Effectiveness of Entomopathogenic Nematodes in an Alginate Gel Formulation Against Lepidopterous Pests

An improved calcium alginate gel formulation was developed and tested as a carrier for entomopathogenic nematodes against Spodoptera littoralis and Helicoverpa armigera larvae. Mortality of 100% was caused in 4th instar larvae of the two insects by feeding them on 1000 infective juveniles (IJ) g -1 of Steinernema carpocapsae (ALL strain) in the gel for 24 h. Exposing 2nd to 5th instars of H. armigera and 3rd to 6th of S. littoralis to 500 IJ g -1 of S. carpocapsae (ALL strain) resulted in 70-100% larval mortality. Mature larvae were less susceptible to the nematodes. Mortality of larvae exposed to 500 IJg -1 of S. carpocapsae (ALL strain) ranged from about 45-55% at 4 h to 90-95% at 48 h. Fourth instar larvae fed for 24 h with 250 IJ g -1 of nematode strains in gel showed in S. littoralis ranges of susceptibility in the following descending order: S. feltiae (IS -7 strain) = S. carpocapsae (DT strain) = S. feltiae (IS-6 strain) > S. carpocapsae (Mexican strain) = S. carpocapsae (ALL strain) = Heterorhabditis bacteriophora (HP-88 strain) = H sp. (IS-5 strain) > S. riobravae (Texas strain); in H. armigera the rating was: S. feltiae (IS-7 strain) = H. bacteriophora (HP88 strain) > S. carpocapsae (ALL strain) = S. feltiae (IS-6 strain ) = Heterorhabditis sp. (IS5 strain) > S. carpocapsae (Mexican strain) > S. riobravae (Texas strain) . The number of nematodes per larval cadaver increased with mortality rates. In greenhouse tests at 28 &#45 2°C and 90% relative humidity, gel discs containing 500 IJ g -1 of nematodes were pinned to leaves of potted plants of cotton ( Gossypium hirsutum ) (Acala SJ2) and the plants were offered to S. littoralis larvae. Larval mortality of 89 &#45 12.7% was caused by S. feltiae (IS-7 strain) and most of the plant leaves were protected against the larvae by the nematodes. In the control, larval mortality was 3.3 &#45 0.05% and the plants were almost completely defoliated. Possibilities of using the gel-nematode formulation to protect sheltered crops against insect pests are discussed     

Dehydration-specific induction of hydrophilic protein genes in the anhydrobiotic nematode Aphelenchus avenae

Some organisms can survive exposure to extreme desiccation by entering a state of suspended animation known as anhydrobiosis. The free-living nematode Aphelenchus avenae can be induced to enter the anhydrobiotic state by exposure to a moderate reduction in relative humidity. During this preconditioning period, the nematode accumulates large amounts of the disaccharide trehalose, which is thought to be necessary, but not sufficient, for successful anhydrobiosis. To identify other adaptations that are required for anhydrobiosis, we developed a novel SL1-based mRNA differential display technique to clone genes that are upregulated by dehydration in A. avenae. Three such genes, Aav-lea-1, Aav-ahn-1, and Aav-glx-1, encode, respectively, a late embryogenesis abundant (LEA) group 3 protein, a novel protein that we named anhydrin, and the antioxidant enzyme glutaredoxin. Strikingly, the predicted LEA and anhydrin proteins are highly hydrophilic and lack significant secondary structure in the hydrated state. The dehydration-induced upregulation of Aav-lea-1 and Aav-ahn-1 was confirmed by Northern hybridization and quantitative PCR experiments. Both genes were also upregulated by an osmotic upshift, but not by cold, heat, or oxidative stress. Experiments to investigate the relationship between mRNA levels and protein expression for these genes are in progress. LEA proteins occur commonly in plants, accumulating during seed maturation and desiccation stress; the presence of a gene encoding an LEA protein in an anhydrobiotic nematode suggests that some mechanisms of coping with water loss are conserved between plants and animals.     

