Cryopreservation of Pratylenchys spp.

Abstract: Optimal conditions for cryopreserving of populations of root lesion nematode (Pratylenchus spp.) were determined. Nematode survival was achieved using glycerol pre-treatments in the range of 14-17% (w/w). Increasing duration of the incubation in glycerol (up to 5 days) before immersion in liquid nitrogen significantly influenced nematode survival The highest mean survival for P. thornei was 76% after incubation in glycerol for 5 days. Nematodes were able to reproduce and infect carrot disc cultures after cryopreservation. This technique has valuable applications for long-term germplasm storage and maintenance of genetic lines.     

Isolation and identification of entomopathogenic nematodes and their symbiotic bacteria from Hérault and Gard (Southern France)

Isolation and identification of native nematode-bacterial associations in the field are necessary for successful control of endemic pests in a particular location. No study has yet been undertaken to recover and identify EPN in metropolitan France. In the present paper, we provide results of a survey of EPN and their symbiotic bacteria conducted in Hérault and Gard regions in Southern France. Molecular characterization of isolated nematodes depicted three different Steinernema species and one Heterorhabditis species, H. bacteriophora. Steinernema species recovered were identified as: S. feltiae and S. affine and an undescribed species. Xenorhabdus symbionts were identified as X. bovienii for both S. feltiae and S. affine. Phylogenetic analysis placed the new undescribed Steinernema sp. as closely related to S. arenarium but divergent enough to postulate that it belongs to a new species within the “glaseri-group”. The Xenorhabdus symbiont from this Steinernema sp. was identified as X. kozodoii. All Heterorhabditis isolates recovered were diagnosed as H. bacteriophora and their bacterial symbionts were identified as Photorhabdus luminescens. Molecular characterization of these nematodes enabled the distinction of two different H. bacteriophora strains. Bacterial symbiontic strains of these two H. bacteriophora strains were identified as P. luminescens ssp. kayaii and P. luminescens ssp. laumondii.     

Identification of Entomopathogenic Nematodes in the Steinernematidae and Heterorhabditidae (Nemata: Rhabditida)

This paper contains taxonomic keys for the identification of species of the genera Steinernema and Heterorhabditis. Morphometrics of certain life stages are presented in data tables so that the morphometrics of species identified using the keys can be checked in the tables. Additionally, SEM photographs and diagnoses of the families and genera of Steinernematidae and Heterorhabditidae are presented.     

Effect of Entomopathogenic Nematode Concentration on Survival during Cryopreservation in Liquid Nitrogen

Entomopathogenic nematodes are used for biological control of insect pests. A method for improved cryopreservation of infective juvenile stage nematodes has been developed using Steinernema carpocapsae and Heterorhabditis bacteriophora. Optimum survival for both species was achieved with 12,000 infective juveniles/ml in glycerol and 7,500/ml in Ringer''s solution. For S. carpocapsae, maximum survival also was observed with 60,000 infective juveniles/ml in glycerol and 25,000/ml in Ringer''s solution. These concentrations resulted in 100% post-cryopreservation survival of S. carpocapsae and 100% retention of original virulence to Galleria mellonella larvae. This is the first report of achieving 100% survival of an entomopathogenic nematode after preservation in liquid nitrogen. Maximum survival of H. bacteriophora following cryopreservation was 87%.     

Cryopreservation of Steinernema carpocapsae and Heterorhabditis bacteriophora

A method for the cryopreservation of third-stage infective juveniles (IJ) of Steinernema carpocapsae and Heterorhabiditis bacteriophora was developed. Cryoprotection was achieved by incubating the nematodes in 22% glycerol (S. carpocapsae) or 14% glycerol (H. bacteriophora) for 24 hours, followed by 70% methanol at 0 C for 10 minutes. The viability of S. carpocapsae frozen in liquid nitrogen as 20 μl volumes spread over cover slip glass was > 80%. Survival of H. bacteriophora frozen on glass varied from 10 to 60% but was improved to > 80% by replacing the glass with filter paper. Cryopreservation and storage of 1-ml aliqots of S. carpocapsae IJ resulted in > 50% survival after 8 months; pathogenicity was retained and normal in vitro development took place. Trehalose and glycerol levels increased and glycogen levels decreased during incubation of S. carpocapsae IJ in glycerol. Normal levels of trehalose, glycerol and glycogen were restored during post freezing rehydration.     

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.     

