Resistance of Anhydrobiotic Aphelenchus avenae to Methyl Bromide Fumigation

The effect of methyl bromide (MB) was tested on active and anhydrobiotic Aphelenchus avenae. A. avenae was induced into anhydrobiosis by three different techniques. Both active and anhydrobiotic nematodes were subjected to 3,000 μ1 MB/liter air for 14 periods from 0 to 82 h. Anhydrobiotic nematodes were more resistant to fumigation than active nematodes, regardless of the technique used to induce anhydrobiosis. The percent survival decreased with increasing MB exposures (μ1 MB × h). For an LD₉₅ of 45,000-54,000 μ1/1 × h were required for active nematodes and >279,000 μ1/1 × h for anhydrobiotic nematodes.     

Studies on Anhydrobiosis of Pratylenchus thornei

Large populations of Pratylenchus thornei, a winter pest of cereals, legumes, and potatoes in the northern Negev region of Israel, survive 7-8 months of summer drought and return to full activity at the beginning of the rainy season. To demonstrate that it survives the summer in an anhydrobiotic state, all developmental stages of P. thornei were exposed to gradually reduced relative humidity (RH) using glycerin water solutions. At 97.7% RH the nematodes were coiled and able to survive exposure to 0% RH. About 40% of artificially desiccated nematodes could be reactivated by gradually increasing the humidity to the final water environment. Desiccated nematodes could withstand temperatures up to 40 C. Reactivated individuals showed intestines apparently devoid of reserve materials. Only 3% survived three cycles of desiccation and reactivation. P. thornei reactivated after anhydrobiosis multiplied twice as much within Vicia sativa roots as did fresh nematodes.     

A Free-living Panagrolaimus sp. from Armenia Can Survive in Anhydrobiosis for 8.7 Years

Although the ability of plant-parasitic nematodes to survive in a dehydrated or anhydrobiotic state for long periods of time has been well documented, the ability of free-living nematodes has not. Here we report on the survival of a free-living nematode, Panagrolaimus sp., from Armenia in the anhydrobiotic state for 8.7 years. This Panagrolaimus sp. can be cultured and maintained readily and may provide a good system for studying anhydrobiosis in nematodes.     

Anhydrobiotic Coiling of Nematodes in Soil

Nematodes of three genera (Acrobeloides sp., Aphelenchus avenae, and Scutellonema brachyurum) were induced to coil and enter anhydrobiosis in drying soil of two types: sandy loam and loamy sand. Coiling was studied in relationship to soil moisture characteristics. Coiling and the physiological state of anhydrobiosis occurred before the water in sandy soils reached a water potential of -15 bars. Coiling was maximum at 3-6 bars, depending on the soil type and nematode species. It appeared that induction of coiling and anhydrohiosis were determined by the physical forces exerted by the water film surrounding the nematode, which, for these three species, was 6-9 monomolecular layers of water, rather than the % moisture and relative humidity of the soil per se.     

Soil nematodes and desiccation survival in the extreme arid environment of the antarctic dry valleys

Soil nematodes are capable of employing an anhydrobiotic survivalstrategy in response to adverse environmental conditions. TheMcMurdo Dry Valleys of Antarctica represent a unique environmentfor the study of anhydrobiosis because extremes of cold, salinity,and aridity combine to limit biological water availability.We studied nematode anhydrobiosis in Taylor Valley, Antarctica,using natural variation in soil properties. The coiled morphologyof nematodes extracted from dry valley soils suggests that theyemploy anhydrobiosis, and these coiled nematodes showed enhancedrevival when re-hydrated in water as compared to vermiform nematodes.Nematode coiling was correlated with soil moisture content,salinity, and water potential. In the driest soils studied (gravimetricwater content <2%), 20–80% of nematodes were coiled.Soil water potential measurements also showed a high degreeof variability. These measurements reflect microsite variationin soil properties that occurs at the scale of the nematode.We studied nematode anhydrobiosis during the austral summer,and found that the proportion of nematodes coiled can vary diurnally,with more nematodes vermiform and presumably active at the warmesttime of day. However, dry valley nematodes uncoiled rapidlyin response to soil wetting from snowmelt, and most nematodeactivity in the Dry Valleys may be confined to periods followingrare snowfall and melting events. Anhydrobiosis represents animportant temporal component of a dry valley nematode's lifespan. The ability to utilize anhydrobiosis plays a significantrole in the widespread distribution and success of these organismsin the Antarctic Dry Valleys and beyond.     

