Effects of Culture Method and Formulation on the Virulence of Steinernema riobrave (Rhabditida: Steinernematidae) to Diaprepes abbreviatus (Coleoptera: Curculionidae)

The Diaprepes root weevil, Diaprepes abbreviatus, is a pest of vegetables, ornamental plants, sugarcane, and citrus in Florida and the Caribbean. The entomopathogenic nematode, Steinernema riobrave, can reduce larval populations of D. abbreviatus substantially. Efficacy of entomopathogenic nematodes, however, may be affected by culture method and formulation. Using D. abbreviatus as the host, we compared the efficacy of two commercial S. riobrave formulations, a liquid and a waterdispersible granule (WDG), with each other and with in vivo produced S. riobrave. Nematodes in the commercial formulations were produced in vitro through liquid fermentation; the in vivo nematodes were cultured in Galleria mellonella and applied in aqueous suspension. Laboratory experiments measured nematode virulence in plastic cups containing soil and seventh-eighth instar D. abbreviatus. One laboratory experiment was conducted using only fresh nematodes (less than 5 days old); another experiment included WDG nematodes that were stored for 25 days at 10 °C. Two field experiments were conducted in which nematodes were applied either to potted citrus (containing D. abbreviatus larvae) placed beneath mature citrus trees or to soil directly beneath the tree. In the latter experiment, efficacy was determined by measuring mortality of caged D. abbreviatus larvae that were buried beneath the soil surface prior to application. Mortality of D. abbreviatus treated with nematodes ranged from 80-98% and 50-75% in laboratory and field experiments, respectively. In all experiments, we did not detect any significant effects of culture method or formulation.     

A Novel Strain of Steinernema riobrave (Rhabditida: Steinernematidae) Possesses Superior Virulence to Subterranean Termites (Isoptera: Rhinotermitidae)

Subterranean termites are major global pests of wood structures and wood products. Among the most economically important subterranean termite species in the US are Heterotermes aureus, Reticulitermes flavipes, and Coptotermes formosanus. In prior studies, the entomopathogenic nematode, Steinernema riobrave strain 355, exhibited a high level of virulence to H. aureus compared with other nematode species. However, S. riobrave 355 was reported to be poorly or only moderately virulent to R. flavipes and C. formosanus, respectively. We hypothesized that other strains of S. riobrave may possess a high level of virulence to all three termite species. Under laboratory conditions we compared three novel strains of S. riobrave (3-8b, 7-12, and TP) with the 355 strain for virulence to H. aureus, R. flavipes, and C. formosanus workers. H. aureus was very susceptible to all the S. riobrave strains, and termites in all nematode treatments were dead after 4 d. The TP strain of S. riobrave caused greater mortality in R. flavipes and C. formosanus compared to the other nematode strains. Specifically, the TP strain caused 75% and 91% mortality in R. flavipes and C. formosanus, respectively, which was more than 300% and 70% higher than the mortality caused by other strains. Additional studies are warranted to determine the ability of S. riobrave (TP) to control the targeted termite species under field conditions.     

Susceptibility of the boll weevil to Steinernema riobrave and other entomopathogenic nematodes