Perspectives on the Behavior of Entomopathogenic Nematodes from Dispersal to Reproduction: Traits Contributing to Nematode Fitness and Biocontrol Efficacy

The entomopathogenic nematodes (EPN) Heterorhabditis and Steinernema are widely used for the biological control of insect pests and are gaining importance as model organisms for studying parasitism and symbiosis. In this paper recent advances in the understanding of EPN behavior are reviewed. The “foraging strategy” paradigm (distinction between species with ambush and cruise strategies) as applied to EPN is being challenged and alternative paradigms proposed. Infection decisions are based on condition of the potential host, and it is becoming clear that already-infected and even long-dead hosts may be invaded, as well as healthy live hosts. The state of the infective juvenile (IJ) also influences infection, and evidence for a phased increase in infectivity of EPN species is mounting. The possibility of social behavior - adaptive interactions between IJs outside the host - is discussed. EPNs’ symbiotic bacteria (Photorhabdus and Xenorhabdus) are important for killing the host and rendering it suitable for nematode reproduction, but may reduce survival of IJs, resulting in a trade-off between survival and reproduction. The symbiont also contributes to defence of the cadaver by affecting food-choice decisions of insect and avian scavengers. I review EPN reproductive behavior (including sperm competition, copulation and evidence for attractive and organizational effects of pheromones), and consider the role of endotokia matricida as parental behavior exploited by the symbiont for transmission.     

Comparison of Three Methods for Estimating the Number of Entomopathogenic Nematodes Present in Soil Samples

Numbers of Steinernema sp. (CB2B) and S. carpocapsae (Agriotos) exponentially declined after application into a clay loam soil. Over a 35-day sampling period, Steinernema sp. (CB2B) was more persistent than S. carpocapsae (Agriotos). The presence or absence of the second-stage cuticle on the third-stage juveniles (J3) at the time of application did not alter the rate of population decline of Steinernema sp. (CB2B). Nearly all J3 of Steinernema sp. (CB2B) and S. carpocapsae (Agriotos) lost their cuticle within 24 hours of being in soil. Centrifugal flotation recovered the greatest number of nematodes, with a lower variance than either the live bait or Baermann funnel techniques. A strong positive linear relationship was evident between numbers of nematodes present in the soil and the numbers that established in a bait insect. Approximately 40% of Steinernema sp. (CB2B) and 30% of the S. carpocapsae (Agriotos) present in the soil established in Galleria mellonella larvae. The extraction techniques had different efficiencies and gave different relative estimates of persistence for the two species. Persistence and infectivity was best measured using a combination of live bait and flotation techniques.     

Selection of insect parasitic nematodes for biological control of the garden chafer,Phyllopertha horticola

Larvae ofPhyllopertha horticola L. (Coleoptera: Scarabaeidae) cause increasing problems on sports fields and lawns in NW-Europe. A biological control programme using insect parasitic nematodes is being developed. This paper contains the results of bioassays with various species and isolates of the nematode generaHeterorhabditis andSteinernema. In bioassays in small pots with moist sand, most of the nematode isolates gave 30–60% mortality against each of the three larval stages. The susceptibility of the grubs for nematode infection generally increased with larval development.H. bacteriophora, H. heliothidis, H. megidis, a DutchHeterorhabditis isolate NLH-E87.3 andS. glaseri 326 showed the highest mortality rates, with nearly 100% mortality of third instar grubs. The DutchHeterorhabditis isolate NLH-E87.3 andS. glaseri 326 were selected as candidates for further studies on their potential as biological control agents forP. horticola grubs in the field.     

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.     

Ecological characterization of entomopathogenic nematodes isolated in stone fruit orchard soils of Mediterranean areas

Entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae were isolated from stone-fruit orchards in two Mediterranean regions of Spain. A total of 630 soil samples (210 sites) from Catalonia and 90 soil samples (30 sites) from Murcia were evaluated resulting in 5.2% and 20% of the soils testing positive for nematodes, respectively. Ten steinernematid isolates and three heterorhabditid isolates were recovered using the Galleria mellonella baiting method. Based on morphometric data, molecular data, and cross-breeding experiments the nematode species were identified as Steinernemafeltiae and Heterorhabditis bacteriophora. Environmental tolerance to heat, desiccation and hypoxia, the effect of temperature on infectivity and reproduction and nematode migration in sand columns were compared among isolates and one Steinernema carpocapsae strain. Results showed differences among species and a great variability within species. Beneficial traits for each strain were added up to identify a superior candidate to control Mediterranean flat-headed rootborer, Capnodis tenebrionis. When all analyzed factors were considered, three S. feltiae isolates (Bpa, Sor and M116) obtained the best scores, and when hypoxia was removed, two of the strains (Bpa and Sor) continued ranking superior to other strains.     

