd, DD136, and Sal), and S. riobrave (355, 38b, 7-12, and TP). S. carpocapsae exhibited the highest level of desiccation tolerance among species followed by S. feltiae and S. rarum; the heterorhabditid species exhibited the least desiccation tolerance and S. riobrave and S. glaseri were intermediate. No intraspecific variation was observed in desiccation tolerance; S. carpocapsae strains showed higher tolerance than all H. bacteriophora or S. riobrave strains yet there was no difference detected within species. In interspecies comparisons, poor freeze tolerance was observed in H. indica, and S. glaseri, S. rarum, and S. riobrave whereas H. georgiana and S. feltiae exhibited the highest freeze tolerance, particularly in the 24-h exposure period. Unlike desiccation tolerance, substantial intraspecies variation in freeze tolerance was observed among H. bacteriophora and S. riobrave strains, yet within species variation was not detected among S. carpocapsae strains. Correlation analysis did not detect a relationship between freezing and desiccation tolerance.     

Short term increases in the cold tolerance of red osier dogwood stems induced by application of cysteine

Bark tissues of Cornus stolonifera stems, treated with cysteine at 24 hours after treatment, survived exposure to −11 C (the tissue temperature) with little or no injury. An initiation of increase in the cold tolerance was usually observed when plants were treated with cysteine at 12 hours after treatment. Neither plants at 36 or 48 hours after treatment nor plants 12 hours before treatment had shown increases in the cold tolerance. They were killed below −5 C, which was the survival temperature of untreated control plants. Two weeks or more of short day induction before cysteine application were required for a significant effect of short term 5 C increase in the cold tolerance.     

Immune responses of the scallop Chlamys farreri after air exposure to different temperatures

The present study examined the influence of air exposure at different temperatures: a common perturbation associated with aquaculture handling practices, on immune responses in zhikong scallop Chlamys farreri. Scallops were exposed to air for 2 h, 6 h, 12 h and 24 h at 5 °C, 17 °C and 25 °C respectively. Thereafter, a recovery period of 24 h at 17 °C was applied. Haemocyte mortality, phagocytosis and reactive oxygen species (ROS) production of haemocytes, acid phosphatase (ACP) and superoxide dismutase (SOD) activity in haemocyte lysates were chosen as immunomarkers of anoxic stress. The results showed that an increase of haemocyte mortality and a decrease of phagocytosis and ACP activity were observed after 2 h of air exposure for all temperatures tested. Moreover, a significant increase of ROS production occurred following 2 h of air exposure at 25 °C and 24 h of air exposure at 17 °C. Significant differences were also observed in haemocyte mortality, percentage of phagocytic cells and ACP and SOD activity depending on the temperature of air exposure. Finally, after 24 h of recovery at 17 °C, percentage of phagocytic haemocytes and ACP activity did not return to initial values. ROS production was significantly higher than before the recovery period and initial values for scallops subjected to air exposure at 5 °C. In our study, scallops showed a relative low anoxia tolerance under a high temperature. All the scallops air exposed to 25 °C died after the 6 h sampling. In conclusion, air exposure associated to aquaculture practices was demonstrated to strongly affect functional immune activities of scallop haemocytes, and high temperature air exposure caused reduced survival of scallops.     

Escherichia coli viability in an isochoric system at subfreezing temperatures

In comparison with isobaric (constant pressure) freezing, isochoric (constant volume) freezing reduces potential mechanical damage from ice crystals and exposes stored biological matter to a lower extracellular concentration, at the price of increased hydrostatic pressure. This study evaluates the effects of isochoric freezing to low temperatures and high pressures on Escherichia coli (E. coli) survival. The viability of E. coli was examined after freezing to final temperatures between −5 °C and −20 °C for periods from 0.5 h to 12 h, with recovery periods from 0 h to 24 h. Freezing for up to two hours to −10 °C and −15 °C had little effect on the percentage of viable E. coli, relative to the controls. However, after two hours of exposure at −20 °C, when left to recover for 24 h, a 75% reduction in survival is observed. Furthermore, after 12 h of isochoric freezing at −15 °C and −20 °C, E. coli population is reduced by 2.5 logs while freezing to these temperatures in conventional isobaric atmospheric conditions reduces population by only one log. This suggests that the combination of low temperature and high pressure experienced during isochoric freezing close to the triple point may be more detrimental to biological matter survival than the combination of elevated concentration, low temperature, and ice crystallization experienced during conventional freezing, and that this effect may be related to the time of exposure to these conditions.     