The novel gene<Emphasis Type="Italic"> CpEdi-9</Emphasis> from the resurrection plant<Emphasis Type="Italic"> C. plantagineum</Emphasis> encodes a hydrophilic protein and is expressed in mature seeds as well as in response to dehydration in leaf phloem tissues

The resurrection plant Craterostigma plantagineum Hochst. is used as an experimental system to investigate desiccation tolerance in higher plants. A search for genes activated during early stages of dehydration identified the gene CpEdi-9, which is expressed in mature seeds and in response to dehydration in the phloem cells of vascular tissues of leaves. Elements for the tissue-specific expression pattern reside in the isolated promoter of the CpEdi-9 gene, as shown through the analysis of transgenic plants. The CpEdi-9 promoter could be a suitable tool for expressing genes in the vascular system of dehydrated plants. CpEdi-9 encodes a small (10 kDa) hydrophilic protein, which does not have significant sequence homologies to known genes. The predicted protein CpEDI-9 shares some physicochemical features with LEA proteins from plants and a nematode. Based on the unique expression pattern and on the nucleotide sequence we propose that CpEdi-9 defines a new class of hydrophilic proteins that are supposed to contribute to cellular protection during dehydration. This group of proteins may have evolved because desiccation tolerance requires the abundant expression of protective proteins during early stages of dehydration in all tissues.Abbreviations ABA Abscisic acid - ABRE ABA-responsive element - Edi Early dehydration induced - GUS Glucuronidase - LEA Late embryogenesis abundant - MU Methylumbelliferone This article is dedicated to Prof. Dr. Francesco Salamini on the occasion of his 65th birthday and his departure from the Max Planck Institute in Köln     

Anhydrobiotic potential and long-term storage of entomopathogenic nematodes (Rhabditida: Steinernematidae)

Anhydrobiosis is considered to be an important means of achieving storage stability of entomopathogenic nematodes that are used in biological control. This study explored the effects of anhydrobiosis on longevity and infectivity of infective juveniles (IJs) of three species of entomopathogenic nematodes Steinernema carpocapsae, Steinernema feltiae, and Steinernema riobrave at 5 and 25 degrees C. Anhydrobiosis was induced in water-dispersible granules (WG) at 0.966-0.971 water activity and 25 degrees C following a 7-day preconditioning of IJs at 5 degrees C in tap water. Survival and infectivity of the desiccated (anhydrobiotic) IJs was compared with non-desiccated IJs stored in water for different periods. Anhydrobiosis increased longevity of S. carpocapsae IJs by 3 months and of S. riobrave by 1 month in WG at 25 degrees C as compared with IJs stored in water. However, desiccation decreased S. feltiae longevity at 25 degrees C and of all three species at 5 degrees C. These results demonstrate a shelf-life of 5 months for S. carpocapsae at 25 degrees C and 9 months at 5 degrees C in WG with over 90% IJ survival. For S. feltiae, over 90% survival occurred only for 2 months at 25 degrees C and 5 months at 5 degrees C in WG. Steinernema riobrave had over 90% survival only for 1 month at 25 degrees C and the survival dropped below 85% within 1 month at 5 degrees C. Induction of anhydrobiosis in WG resulted in 85, 79 and 76% reduction in oxygen consumption by S. carpocapsae, S. feltiae, and S. riobrave IJs, respectively. Differences in IJ longevity among three species in water at 25 degrees C were related both to the initial lipid content and the rate of lipid utilisation, but not at 5 degrees C. The one-on-one infection bioassays indicated that desiccation had no negative effect on the infectivity of any of the nematode species suggesting no harmful effect on the IJs and/or their symbiotic bacteria. The species differences in IJ longevity and desiccation survival at different temperatures are discussed in relation to their foraging strategy and temperature adaptation.     

A molecular analysis of desiccation tolerance mechanisms in the anhydrobiotic nematode Panagrolaimus superbus using expressed sequenced tags

Background

Some organisms can survive extreme desiccation by entering into a state of suspended animation known as anhydrobiosis. Panagrolaimus superbus is a free-living anhydrobiotic nematode that can survive rapid environmental desiccation. The mechanisms that P. superbus uses to combat the potentially lethal effects of cellular dehydration may include the constitutive and inducible expression of protective molecules, along with behavioural and/or morphological adaptations that slow the rate of cellular water loss. In addition, inducible repair and revival programmes may also be required for successful rehydration and recovery from anhydrobiosis.