The susceptibility of the boll weevil (BW), Anthonomus grandis Boheman, to Steinernema riobrave and other nematode species in petri dishes, soil (Hidalgo sandy clay loam), and cotton bolls and squares was investigated. Third instar weevils were susceptible to entomopathogenic nematode (EN) species and strains in petri dish bioassays at 30 degrees C. Lower LC(50)'s occurred with S. riobrave TX- 355 (2 nematodes per weevil), S. glaseri NC (3), Heterorhabditis indicus HOM-1 (5), and H. bacteriophora HbL (7) than H. bacteriophora IN (13), S. riobrave TX (14), and H. bacteriophora HP88 (21). When infective juveniles (IJs) of S. riobrave were applied to weevils on filter paper at 25 degrees C, the LC(50) of S. riobrave TX for first, second, and third instars, pupae, and 1-day-old and 10-days-old adult weevils were 4, 5, 4, 12, 13, and 11IJs per weevil, respectively. The mean time to death, from lowest to highest concentration, for the first instar (2.07 and 1.27days) and second instar (2.55 and 1.39days) weevils were faster than older weevil stages. But, at concentrations of 50 and 100IJs/weevil, the mean time to death for the third instar, pupa and adult weevils were similar (1.84 and 2.67days). One hundred percent weevil mortality (all weevil stages) occurred 3days after exposure to 100IJs per weevil. Invasion efficiency rankings for nematode concentration were inconsistent and changed with weevil stage from 15 to 100% when weevils were exposed to 100 and 1IJs/weevil, respectively. However, there was a consistent relationship between male:female nematode sex ratio (1:1.6) and nematode concentration in all infected weevil stages. Nematode production per weevil cadaver increased with increased nematode concentrations. The overall mean yield of nematodes per weevil was 7680IJs. In potted soil experiments (30 degrees C), nematode concentration and soil moisture greatly influenced the nematode efficacy. At the most effective concentrations of 200,000 and 400,000IJs/m(2) in buried bolls or squares, higher insect mortalities resulted in pots with 20% soil moisture either in bolls (94 and 97% parasitism) or squares (92 and 100% parasitism) than those of 10% soil moisture in bolls (44 and 58% parasitism) or squares (0 and 13% parasitism). Similar results were obtained when nematodes were sprayed on the bolls and squares on the soil surface. This paper presents the first data on the efficacy of S. riobrave against the boll weevil, establishes the potential of EN to control the BW inside abscised squares and bolls that lay on the ground or buried in the soil.     

Antimicrobial activity of Xenorhabdus sp. RIO (Enterobacteriaceae), symbiont of the entomopathogenic nematode,Steinernema riobrave (Rhabditida: Steinernematidae)

Galleria mellonella larvae infected with Steinernema riobrave soon showed (after 24 h) the typical growth of its Xenorhabdus sp. RIO symbiont and, in parallel, the growth of another Gram negative bacterial species in the body cavity. A population of Entercoccus sp. in the nematode infected larvae collapsed to zero by 96 h. The level of antibiotic and antimycotic activity followed a pattern similar to that of the growth curve to stationary phase of the Xenorhabdus sp. RIO symbiont, over a period of 168 h. The antimycotic activity was composed of exo- and endochitinases as well as other proteinaceous and some small molecule compounds. The changing pH, relatively high growth rate of Xenorhabdus sp. RIO compared with that of other Gram negative bacterial species and of collapse of the Enterococcus sp. population enabled Xenorhabdus sp. RIO to out-compete other species.     

Susceptibility of the peachtree borer, Synanthedon exitiosa, to Steinernema carpocapsae and Steinernema riobrave in laboratory and field trials

The nematode Steinernema carpocapsae (All) strain was significantly more effective against peachtree borer larvae (Synanthedon exitiosa [Lepidoptera: Sesiidae]) than Steinernema riobrave (7-12) strain in field and laboratory experiments. Eighty-eight percent control of peachtree borer larvae was obtained with S. carpocapsae in the field trial when applied at 3 x 10(5) infective juveniles per tree, and 92% mortality was obtained in the lab assay using 50 infective juveniles per larva.     

Ecological characterization of Steinernema anatoliense (Rhabditida: Steinernematidae)

Our study describes the basic ecological characteristics of the entomopathogenic nematode Steinernema anatoliense including its response to temperature, moisture, and host range. The effect of temperature and soil moisture on the infection of Galleria mellonella larvae by S. anatoliense was determined. The temperature range for infectivity was greater than that for development. The optimal temperature for infection and development was 25 degrees C. Although S. anatoliense infected the hosts at 10 degrees C, no reproduction occurred at this temperature. This nematode species that was isolated from a cold region of Turkey exhibited warm-adapted temperature characteristics. Optimum water content of the soil for S. anatoliense to infect the host was 10%.     