Effects of salinity and temperature on the survival and byssal attachment of the lion's paw scallop Nodipecten nodosus at its southern distribution limit

The lion's paw scallop, Nodipecten nodosus, is subject to wide temperature variations on seasonal and short-term scales, and may be exposed to low-salinity events, caused by oceanographic and meteorological processes at its southern distribution limit (Santa Catarina State, Brazil). Such variations may have important implications on the distribution and on aquaculture site selection. The upper and lower temperature tolerances and the percentage of byssal attachment at different temperatures (11 to 35 °C) were studied for spat, juvenile and adult scallops. The lethal and sublethal effects of reduced salinity (13‰ to 33‰) on spat, juvenile and adult scallops were studied at ambient temperature (23.5 °C) and on spat also at low (16 °C) and high (28 °C) temperatures during 96-h bioassays. In addition, the influences of short exposure (1 h) to low salinity (13‰ and 17‰) at different temperatures (16 and 28 °C), and the effects of exposure (2 and 4 h) to high temperature (33 °C) at ambient salinity (33‰) were studied. N. nodosus is a moderately eurythermal but stenohaline tropical species, adults having lower tolerance to high temperature and low salinity than spat. Lethal temperatures for a 48-h exposure (LT₅₀) were 29.8 °C for adult and juveniles, and 31.8 °C for spat. Maximum rate of byssal attachment occurred in a narrower temperature range for juveniles and adults (23 to 27 °C) than for spat (19 to 27 °C), which are suggested as the optimum ranges of temperatures for growth. Lethal salinities (LC₅₀) for a 48-h exposure at ambient temperature were 23.2‰, 23.6‰ and 20.1‰ for adults, juveniles and spat, respectively, but the percent byssal attachment was significantly reduced below salinities of 29‰ indicating that scallops were physiologically stressed. A 1-h exposure to 17‰ was lethal to spat at 28 °C, but at 16 °C there was a 28.5% survival, 96 h after the exposure. Temperatures and salinity in coastal areas of southern Brazil can reach levels leading to sublethal effects, and in some sites, it may surpass the limits of tolerance for the survival of the species.     

Changes in Sugar Content during Cold Acclimation and Deacclimation of Cabbage Seedlings

The relationship between freezing tolerance and sugar contentin cabbage seedlings was investigated. Seedlings exposed tonon-freezing low temperature (5 °C) acquired freezing tolerancedown to -6 °C. The degree of freezing tolerance increasedwith duration of exposure to low temperature (up to 10 d). Sucrose,glucose, fructose and myo -inositol were detected as solublesugars in cabbage leaves, and all soluble sugars, except formyo -inositol, and starch increased gradually during cold acclimationsuch that their levels were positively correlated with the degreeof freezing tolerance. The induced freezing tolerance was attributednot to ontogenetic changes but to cold acclimation. However,the induced freezing tolerance was lost after only 1 d of deacclimationat control temperatures, and this change was associated witha large reduction in sugar content. These results reveal that the sugar content of cabbage leavesis positively correlated with freezing tolerance. Brassica oleracea L.; cabbage; cold acclimation; deacclimation; freezing tolerance; sugars     

Incubation Temperature and Time Effects on Life Stages of Bursaphelenchus xylophilus in Wood Chips

Wood chips of Pinus strobus inoculated with Bursaphelenchus xylophilus were incubated at 3, 12, 30, or 40 C during intervals of 47, 82, and 130 days to determine the effects of incubation temperature and time on total number of nematodes and occurrence of each life stage. Nematodes did not survive at 40 C; the greatest number of nematodes was maintained at 3 C. The number and percentage of juveniles in the propagative cycle were greatest at 3 C after 47 days, but the percentage was greatest at 30 C after 130 days. More third-stage dispersal larvae, with percentages as high as 85%, were extracted at 3 and 12 C than at 30 C by the end of the study. Dauer larvae were extracted from the chips, but percentages never exceeded 5%. The percentage of adults was greater at 30 C than at 3 and 12 C after 82 and 130 days. When a 1-week heat treatment at 30 C was applied to samples at 3 and 12 C,. numbers and percentages of adults increased. Percentages of dauer larvae increased very slightly when the heat treatment was applied after 47 days, but numbers and percentages of juveniles and dispersals were affected erratically.     