Experimentally Induced Repeated Anhydrobiosis in the Eutardigrade Richtersius coronifer

Tardigrades represent one of the main animal groups with anhydrobiotic capacity at any stage of their life cycle. The ability of tardigrades to survive repeated cycles of anhydrobiosis has rarely been studied but is of interest to understand the factors constraining anhydrobiotic survival. The main objective of this study was to investigate the patterns of survival of the eutardigrade Richtersius coronifer under repeated cycles of desiccation, and the potential effect of repeated desiccation on size, shape and number of storage cells. We also analyzed potential change in body size, gut content and frequency of mitotic storage cells. Specimens were kept under non-cultured conditions and desiccated under controlled relative humidity. After each desiccation cycle 10 specimens were selected for analysis of morphometric characteristics and mitosis. The study demonstrates that tardigrades may survive up to 6 repeated desiccations, with declining survival rates with increased number of desiccations. We found a significantly higher proportion of animals that were unable to contract properly into a tun stage during the desiccation process at the 5^th and 6^th desiccations. Also total number of storage cells declined at the 5^th and 6^th desiccations, while no effect on storage cell size was observed. The frequency of mitotic storage cells tended to decline with higher number of desiccation cycles. Our study shows that the number of consecutive cycles of anhydrobiosis that R. coronifer may undergo is limited, with increased inability for tun formation and energetic constraints as possible causal factors.     

Escape of Steinernema feltiae from Alginate Capsules Containing Tomato Seeds

The entomogenous nematode Steinernema feltiae was encapsulated in an alginate matrix containing a tomato seed. When these capsules were placed on 0.8% agar for 7 days, the seed germinated and ca. 20% of the nematodes escaped from the capsules, whereas only 0.1% escaped from capsules without seeds. When capsules containing nematodes and a seed were planted into sterilized or nonsterilized soil, nematodes escaped to infect Galleria mellonella larvae. When seed in capsules containing ca. 274 nematodes per capsule were planted in nonsterilized soil, Galleria mortality was 90% 1 week later. Galleria mortality declined to 27%, 23%, and 0% in weeks 2, 4, and 8 postplant, respectively. In sterilized soil, Galleria mortality was 96% and did not differ significantly from the nonsterilized soil in week 1, but was significantly higher in sterilized soil over nonsterilized soil for week 2 (81%) and week 4 (51%). When capsules containing nematodes only were used, Galleria mortality was 71% in sterilized soil 1 week after planting and 58%, 33%, and 12% in weeks 2, 4, and 8 postplant, respectively. In nonsterilized soil, Galleria mortality was 8%, 30%, 21%, and 28% after 1, 2, 4, and 8 weeks, respectively, using only encapsulated nematodes. When the number of nematodes per capsule was increased to ca. 817, Galleria mortality was 92 % or higher in sterilized soil from week 1 to week 4.     

Infection Behavior and Overwintering Survival of Foliar Nematodes, Aphelenchoides fragariae, on Hosta

We studied the pathogenicity and overwintering survival of the foliar nematode, Aphelenchoides fragariae, infecting Hosta spp. Nematodes applied to either lower or upper sides of noninjured and injured hosta leaves were able to infect and produce typical symptoms on nine cultivars. Leaves of only four cultivars (Borschi, Fragrant Blue, Patomic Pride, and Olive Bailey Langdon) showed no symptoms of nematode infection. The nematodes overwintered as juveniles and adults in soil, dry leaves, and dormant buds, but not in roots. Nematode winter survival was higher in dormant buds and soil from the polyhouse than in an open home garden. Of the nematodes found in the dormant buds, 35% to 79% were located between the first two outside layers of the buds. The nematodes tolerated 8 hr exposure to 40°C and −80°C in leaf tissues. Relative humidity influenced nematode migration from soil to leaves. The presence of nematodes only on the outer surface of foliage (leaves and petioles) confirmed the migration of A. fragariae on the surface of the plants. Of the total number of nematodes found on the foliage, 25% to 46% and 66% to 77% were alive at 90% and 100% relative humidity, respectively, suggesting that high moisture is required for the survival and upward movement of nematodes. We conclude that A. fragariae can overwinter in soil, infected dry leaves, and dormant buds and migrate in films of water on the outer surface of the plant during spring to leaves to initiate infection.     