Steinernema akhursti sp. n. (Nematoda: Steinernematidae) from Yunnan, China

A new species of entomopathogenic nematode, herein described as Steinernema akhursti sp. n., was recovered from soil samples collected from Yunnan Province, the People's Republic of China. Both morphological and molecular data show congruently that S. akhursti sp. n. belongs to the Steinernema feltiae group. It can be separated from all described Steinernema species by the combined morphological and morphometrical characters of various stages of the nematodes. For the first generation male, the new species can be recognized by spicule length 90 +/- 4.6 microm, spicule tip blunt with an aperture on the ventral side, gubernaculum with a long and needle-shaped cuneus, and tail conoid with a prominent mucron on the tip and a concave on ventral side. For the infective juvenile, the combination of the following characters: body length 812 +/- 19 microm, distance from anterior end to excretory pore 59 +/- 1.5 microm, tail length 73 +/- 2.9 microm, E% 77 +/- 4.5, lateral field with six evenly distributed and identical ridges at the middle body portion, and tail with long and slightly constrict hyaline portion can be used to separate the new species from other nematodes. For the female, the new species is characterized by: tail conoid with a short mucron and slightly swelling anal portion and a symmetrical, slightly protruding vulva with conspicuous double-flapped epiptygma. The nematode can be separated from other described species of Steinernema by DNA sequences of either a partial 28S rDNA or the internal transcribed spacer regions of rDNA and from the closely related species S. feltiae and Steinernema oregonense by cross-breeding tests.     

Steinernema biddulphi n. sp., a New Entomopathogenic Nematode (Nematoda: Steinernematidae) from South Africa

A new species of entomopathogenic nematode (EPN), Steinernema biddulphi n. sp., was isolated from a maize field in Senekal, Free State Province of South Africa. Morphological and molecular studies indicated the distinctness of S. biddulphi n. sp. from other Steinernema species. Steinernema biddulphi n. sp. is characterized IJs with average body length of 663 μm (606–778 μm), lateral fields with six ridges in mid-body region forming the formula 2,6,2. Excretory pore located anterior to mid-pharynx (D% = 46). Hyaline layer occupies approximately half of tail length. Male spicules slightly to moderately curved, with a sharp tip and golden brown in color. The first generation of males lacking a mucron on the tail tip while the second generation males with a short filamentous mucron. Genital papillae with 11 pairs and one unpaired preanal papilla. The new species is further characterized by sequences of the internal transcribed spacer (ITS) and partial 28S regions (D2-D3) of the ribosomal DNA (rDNA). Phylogenetic data show that S. biddulphi n. sp. belongs to the “bicornutum” clade within the Steinernematidae family.     

Effect of Soil Texture on the Distribution and Infectivity of Neoaplectana glaseri (Nematoda: Steinernematidae)

The vertical migration of infective juveniles of Neoaplectana glaseri applied to the soil surface or introduced 16 cm below the soil surface was studied in pure silica sand, coarse sandy loam, silty clay loam, and clay. The number of juveniles that migrated and infected wax moth pupae placed in the soil decreased as the proportion of clay and silt increased. The majority of nematodes moved downwards 2-6 cm from the surface, but some penetrated to a depth of 14 cm in pure silica sand and coarse sandy loam. In pure silica sand and coarse sandy loam, nematodes introduced 16 cm below the soil surface were able to infect wax moth pupae located at depths of 0-4 cm and 28-32 cm. Nematodes showed a greater tendency to disperse downwards from the point of application. Movement of the nematode was least in clay soil and limited in silty clay loam soil. The number of migrating nematodes was greatest when wax moth pupae were present.     

Interaction of microbial populations in Steinernema (Steinernematidae,Nematoda) infected Galleria mellonella larvae

Infection of Galleria mellonella larvae with the entomopathogenic nematodes Steinernema feltiae (A21 and R strains) and Steinernema glaseri (Dongrae) resulted in several species of bacteria, including the respective bacterial symbiont, Xenorhabdus spp., growing in the infected insect cadavers. These other bacteria were Enterococcus in all three nematode infections studied and Acinetobacter in the S. feltiae infections. The respective populations of these bacteria changed with time. Following infection of G. mellonella larvae with any one of the Steinernema sp., only Enterococcus bacteria were detected initially in the dead larvae. Between 30 and 50h post-infection Xenorhabdus bacteria were detected and concurrent with this Enterococcus population declined to zero. This was probably due to secondary metabolites with antibacterial properties that were produced by Xenorhabdus. In the S. feltiae (both R and A21 strains) infections a third bacterium, Acinetobacter, appeared at about 130h (in S. feltiae A21 infections) or 100h (in S. feltiae R infections) and increased in population size to approximately that of Xenorhabdus. It was demonstrated that Enterococcus, orginating from the G. mellonella digestive tract, was sensitive to the organically soluble antimicrobials produced by Xenorhabdus but Acinetobacter, which was carried by the nematode, was not.     