Morphometrics of Infective Juveniles of Steinernema spp. and Heterorhabditis bacteriophora (Nemata: Rhabditida)

Selected morphometrics of Heterorhabditis bacteriophora and seven species of Steinernema from in vivo culture were compared in relation to time of harvest. In addition, five Steinernema species were reared in vitro and their morphometrics were compared with those from in vivo culture. With in vivo culture, there was generally a negative linear relationship between body length of infective juveniles (IJ) and time of harvest. The distance from the anterior end to the excretory pore (EP) and the tail length (T) of IJ also varied with time of harvest. The E percentage (= EP/T x 100) was the least variable. Body lengths of IJ reared in vitro were much less than those of IJ reared in vivo. The study suggests that IJ harvested from in vivo culture within 1 week of emergence from cadavers are best for species identification. Infective juveniles from in vitro culture should not be used for species identification.     

Persistence of Four Heterorhabditis spp. Isolates in Soil: Role of Lipid Reserves

Infective juveniles of four Heterorhabditis isolates (H. bacteriophora HI, H. megidis UK211 and HF85, and H. downesi M245) were stored in moist (pF 1.7) and dry (pF 3.3) loam soil at 20°C for up to 141 days. Survival, assessed by the number of nematodes extracted by centrifugal flotation, declined over time, reaching fewer than 18% alive by day 141 for all but one treatment (H. bacteriophora HI in dry soil). The infectivity of nematodes in soil for Tenebrio molitor also declined over time, roughly in accordance with the decline in numbers of nematodes. Energy reserves of extracted nematodes were assessed by image analysis densitometry. There were differences among isolates both in survival and in the depletion of reserves, and there was a significant correlation between these two parameters, suggesting that the extent to which energy reserves are depleted affects survival or that a common factor influences both. However, significant nematode mortality occurred while levels of reserves remained high, and the maximum reduction in utilizable body content for any treatment was 51%, well above starvation level. Therefore, the decline in numbers of living nematodes and the reduced nematode infectivity in soil cannot directly result from starvation of the nematodes. Survival and infectivity declined more rapidly in moist than in dry soil; one isolate, H. downesi M245, was less affected by soil moisture content than the other three isolates.     

Assessing Nuclear and Mitochondrial DNA Sequence Variation Within Steinernema (Rhabditida: Steinernematidae)

DNA sequence analysis was used to characterize the nuclear ribosomal DNA ITS1 region and a portion of the COII and 16S rDNA genes of the mitochondrial genome from Steinernema entomopathogenic nematodes. Nuclear ITS1 nucleotide divergence among seven Steinernema spp. ranged from 6 to 22%, and mtDNA divergence among five species ranged from 12 to 20%. No intraspecific variation was observed among three S. feltiae strains. Phylogenetic analysis of both nuclear and mitochondrial DNA sequences confirms the existing morphological relationships of several Steinernema species. Both the rDNA ITS1 and mtDNA sequences were useful for resolving relationships among Steinernema taxa.     

Behavioural and physiological responses of infective juveniles of the entomopathogenic nematode Heterorhabditis to desiccation

Infective juveniles (IJs) of entomopathogenic nematodes (EPNs) are susceptible to a wide variety of environmental factors, including desiccation, which limit their usefulness as biocontrol agents. Although EPNs can be subjected to a gradual loss of water in their natural environment they are not full anhydrobiotes, being able to survive only moderate levels of desiccation at high relative humidities (rh). We investigated the desiccation tolerance of IJs of several Heterorhabditisspecies and strains when exposed to fast and slow desiccation regimes. We also investigated the behavioural and biochemical responses of Heterorhabditis IJs when exposed to 98% rh for 4 days. IJs of H. megidis UK211 (but not IJs of H. indica) aggregate into large clumps when desiccated at high rh, but unlike Steinernema spp., neither H. megidis nor H. indica IJs showed any tendency to coil. Preincubation of H. megidis UK211 IJs at high (98%) rh enhances their ability to survive for 150 min at 57% rh. We show that preincubation of H. megidis and H. indica at 98% rh induces the synthesis of glycerol but not of trehalose, whereas identical preincubation conditions do induce trehalose synthesis in Steinernema carpocapsae and Aphelenchus avenae. The biosynthesis of glycerol rather than trehalose by IJs of two species of Heterorhabditis in response to moderate levels of desiccation indicates that Heterorhabditis is unlikely to have the necessary metabolic responses to desiccation required to enable it to enter into a fully anhydrobiotic state.     

Characterization of Heterorhabditis Isolates by PCR Amplification of Segments of mtDNA and rDNA Genes

Restriction digests of amplified DNA from the mitochondrial genome and the nuclear ribosomal internally transcribed spacer region have been evaluated as genetic markers for species groups in Heterorhabditis. Six RFLP profiles have been identified. These profiles supported groupings determined by cross-breeding studies and were in agreement with less definitive groupings based on other biochemical and molecular methods. Digestion patterns of both amplification products provided strong evidence for the recognition of species groups, which include Irish, NW European, tropical, and a H. bacteriophora complex. The H. bacteriophora complex could be further resolved into three genotypes represented by H. zealandica, the H. bacteriophora, Brecon (Australian) type isolate for H. bacteriophora, and a grouping composed of isolates NC1, V16, HI82, and HP88. All cultures obtained of the H. megidis isolate were identical to the NW European group. These results could be used to aid monitoring of field release of Heterorhabditis as well as allowing a rapid initial assessment of taxonomic grouping.     