A Realistic Appraisal of Methods to Enhance Desiccation Tolerance of Entomopathogenic Nematodes

Understanding the desiccation survival attributes of infective juveniles of entomopathogenic nematodes (EPN) of the genera Steinernema and Heterorhabditis, is central to evaluating the reality of enhancing the shelf-life and field persistence of commercial formulations. Early work on the structural and physiological aspects of desiccation survival focused on the role of the molted cuticle in controlling the rate of water loss and the importance of energy reserves, particularly neutral lipids. The accumulation of trehalose was also found to enhance desiccation survival. Isolation of natural populations that can survive harsh environments, such as deserts, indicated that some populations have enhanced abilities to survive desiccation. However, survival abilities of EPN are limited compared with those of some species of plant-parasitic nematodes inhabiting aerial parts of plants. Research on EPN stress tolerance has expanded on two main lines: i) to select strains of species, currently in use commercially, which have increased tolerance to environmental extremes; and ii) to utilize molecular information, including expressed sequence tags and genome sequence data, to determine the underlying genetic factors that control longevity and stress tolerance of EPN. However, given the inherent limitations of EPN survival ability, it is likely that improved formulation will be the major factor to enhance EPN longevity and, perhaps, increase the range of applications.     

Studies on the Host-finding Mechanisms of Neotylenchus linfordi

The plant-parasitic nematode, Neotylenchus linlordi, congregated around colonies or filtrates from mycelia of Gliocladium roseum, Rhizoctonia solani, Pyrenochaeta terrestris and Chaetomium indicum. The average time required for the nematodes to reach the fungal colonies ranged from less than 4 hr for G. roseum to 20 hr for R. solani. Nematodes first circled near the point of introduction, then moved toward the fungus or filtrate. Several methods of measuring the response of N. linfordi to G. roseum culture filtrate were evaluated. The response was strongest when the test materials were assayed on an agar disk submerged in water agar and the introduced nematodes suspended in agar in a center well midway between the test materials. Filtrates obtained from cultures of G. roseum incubated between 12 and 21 days in potato dextrose broth, were most active. The attractants were small thermostable molecules, soluble in methyl alcohol and unaffected by pH. A yellow pigment with properties similar to a mixture of aurantiogliocladin, rubrogliocladin, and gliorosein was shown to be one of the active materials. The response of N. linfordi to the G. roseum filtrate was not associated with any nutritive factors which would result in reproduction.     

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.     

Anhydrobiosis increases survival of trichostrongyle nematodes

This study demonstrates that infective-stage larvae of 2 trichostrongyle ruminant gastrointestinal nematodes, Haemonchus contortus and Trichostrongylus colubriformis, can enter into anhydrobiotic states when completely desiccated. Larvae of control trichostrongyle species, Heligmosomoides polygyrus and Nippostrongylus brasiliensis, that infect mice were unable to survive desiccation or to enter into anhydrobiosis. Ruminant larvae were able to survive up to 7 desiccation/rehydration cycles, and, during anhydrobiosis, metabolic activity was decreased and survival of the larvae was prolonged both in the laboratory and in the field. Relative humidity had no effect on ruminant larval survival after anhydrobiosis compared with controls. Temperature had a significant effect, 85.8 +/- 2.3% of larvae in anhydrobiosis could survive low temperatures (0 C) that killed all control larvae. Metabolic activity, measured by changes in lipid content and CO2 respiration, was significantly lower in larvae that entered anhydrobiosis compared with controls (P < 0.05). In field experiments using open-meshed chambers under ambient environmental conditions, larvae in anhydrobiosis had significantly higher survival rates in the field compared with controls (P < 0.05) during summer and winter trials. These data suggest that anhydrobiosis in ruminant larvae promotes survival at freezing temperatures, decreases metabolic activity, and prolongs survival under natural field conditions.     