Effect of Soil Texture on the Distribution and Infectivity of Neoaplectana carpocapsae (Nematoda: Steinernematidae)

The vertical migration of N. carpocapsae infective juveniles applied to the soil surface or introduced 14 cm below the soil surface was studied in four different soil types (pure silica sand, coarse sandy loam, silty clay loam, and clay). The percentage of juveniles able to migrate and infect wax moth pupae placed in the soil decreased as the percentage of clay and silt increased. Most nematodes placed on the soil surface remained within 2 cm of the surface, but some penetrated to a depth of 10 cm in pure silica sand and coarse sandy loam to infect pupae. Some pupae at the same depth were also infected with nematodes in silty clay loam soil. In pure silica sand and coarse sandy loam, nematodes introduced 14 cm below the soil surface were able to infect wax moth pupae located between 4 and 24 cm. Movement was least in clay soil and limited in silty clay loam. Nematodes showed a tendency to disperse upwards from the point of application. In all cases the number of migrating nematodes was greatest when wax moth pupae were present.     

First Record of Steinernema kraussei (Rhabditida: Steinernematidae) from Turkey and Its Virulence against Agrotis segetum (Lepidoptera: Noctuidae)

During a survey of entomopathogenic nematodes (EPNs) in the eastern Black Sea region of Turkey in 2009–2012, a steinernematid species was recorded and isolated using the Galleria-baiting method. The isolate was identified as Steinernema kraussei based on its morphological and molecular properties. The analysis of the ITS rDNA sequence placed the Turkish population of S. kraussei in the “feltiae-kraussei” group in the clade that contains different isolates of the species. This is the first record of S. kraussei from Turkey. The efficacy of S. kraussei was tested on Agrotis segetum (Lepidoptera: Noctuidea) larvae at different densities (100, 300, and 500 infective juveniles (IJs) g⁻¹ dry sand ) in laboratory conditions at 25 °C. The highest mortality (98%) was obtained with 500 IJs g⁻¹ dry sand within 7 d after inoculation. Our results indicate that the new isolate is a highly promising biological control agent against A. segetum, one of the most serious soil pests of agricultural area and fruits worldwide.     

Steinernema diaprepesi n. sp. (Rhabditida: Steinernematidae), a Parasite of the Citrus Root Weevil Diaprepes abbreviatus (L) (Coleoptera: Curculionidae)

A nematode collected from Diaprepes abbreviatus is identified and described as a new species, Steinernema diaprepesi n. sp. The new species is closely related to S. feltiae, S. glaseri, and S. oregonense and can be distinguished from these species by the following characteristics: Males: Spicule averaging 79 (71-90) µm and spicule shape; D% (distance from anterior end to excretory pore/ esophagus length × 100) about 80; the ratio SW (spicule length/anal body width) about 1.8. Females: Vulva with short, double- flapped epiptygma; tail terminus usually with 5 papillae-like structures. Infective juveniles: Body averaging 1,002 (880-1,133) µm, EP (distance from anterior end to excretory pore) = 74 (66-83) µm; tail length = 83 (65-91) µm, and E% (EP/tail length × 100) = 89.6 (78-114). Lateral field pattern variable, the formula for the arrangement of ridges from head to tail is: 2, 6, 7, 8, 4, 2. The portion with eight ridges is the longest. This new species can be differentiated further from three closest species (S. feltiae, S. glaseri, and S. oregonense) by characteristic sequences of their ITS regions, including sequence lengths, ratios of similarity, composition, and differences in base characters in sequence alignment.     