Nematostatic Activity of Oxamyl and N,N-Dimethyl-1-cyanoformamide (DMCF) on Meloidogyne incognita Juveniles

The nematostatic activity of oxamyl, methyl-N'',N''-dimethy]-N-hydroxy-l-thiooxamimidate (oxamyl-oxime) and N,N-dimethyl-l-cyanoformamide (DMCF) was studied by immersing 10 Meloidogyne incognita second-stage juveniles into aqueous solutions of various concentrations of each chemical. At concentrations of 500 to 8,000 μg/ml, oxamyl quickly immobilized immersed juveniles. In all other concentrations studied (down to 4 μg/ml), oxamyl stopped or reduced movement of juveniles within 24 hours. DMCF also quickly immobilized juveniles at concentrations of 4,000 and 8,000 μg/ml and reduced movement at 2,000 μg/ml. Lower concentrations had no observed effect on movement. In solutions of the oxime from 2,000 to 8,000 μg/ml, some reduction of movement was observed, but most juveniles maintained some motion over a period of 24 hours. Juveniles were transferred to water from 4,000 μg/ml solutions of oxamyl and DMCF after various intervals of time in order to determine the effect of duration of exposure to the chemicals on the ability of the immobilized juveniles to recover normal motion. Some recovery was observed even after 24 hours of exposure to DMCF, but none after exposure to oxamyl for longer than 40 minutes.     

Influence of Photoperiod and Temperature on Migrations of Meloidogyne Juveniles

Photoperiod influences the migration of M. incognita juveniles toward tomato roots. Approximately 33% migrated vertically 20 cm in 7 days to roots when 12 h dark were alternated with 12 h light. Only 7% migrated when light was constant for 24 h. Vertical migration of M. incognita juveniles was studied at 14, 16, 18, 20, and 22 C. The migration of M. incognita juveniles begins at about 18 C and reaches its maximum at 22 C. The migration of M. hapla and M. incognita juveniles were compared at 14, 18, and 22 C. Juveniles of M. hapla were able to migrate at a lower temperature than those of M. incognita. With M. hapla, there was no significant difference in migration between 18 and 22 C.     

Heat shock protects germinating conidiospores of Neurospora crassa against freezing injury.

Germinating conidiospores of Neurospora crassa that were exposed to 45 degrees C, a temperature that induces a heat shock response, were protected from injury caused by freezing in liquid nitrogen and subsequent thawing at 0 degrees C. Whereas up to 90% of the control spores were killed by this freezing and slow thawing, a prior heat shock increased cell survival four- to fivefold. Survival was determined by three assays: the extent of spore germination in liquid medium, the number of colonies that grew on solid medium, and dry-weight accumulation during exponential growth in liquid culture. The heat shock-induced protection against freezing injury was transient. Spores transferred to normal growth temperature after exposure to heat shock and before freezing lost the heat shock-induced protection within 30 min. Spores subjected to freezing and thawing stress synthesized small amounts of the heat shock proteins that are synthesized in large quantities by cells exposed to 45 degrees C. Pulse-labeling studies demonstrated that neither chilling the spores to 10 degrees C or 0 degrees C in the absence of freezing nor warming the spores from 0 degrees C to 30 degrees C induced heat shock protein synthesis. The presence of the protein synthesis inhibitor cycloheximide during spore exposure to 45 degrees C did not abolish the protection against freezing injury induced by heat shock. Treatment of the cells with cycloheximide before freezing, without exposure to heat shock, itself increased spore survival.     

Penetration of Celery and Alfalfa Roots by Pratylenchus penetrans as Affected by Temperature

A greater percentage of females than juveniles or males of P. penetrans penetrated celery roots grown in infested soil at 5, 18, or 30 C; the difference was greatest at 5 C. The time of initial penetration of alfalfa seedlings inoculated with single nematodes on water agar varied with temperature. Females penetrated the seedlings earlier and over a wider range of temperatures than did males or juveniles. The rate of penetration was highest for females. After initial penetration, the penetration rate decreased with time. At 13-28 C, approximately 80% of roots were penetrated by females and only 25-30% by males and juveniles by the end of the experiment.     

A Leech Capable of Surviving Exposure to Extremely Low Temperatures

It is widely considered that most organisms cannot survive prolonged exposure to temperatures below 0°C, primarily because of the damage caused by the water in cells as it freezes. However, some organisms are capable of surviving extreme variations in environmental conditions. In the case of temperature, the ability to survive subzero temperatures is referred to as cryobiosis. We show that the ozobranchid leech, Ozobranchus jantseanus, a parasite of freshwater turtles, has a surprisingly high tolerance to freezing and thawing. This finding is particularly interesting because the leach can survive these temperatures without any acclimation period or pretreatment. Specifically, the leech survived exposure to super-low temperatures by storage in liquid nitrogen (−196°C) for 24 hours, as well as long-term storage at temperatures as low as −90°C for up to 32 months. The leech was also capable of enduring repeated freeze-thaw cycles in the temperature range 20°C to −100°C and then back to 20°C. The results demonstrated that the novel cryotolerance mechanisms employed by O. jantseanus enable the leech to withstand a wider range of temperatures than those reported previously for cryobiotic organisms. We anticipate that the mechanism for the observed tolerance to freezing and thawing in O. jantseanus will prove useful for future studies of cryopreservation.