Anhydrobiosis in Pratylenchus penetrans

Anhydrobiotic survival of Pratylenchus penetrans was compared in several soil moisture regimes. Bodies of anhydrobiotic nematodes were coiled. In slow-dried soils, Vineland silt loam (VSL) and Fox loamy sand (FLS), 70 and 58% of the total P. penetrans populations were anhydrobiotic when soil moistures reached ca. 3% and water potential 15 kPa or greater. Coiling began at a much lower water potential in FLS than in VSL. In fast-dried soils, only 31 and 22% of the P. penetrans populations in the same two soil types had entered the anhydrobiotic state at comparable moistures. In the above soils, 76-96% of the P. penetrans were alive immediately after entering the anhydrobiotic state. In slow-dried VSL, some nematodes (1%) survived 770 days. In the other soils, all anhydrobiotic nematodes were dead after 438 days. Anhydrobiosis increased the ability of nematodes to survive subzero temperatures, but it did not increase their ability to survive temperatures above 40 C. Infectivity and reproductivity of rehydrated P. penetrans were not affected by anhydrobiosis.     

Nematicidal Effects of Silver Nanoparticles on Root-knot Nematode in Bermudagrass

Certain nematodes are common soilborne organisms found in turfgrass in the United States that cause significant economic damage to golf course turf. One of the most prevalent plant-parasitic nematodes infesting turfgrass are root-knot nematodes (Meloidogyne spp.). Chemical treatment options for root-knot nematodes in turfgrass are limited, and there is a need for new nematicidal active ingredients to address this problem. In this study, we evaluated the use of silver nanoparticles (AgNP) as a potential nematicide in laboratory and field experiments. AgNP was synthesized by a redox reaction of silver nitrate with sodium borohydride using 0.2% starch as a stabilizer. When J2 of M. incognita were exposed to AgNP in water at 30 to 150 μg/ml, >99% nematodes became inactive in 6 hr. When turfgrass and soil composite samples infested with M. graminis were treated with 150 μg/ml AgNP, J2 were reduced in the soil samples by 92% and 82% after 4- and 2-d exposures, respectively, in the treated compared to the nontreated soil samples. Field trials evaluating AgNP were conducted on a bermudagrass (Cynodon dactylon × C. transvaalensis) putting green infested with M. graminis. Biweekly application of 90.4 mg/m² of AgNP improved turfgrass quality in one year and reduced gall formation in the roots in two years without phytotoxicity. The AgNP application did not significantly reduce the number of M. graminis J2 in plots during the growing season. The laboratory assays attested to the nematicidal effect of AgNP, and the field evaluation demonstrated its benefits for mitigating damage caused by root-knot nematode in bermudagrass.     

Nematodes Associated with Plants from Naturally Acidic Wetlands Soil

Four plants, Cyperus ochraceus, Eriocaulon compressum, Lythrum alatum, and Xyris jupicai, growing along the shoreline of an oligotrophic lake in north central Florida were sampled for nematodes. The nematodes recovered were placed in four trophic groups: bacterivores, herbivores, omnivores, and predators. When the nematodes on all plants were considered, 27% were bacterivores, 23% were herbivores, 7% were omnivores, and 43% were predators. Tripyla was the dominant predator and the dominant genus of all nematodes, and Malenchus was the dominant herbivore. Dominance was not clearly pronounced in the other trophic groups.     

Granular Formulations of Steinernema carpocapsae (strain All) (Nematoda: Rhabditida) with Improved Shelf Life

Shelf life (nematode survival) of Steinernema carpocapsae (strain All) nematodes at 21 C in "Pesta" granules, made by a pasta-like process, was increased from 8 to 26 weeks by incorporating low concentrations of formaldehyde. Pesta samples containing an average of 427,000 nematodes/g were prepared with wheat flour (semolina or bread flour), kaolin, bentonite, peat moss, nematode slurry, and formaldehyde (0-1.4% w/w) and were dried to a water content of 23.6-26.9%. Nematodes emerged from Pesta (S. carpocapsae) granules when placed in water or on moist filter paper. Incorporation of 0.2% w/w formaldehyde (nominal; 0.05% by analysis) was optimum for increasing nematode survival in semolina-based Pesta, and also inhibited fungal growth on the granules. Bread flour Pesta samples prepared by formaldehyde addition to the nematode slurry prior to dough preparation, rather than by addition to a mixture of dry ingredients, had longer shelf life. Nematodes recovered from granules made with 0.2% formaldehyde and stored 20 weeks at 21 C caused 100% mortality of wax moth (Galleria mellonella) larvae.     