Vertical Dispersal of Steinernema scapterisci

When infective juveniles ofSteinernema scapterisci Nguyen &Smart were released on the soil surface in the field and in the laboratory, some of them moved downward through the soil at least 10 cm in 5 days and infected and killed mole crickets. When released 2 cm below the soil surface, most of the juveniles moved into the upper 2 cm layer of soil, but some moved downward 10 cm. When placed at the center of a 16-cm soil column, infective juveniles moved in both directions with three times more moving downward than upward. Infective juveniles were more efficient in killing mole crickets in the field than in the laboratory.     

Steinernema scapterisci n. sp. (Rhabditida: Steinernematidae)

Steinernema scapterisci n. sp., isolated in Uruguay from the mole cricket Scapteriscus vicinus, can be distinguished from other members in the genus by the presence of prominent cheilorhabdions, an elliptically shaped structure associated with the excretory duct, and a double-flapped epitygma in the first-generation female. The spicules of the male are pointed, tapering smoothly to a small terminus, and the shaft (calomus) is long, bearing a sheath. The gubernaculum has a long, upward-bent anterior part. The ratio of head to excretory pore divided by tail length of the third-stage juvenile is greater for S. scapterisci n. sp. than for S. carpocapsae. Steinernema scapterisci n. sp. did not hybridize with S. carpocapsae strain Breton. In laboratory tests, S. scapterisci n. sp. killed 10% or less of non-orthopteran insects, including the wax moth larva, a universal host for other species of Steinernema.     

Steinernema aciari sp. n. (Nematoda: Steinernematidae), a new entomopathogenic nematode from Guangdong, China

A new species of entomopathogenic nematode, Steinernema aciari sp. n. was described. It was recovered from a soil sample collected from Haimen town, Shantou district in the eastern coast of Guangdong province, the People's Republic of China during a survey for entomopathogenic nematodes. S. aciari sp. n. belongs to the Steinernema glaseri group. It can be separated from all described Steinernema species by the combined morphological and morphometrical characters of various stages of the nematodes. For male, the new species can be recognized by spicule length (86+/-6.3 microm); spicule tip blunt with a hook-like structure; gubernaculum with a short and Y-shaped cuneus and corpus well-separated posteriorly. For infective juvenile, the combination of the following characters: body length (1113+/-68 microm), distance from anterior end to excretory pore (95+/-3.7 microm), tail length (78+/-5.2 microm), and E % (123+/-7) can be used to differentiate the new species from other nematodes. For female, the tail (conoid with a long mamillate terminus and a distinct postanal swelling) and vulva (slightly protruding from body surface with conspicuous double flapped epiptygma) shapes can be used as diagnostic characters for the new species. The new species can also be distinguished from other Steinernema species by DNA sequences of either a partial 28S rDNA or the internal transcribed spacer regions of rDNA, and from the close related species S. glaseri, Steinernema longicaudum CWL05, and Steinernema guangdongense by cross-breeding test.     

Steinernema ichnusae sp. n. (Nematoda: Steinernematidae) a new entomopathogenic nematode from Sardinia Island (Italy)

A new Steinernema species was isolated from three different sandy soil samples along the Platamona Beach, in the north-west coast of Sardinia Island (Italy). This new species is characterized by the following morphological characters: infective third-stage juvenile with a body length of 866 ± 61 (767-969) μm, distance from head to excretory pore of 63 ± 2.7 (59-68) μm, tail length of 81 ± 3.2 (76-89) μm, ratio E (%) 77 ± 3.4 (68-83); male tail with a mucron only in the second generation, spicule length of 66 ± 1.4 (64-67) μm and gubernaculum length of 44 ± 1.4 (43-46) μm in the first generation male; female of first generation with a slight vulval protrusion and ratio D (%) of 53 ± 4.0 (47-63). The new species differs distinctly from the related species (S. feltiae, S. kraussei, S. litorale, S. oregonense and S. cholashanense) in some morphometric values such as percentage of hyaline portion, ratios of gubernaculum/spicule length, spicule head length/width. The DNA analyses of the internal transcribed spacers and D2D3 regions show that the studied nematode isolates are a new species. Cross hybridisation tests with S. feltiae, S. kraussei, S. litorale, S. weiseri and S. oregonense showed that these species were reproductively isolated.     