Results

To identify constitutively expressed candidate anhydrobiotic genes we obtained 9,216 ESTs from an unstressed mixed stage population of P. superbus. We derived 4,009 unigenes from these ESTs. These unigene annotations and sequences can be accessed at http://www.nematodes.org/nembase4/species_info.php?species=PSC. We manually annotated a set of 187 constitutively expressed candidate anhydrobiotic genes from P. superbus. Notable among those is a putative lineage expansion of the lea (late embryogenesis abundant) gene family. The most abundantly expressed sequence was a member of the nematode specific sxp/ral-2 family that is highly expressed in parasitic nematodes and secreted onto the surface of the nematodes' cuticles. There were 2,059 novel unigenes (51.7% of the total), 149 of which are predicted to encode intrinsically disordered proteins lacking a fixed tertiary structure. One unigene may encode an exo-??-1,3-glucanase (GHF5 family), most similar to a sequence from Phytophthora infestans. GHF5 enzymes have been reported from several species of plant parasitic nematodes, with horizontal gene transfer (HGT) from bacteria proposed to explain their evolutionary origin. This P. superbus sequence represents another possible HGT event within the Nematoda. The expression of five of the 19 putative stress response genes tested was upregulated in response to desiccation. These were the antioxidants glutathione peroxidase, dj-1 and 1-Cys peroxiredoxin, an shsp sequence and an lea gene.

Conclusions

P. superbus appears to utilise a strategy of combined constitutive and inducible gene expression in preparation for entry into anhydrobiosis. The apparent lineage expansion of lea genes, together with their constitutive and inducible expression, suggests that LEA3 proteins are important components of the anhydrobiotic protection repertoire of P. superbus.     

Sequence analysis of expressed sequence tags from an ABA-treated cDNA library identifies stress response genes in the moss Physcomitrella patens.

Partial cDNA sequencing was used to obtain 169 expressed sequence tags (ESTs) in the moss, Physcomitrella patens. The source of ESTs was a random cDNA library constructed from 7 day-old protonemata following treatment with 10(-4) M abscisic acid (ABA). Analysis of the ESTs identified 69% with homology to known sequences, 61% of which had significant homology to sequences of plant origin. More importantly, at least 11 ESTs had significant similarities to genes which are implicated in plant stress-responses, including responses which may involve ABA. These included a cDNA associated with desiccation tolerance, two heat shock protein genes, one cold acclimation protein cDNA and five others that may be involved in either oxidative or chemical stress or both, i.e., Zn/Cu-superoxide dismutase, NADPH protochlorophyllide oxidoreductase (PorB), selenium binding protein, glutathione peroxidase and glutathione S transferase. Analysis of codon usage between P. patens and seed plants indicated that although mosses and higher plants are to a large extent similar, minor variations also exists that may represent the distinctiveness of each group.     

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.     

Gene expression pattern at desiccation in the anther of <Emphasis Type="Italic">Lilium longiflorum</Emphasis>

Although gene expression profile of pollen has been described, there is limited information regarding a particular phase during anther/pollen development. This work characterizes gene expression pattern at desiccation in lily (Lilium longiflorum Thunb. cv Snow Queen) anthers. We have applied a suppression-subtractive hybridization (SSH) strategy, through which 90 clones were identified and sequenced. These clones resulted in the identification of 42 individual cDNAs among which 33 genes were specifically expressed at the desiccation phase of anthers of >150-mm buds. Fourteen cDNAs were chosen for further examination. Six genes were both dehydration- and abscisic acid (ABA)-inducible whereas the other eight genes were apparently dehydration-irrelevant. The group of dehydration- and ABA-induced genes was also induced by desiccation that developmentally occurs in the anther. The application of fluridone has a significant effect of inhibition on mRNA accumulation of these genes in maturing anthers during which desiccation occurs. Pollen germination analysis indicated that, of those dehydration-irrelevant genes, three were ABA-responsive and the other five were not. Thus, three separate signal pathways that function in the activation of late genes at desiccation during anther development are established. The first is the ABA-dependent pathway induced by environmental stress of dehydration. The other two pathways of signaling triggered by developmental cues, through which one is ABA-dependent and another is ABA-independent. The 14 gene proteins showed spatial and temporal expression patterns and may participate in membrane/cell wall synthesis, cytoskeletal organization, signaling, RNA binding, ubiquitin-mediated degradation and transportation during germination and tube growth. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Rate of dehydration and cumulative desiccation stress interacted to modulate desiccation tolerance of recalcitrant cocoa and ginkgo embryonic tissues