Tolerance of Sargassum thunbergii germlings to thermal, osmotic and desiccation stress

The construction of artificial seaweed beds in the intertidal zone is a challenge due to extreme levels of physical stress. In order to provide a basis for the construction using the dispersal of microscopic juveniles, a three-way factorial experimental design was used to evaluate the tolerance of Sargassum thunbergii germlings shortly released from fertile thalli to temperature, salinity and desiccation in this study. Results revealed that temperature, salinity and desiccation significantly affected the growth and survival of germlings. Germlings showed rapid growth with relative growth rate (RGR, % day⁻¹) over 16% when cultured at 25 °C and full immersion in normal seawater. Although growths of germlings subjected to moderate conditions were significantly inhibited, RGRs over 13% were obtained. The RGRs of germlings below 10% were observed only at 35 °C and 9 h desiccation treatments. In comparison to growth, survival was less affected by physical stress. Germlings showed low mortalities below 10% under appropriate conditions (25 °C and 30 °C combined with full immersion), and below 60% under moderate conditions, by the end of experiment. However, the mortality rates increased to over 90% under extreme conditions (9 h desiccation and 35 °C combined with full immersion in salinity of 12). These results showed that S. thunbergii germlings had high tolerance to physical stresses. In addition to the main effects, both two-way and three-way interactions between temperature, salinity and desiccation were significant. Based on the magnitude of effect, desiccation was the predominant factor affecting both growth and survival. According to the results, construction of artificial tanks in natural habitat to minimize desiccation may be an effective strategy for S. thunbergii restoration using germlings.     

Enhanced Trehalose Accumulation and Desiccation Survival of Entomopathogenic Nematodes Through Cold Preacclimation

Limited storage stability is a major obstacle to further expansion of the use of entomopathogenic nematodes for pest control. Progress has been made that Steinernema carpocapsae can now be stored under partial anhydrobiosis for up to 6 months at 25°C and 10 months at 5°C in a water-dispersible granular (WG) formulation. However, other species have been more difficult to store in the WG formulation due to migration of nematodes out of the granules and sensitivity of some species to desiccation directly at cold temperatures. As acclimation to cold induces trehalose accumulation (a major cryo- and desiccation protectant) in many invertebrates, it was hypothesized that cold preacclimation of entomopathogenic nematodes will enhance their survival in the WG formulation at cold temperatures. This hypothesis was tested using a temperate species Steinernema feltiae , a subtropical species S. carpocapsae , and a tropical species Steinernema riobrave possessing different thermal niche breadths and reproduction temperature optima. Cold acclimation of infective juveniles increased trehalose accumulation in all three species and the amount of trehalose accumulated was both temperature and species dependent. Trehalose content reached at its peak after 6 days at 5°C in S. feltiae (82.28 μg/mg dry weight), after 10 days at 10°C in S. carpocapsae (94.16 μg/mg dry weight) and after 6 days at 15°C in S. riobrave (47.58 μg/mg dry weight). Cold preacclimation at 5°C for 2 days enhanced desiccation survival of S. feltiae in 25% glycerol (osmotic desiccation) at both 5 and 25° and of S. carpocapsae and S. riobrave only at 5°C. Non-cold acclimated S. carpocapsae and S. riobrave were extremely sensitive to desiccation directly at 5°C in 25% glycerol, resulting in over 98% mortality within 6 days, but S. feltiae was more sensitive to desiccation at 25°C than at 5°C. Cold preacclimation increased survival of all the three species in the WG formulation at both 5 and 25°C. The survival of S. riobrave at 5°C in the WG formulation was positively correlated with the length of preacclimation period at 5°C (R 2 = 0.99) and with the amount of trehalose accumulated during cold preacclimation (R 2 = 0.81). These results support the hypothesis that cold preacclimation enhances desiccation survival of entomopathogenic nematodes at cold temperatures and the increased survival correlates well with the increased trehalose accumulation. Results also demonstrate that cold preacclimation can be used as a tool to enhance survival of nematodes in the formulations with reduced water activity.