The bacterium associated with the entomopathogenic nematode Steinernema abbasi (Nematoda: Steinernematidae) isolated from Taiwan

A symbiotic bacterium of the entomopathogenic nematode, Steinernema abbasi, isolated from Taiwan, determined to be a species of Xenorhabdus based on its physiological and biochemical characteristics has been determined to be similar to Xenorhabdus indica of S. abbasi Oman isolate as based on sequence analyses of 16S rDNA.     

Influence of nematode age and culture conditions on morphological and physiological parameters in the bacterial vesicle of Steinernema carpocapsae (Nematoda: Steinernematidae)

Steinernema spp. third-stage infective juveniles (IJs) play a key role in the symbiotic partnership between these entomopathogenic nematodes and Xenorhabdus bacteria. Recent studies suggest that Steinernema carpocapsae IJs contribute to the nutrition and growth of their symbionts in the colonization site (vesicle) [Martens, E.C. and Goodrich-Blair, H., 2005. The S. carpocapsae intestinal vesicle contains a sub-cellular structure with which Xenorhabdus nematophila associates during colonization initiation. Cellular Microbiol. 7, 1723-1735.]. However, the morphological and physiological interactions between Xenorhabdus symbionts and Steinernema IJs are not understood in depth. This study was undertaken to assess the influence of culture conditions and IJ age on the structure, nutrition, and symbiont load (colonization level) of S. carpocapsae vesicles. Our observations indicate the vesicles of axenic IJs are shorter and wider than those of colonized IJs. Moreover, as colonized IJs age the vesicle becomes shorter and narrower and bacterial load declines. The colonization proficiency of several bacterial metabolic mutants was compared between two cultivation conditions: in vitro on lipid agar and in vivo in Galleria mellonella insects. Colonization defects were generally less severe in IJs cultivated in vivo versus those cultivated in vitro. However, IJs from both cultivation conditions exhibited similar declining bacterial load over time. These results suggest that although the vesicle forms in the absence of bacteria, the presence of symbionts within the vesicle may influence its fine structure. Moreover, these studies provide further evidence in support of the concept that the conditions under which steinernematid nematodes are cultivated and stored affect the nutritive content of the vesicle and the bacterial load, and therefore have an impact on the quality of the nematodes for their application as biological control agents.     

Effect of Entomopathogenic Nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) on Meloidogyne mayaguensis Rammah and Hirschmann (Tylenchida: Meloidoginidae) Infection in Tomato Plants

Some studies suggest that entomopathogenic nematodes (EPN) affect plant-parasitic nematode populations. Here, the effects of live and dead IJ of Heterorhabditis bacteriophora JPM4, H. baujardi LPP7, Steinernema feltiae SN and S. carpocapsae All were evaluated against eggs and J2 of the plant-parasitic nematode Meloidogyne mayaguensis. According to treatment, 100 IJ were applied with 350 eggs, 350 J2 or 175 eggs + 175 J2 to tomato plants. Bioassays were conducted in March to May and repeated in September to November 2005. Both experiments lasted 9 weeks, and the variable evaluated was number of galls per plant. When eggs were used for infections in the first trial, plants exhibited lower gall number compared to control when live and dead H. baujardi IJ and live S. feltiae IJ were added (9.7, 4.5, 7.3 and 85.7 galls, respectively). In the second trial, live S. feltiae and S. carpocapasae IJ influenced gall formation compared to control (14.33, 14.57 and 168.02 galls, respectively). When J2 were used for infections, plants with live H. baujardi IJ presented less galls when compared to control in both trials (38.3 and 355.7 galls in the first trial and 145.2 and 326.2 in the second one, respectively). Infection with a mixture of J2 and eggs resulted in fewer galls than when live S. feltiae IJ were present in both trials, compared to control (38.3 and 44.2 galls vs. 275.3 and 192.2 galls, respectively). We conclude that H. baujardi and S. feltiae apparently may be inhibiting egg hatching and J2 infection.