Biochemistry of Anhydrobiosis in Beddingia siricidicola,a Biological Control Agent of Sirex noctilio

Proto-anhydrobiosis of the nematode, Beddingia siricidicola, was achieved by incubation in polyethylene glycol or various concentrations up to 4 M of glycerol. The associated changes in the levels of glycerol, unbound proline, trehalose, lipids, and glycogen were determined by alkylation strategies, followed by gas chromatography or gas chromatography/mass spectrometry. The level of glycerol reached 8.9% of dry weight, proline 2.4% of dry weight, and trehalose 8.0% of dry weight within B. siricidicola that were incubated in 1.5 M glycerol over 6 d, while glycerol reached 17.9% of dry weight after incubation for the same period in 4 M glycerol. Movement was thereby reduced but the nematodes from 1.5 M glycerol revived after a few minutes upon rehydrating and they were able to avoid osmotic damage by rapidly excreting the glycerol, much of it being expelled within the first hour. The potential for storage and transport of this nematode for the biological control of the pine-killing wasp, Sirex noctilio, was greatly improved when nematode suspensions were maintained in 1.5 M glycerol under refrigeration.     

Diversity of Meloidogyne spp. on Musa in Martinique,Guadeloupe, and French Guiana

Ninety-six isolates of Meloidogyne species collected from banana fields from Martinique, Guadeloupe, and French Guiana, were examined using esterase (Est) and malate dehydrogenase (Mdh) phenotypes. Adult females identified as M. arenaria, M. incognita, M. javanica, M. cruciani, M. hispanica, and Meloidogyne sp. showed species-specific phenotypes only for the esterase enzymes. Intraspecific variability among isolates of M. arenaria, M. incognita, and M. javanica was detected using Est and Mdh. Perineal patterns were used as a complementary tool together with enzyme characterization and were essential for checking the morphological consistency of the identification. The major species of M. arenaria and M. incognita were detected at 61.9% and 34.3% of the total number of isolates, respectively, and the other minor species at 3.8%. The mixed Meloidogyne species were detected in 45.1% of the samples. Genetic analysis was conducted using RAPD markers, which alone or in combination provided reliable polymorphisms both between and within species. RAPD analysis of the data resulted in clustering of species and isolates congruent with esterase phenotype characterization. The intraspecific variability in M. incognita and in M. arenaria represented 14.9% and 61.6% of the amplified polymorphic fragments, respectively. This high level of variation in M. arenaria isolates may indicate multiple origins for populations classified as M. arenaria or more than one species inside the same group, but more detailed morphological and DNA studies will be necessary to test this hypothesis.     

Isolation and identification of entomopathogenic nematodes from citrus orchards in South Africa and their biocontrol potential against false codling moth

A survey was conducted to determine the diversity and frequency of endemic entomopathogenic nematodes (EPN) in citrus orchards in the Western Cape, Eastern Cape and Mpumalanga provinces of South Africa. The main aim of the survey was to obtain nematodes as biological control agents against false codling moth (FCM), Thaumatotibia leucotreta, a key pest of citrus in South Africa. From a total of 202 samples, 35 (17%) tested positive for the presence of EPN. Of these, four isolates (11%) were found to be steinernematids, while 31 (89%) were heterorhabditids. Sequencing and characterisation of the internal transcribed spacer (ITS) region was used to identify all nematode isolates to species level. Morphometrics, morphology and biology of the infective juvenile (IJ) and the first-generation male were used to support molecular identification and characterisation. The Steinernema spp. identified were Steinernema khoisanae, Steinernema yirgalemense and Steinernema citrae. This is the first report of S. yirgalemense in South Africa, while for S. citrae it is the second new steinernematid to be identified from South Africa. Heterorhabditis species identified include Heterorhabditis bacteriophora, Heterorhabditis zealandica and an unknown species of Heterorhabditis. Laboratory bioassays, using 24-well bioassay disks, have shown isolates of all six species found during the survey, to be highly virulent against the last instar of FCM larvae. S. yirgalemense, at a concentration of 50 IJs/FCM larva caused 100% mortality and 74% at a concentration of 200 IJs/pupa. Using a sand bioassay, S. yirgalemense gave 93% control of cocooned pupae and emerging moths at a concentration of 20 IJs/cm². This is the first report on the potential use of EPN to control the soil-borne life stages of FCM, which includes larvae, pupae and emerging moths. It was shown that emerging moths were infected with nematodes, which may aid in control and dispersal.