Rate of dehydration greatly affects desiccation tolerance of recalcitrant seeds. This effect is presumably related to two different stress vectors: direct mechanical or physical stress because of the loss of water and physicochemical damage of tissues as a result of metabolic alterations during drying. The present study proposed a new theoretic approach to represent these two types of stresses and investigated how seed tissues responded differently to two stress vectors, using the models of isolated cocoa (Theobroma cacao) and ginkgo (Ginkgo biloba) embryonic tissues dehydrated under various drying conditions. This approach used the differential change in axis water potential (DeltaPsi/Deltat) to quantify rate of dehydration and the intensity of direct physical stress experienced by embryonic tissues during desiccation. Physicochemical effect of drying was expressed by cumulative desiccation stress [integralf(psi,t)], a function of both the rate and time of dehydration. Rapid dehydration increased the sensitivity of embryonic tissues to desiccation as indicated by high critical water contents, below which desiccation damage occurred. Cumulative desiccation stress increased sharply under slow drying conditions, which was also detrimental to embryonic tissues. This quantitative analysis of the stress-time-response relationship helps to understand the physiological basis for the existence of an optimal dehydration rate, with which maximum desiccation tolerance could be achieved. The established numerical analysis model will prove valuable for the design of experiments that aim to elucidate biochemical and physiological mechanisms of desiccation tolerance.     

The isolation of genes from the resurrection grass Sporobolus stapfianus which are induced during severe drought stress

A modification of the ‘cold plaque’ screening technique (Hodge et al., Plant Journal1992, 2, 257–260) was used to screen a cDNA library constructed from drought‐stressed leaf tissue of the desiccation tolerant (‘resurrection’) grass Sporobolus stapfianus. This technique allowed a large number of clones representing genes expressed at low abundance to be isolated. An examination of expression profiles revealed that several of these genes are induced in desiccation‐tolerant tissue experiencing severe drought stress. Further characterization indicated that the gene products encoded include an eIF1 protein translation initiation factor and a glycine‐ and proline‐rich protein which have not previously been associated with drought stress. In addition, genes encoding a serine/threonine phosphatase type 2C, a tonoplast‐intrinsic protein (TIP) and an early light‐inducible protein (ELIP) were isolated. A number of these genes are expressed differentially in desiccation‐tolerant and desiccation‐sensitive tissues, suggesting that they may be associated with the desiccation tolerance response of S. stapfianus. The results indicate that there may be unique gene regulation processes occurring during induction of desiccation tolerance in resurrection plants which allow different drought‐responsive genes to be selectively expressed at successive levels of water loss.     

Analysis of expressed sequence tags from roots of resistant soybean infected by the soybean cyst nematode.

The soybean cyst nematode (SCN) Heterodera glycines is the most devastating pest of soybean in the U.S.A. The resistance response elicited by SCN in soybean is complex, and genes involved in the response to a large extent are unknown and not well characterized. We constructed cDNA libraries made from mRNA extracted from roots of the resistant soybean Glycine max L. Merr. 'Peking' at 12 h, 2 to 4 days, and 6 to 8 days post inoculation with the soybean cyst nematode, population NL1-RHp, similar to race 3. Expressed sequence tag analysis of the libraries provides rapid discovery of genes involved in the response of soybean to the nematode. A total of 3454 cDNA clones were examined from the three libraries, of which 25 cDNAs were derived from nematode RNA. The levels of certain stress-induced genes such as SAM22 and glutathione S-transferase (GST8) were elevated in the SCN-infected roots relative to uninoculated roots. Early defense response genes, particularly ascorbate peroxidase and lipoxygenase, were abundant in the 12-h library. By 6-8 days, the expression of most of those genes was not as abundant, whereas genes coding for unknown proteins and stress-induced proteins continued to be highly expressed. These ESTs and associated information will be useful to scientists examining gene and protein interactions between nematodes and plants.     

Directional movement of entomopathogenic nematodes in response to electrical field: effects of species, magnitude of voltage, and infective juvenile age

Entomopathogenic nematodes respond to a variety of stimuli when foraging. Previously, we reported a directional response to electrical fields for two entomopathogenic nematode species; specifically, when electrical fields were generated on agar plates Steinernema glaseri (a nematode that utilizes a cruiser-type foraging strategy) moved to a higher electric potential, whereas Steinernema carpocapsae, an ambush-type forager, moved to a lower potential. Thus, we hypothesized that entomopathogenic nematode directional response to electrical fields varies among species, and may be related to foraging strategy. In this study, we tested the hypothesis by comparing directional response among seven additional nematode species: Heterorhabditis bacteriophora, Heterorhabditis georgiana, Heterorhabditis indica, Heterorhabditis megidis, Steinernema feltiae, Steinernema riobrave, and Steinernema siamkayai. S. carpocapsae and S. glaseri were also included as positive controls. Heterorhabditids tend toward cruiser foraging approaches whereas S. siamkayai is an ambusher and S. feltiae and S. riobrave are intermediate. Additionally, we determined the lowest voltage that would elicit a directional response (tested in S. feltiae and S. carpocapsae), and we investigated the impact of nematode age on response to electrical field in S. carpocapsae. In the experiment measuring diversity of response among species, we did not detect any response to electrical fields among the heterorhabditids except for H. georgiana, which moved to a higher electrical potential; S. glaseri and S. riobrave also moved to a higher potential, whereas S. carpocapsae, S. feltiae, and S. siamkayai moved to a lower potential. Overall our hypothesis that foraging strategy can predict directional response was supported (in the nematodes that exhibited a response). The lowest electric potential that elicited a response was 0.1 V, which is comparable to electrical potential associated with some insects and plant roots. The level of response to electrical potential diminished with nematode age. These results expand our knowledge of electrical fields as cues that may be used by entomopathogenic nematodes for host-finding or other aspects of navigation in the soil.     

Effect of Neem and Selected Fungicides on Viability and Virulence of the Entomopathogenic Nematode Steinernema feltiae

Entomopathogenic nematodes are often used in conjunction with other pest management tactics and the lack of compatibility information is a major impediment in further expansion of their use. We evaluated the effects of different formulations of neem and selected fungicides commonly used in greenhouses on Steinernema feltiae which is used for the control of fungus gnats. Neem as pure oil at the field recommended concentrations (5- 10 mL L -1 ) had no effect on the viability and virulence of S. feltiae up to 120 h incubation. However the neem formulation, Nimbecidine and neem oil when mixed with a bactericidal soap (commonly used as a surfactant with neem oil) caused 13- 25% mortality of S. feltiae. This toxic effect was entirely due to the soap that alone caused about 24% mortality. Neither neem oil, Nimbecidine or soap had any effect on nematode virulence. The fungicide cinnamaldehyde (Cinnamate) was highly toxic, resulting in 100% nematode mortality after 4 h of incubation, followed by hydrogen dioxide/peroxyacetic acid mixture (ZeroTol) that caused 100% mortality after 120 h of incubation. Another fungicide, azoxystrobin (Abound) caused no nematode mortality. This investigation concludes that neem and the fungicide azoxystrobin (Abound) can be safely tank mixed at the field recommended concentrations with the infective juveniles of S. feltiae for application, but cinnamaldehyde (Cinnamate) and hydrogen dioxide/peroxyacetic mixture (ZeroTol), are incompatible. Also the surfactants that are usually recommended as 'tank-mix' applications can be toxic to the nematodes and should therefore be evaluated for compatibility prior to use.