Photorhabdus luminescens LN2转座突变菌株的研究

发光杆菌属Photorhabdus细菌与异小杆属Heterorhabditis昆虫病原线虫的共生关系是这类生物杀虫剂产业化生产和田间应用的基础。本文采用Tn5转座方法构建了共生细菌P. luminescens LN2突变体库;从中筛选出一个对其共生线虫H. indica LN2的生长繁殖有显著促进作用的突变菌株（LN2-M2716）;测定了该突变菌株的菌落特征、对大蜡螟Galleria mellonella及非特异共生线虫H. bacteriophora H06的毒性、对线虫产量的影响。结果显示,LN2-M2716菌株在菌落形态、色素分泌、过氧化氢酶反应、荧光、食物信息作用以及对大蜡螟毒力等方面与野生型菌株差异不明显;但对非特异共生线虫H. bacteriophora H06的毒性及对特异共生线虫H. indica LN2生长繁殖的促进作用方面均明显高于野生型菌株。论文结果为构建支持线虫高产的菌株提供了关键技术。     

Identification and Characterization of a Novel Gene Involved in the trans-Specific Nematicidal Activity of Photorhabdus luminescens LN2

Photorhabdus luminescens subsp. akhurstii LN2 from Heterorhabditis indica LN2 showed nematicidal activity against axenic Heterorhabditis bacteriophora H06 infective juveniles (IJs). Transposon mutagenesis identified an LN2 mutant that supports the growth of H06 nematodes. Tn5 disrupted the namA gene, encoding a novel 364-residue protein and involving the nematicidal activity. The green fluorescent protein-labeled namA mutant was unable to colonize the intestines of H06 IJs.Entomopathogenic Heterorhabditis and Steinernema nematodes are safe and effective bioinsecticides for the biological control of many economically important pests (9). The infective juveniles (IJs) of these nematodes harbor Photorhabdus or Xenorhabdus bacteria as symbionts in their intestines. The IJ nematodes properly maintain and carry the bacteria needed for killing insects and providing a suitable environment for the reproduction of new vectors (5, 8). Different bacterial isolates differ in their ability to support in vitro monoxenic cultures of nonhost nematodes (2, 7, 13) and to retain the bacterial cells in the IJ intestines (2, 8, 11).Strains of Photorhabdus and Xenorhabdus spp. not only show insecticidal activities toward different insects (3, 4, 21) but also exhibit nematicidal activities against nematodes (14, 16, 17). The trans-specific nematicidal activity of Photorhabdus luminescens subsp. akhurstii LN2, a normal symbiont of Heterorhabditis indica LN2 against Heterorhabditis bacteriophora H06, was previously observed (12). The LN2 bacteria may secrete unidentified toxic factors that are lethal to the H06 nematodes. However, the genes of these bacteria involved in the trans-specific nematicidal activities have not been reported.This paper describes the identification, through Tn5 mutagenesis and characterization, of a novel P. luminescens LN2 gene involved in nematicidal activity against the H06 IJs. The colonization of the green fluorescent protein (GFP)-labeled mutant cells in H06 IJ intestines was examined.     

昆虫病原线虫共生细菌共生性的分子生物学研究进展

嗜线虫致病杆菌属Xenorhabdus和发光杆菌属Photorhabdus细菌隶属肠杆菌科Enterobacteriaceae,对多种害虫致病能力强,分别与斯氏属Steinernema和异小杆属Heterorhabditis昆虫病原线虫互惠共生。该两属共生细菌既存在对昆虫寄主的病原性,又存在与线虫寄主的共生性。共生细菌与其线虫寄主的共生性主要表现以下4方面：（1）细菌产生食物信号诱导滞育不取食的感染期线虫恢复;（2）细菌为线虫生长与繁殖提供营养;（3）细菌能于感染期线虫的肠道定殖与生长;（4）细菌产生杀线虫毒素杀死非共生线虫。本文综述了共生菌以上4方面的共生性及其相关的分子机制。     

Entomopathogenic nematodes: natural enemies of root-feeding caterpillars on bush lupine

A new species of soil-dwelling entomopathogenic nematode Heterorhabditis hepialus killed up to 100% (mean=72%) of root-boring caterpillars of a ghost moth Hepialus californicus in coastal shrub lands. When unchecked, ghost moth caterpillars killed bush lupine, Lupinus arboreus. Here we describe this strange food chain. Although unappreciated by ecologists, entomopathogenic nematodes are widespread and probably one of the most important groups of natural enemies for underground insects. The free-living infective juvenile (IJ) of entomopathogenic nematodes searches for host insects in the soil. A single IJ can kill a host, although several often invade together. After entering the host through a spiracle or other orifice, the IJ regurgitates its symbiotic bacterium, Photorhabdus luminescens, which kills the host within 48 h. The bacteria digest the cadaver and provide food for the exponentially growing nematode population inside. The bacteria produce antibiotics and other noxious substances that protect the host cadaver from other microbes in the soil. When the cadaver is exhausted of resources, IJs break the host integument and can disperse. As many as 420,000 IJs can be produced within a large ghost moth caterpillar. Surface soil of the lupine rhizosphere is the primary habitat of IJs of H. hepialus. Attracted to waste gases emitted by insects, the 0.5-mm-long IJs can move 6 cm/day through moist soil. Prevalences of H. hepialus ranged from as high as 78% of rhizospheres in some lupine stands to almost zero in others, but it was absent from no stand at our study site. Field intensities ranged from 0.003 IJs/cm³ of soil to 7.5 IJs/cm³, and correlated roughly with prevalences among sites. Few ghost moth caterpillars (mean=6.7) succeeded in entering lupine roots where prevalence of H. hepialus was highest, and this stand had lowest mortality (0.02) of mature bush lupine. In the three stands with lowest prevalence (mean = 2%) of this nematode, many caterpillars (mean = 38.5) entered roots, and lupine mortality was high (range = 0.41–1.0). Old aerial photographs indicate that the stands with highest recent nematode prevalence have had little or no mass die-off of lupine over the past 40 years. The photos depict repeated die-offs of lupine during the past four decades in stands with lowest recent prevalence of the nematode. This pattern leads us to entertain the hypothesis that the nematode affects vegetation dynamics indirectly through a trophic cascade. Dispersal of entomopathogenic nematodes is little understood. We found that air drying of soil extirpates H. hepialus and speculate that this nematode is dispersed during the wet season in moist soil bits on the exterior of fossorial insects and mammals. H. hepialus colonized some previously unoccupied lupine rhizospheres during the wet winter-spring season and, obversely, became extinct from some rhizosperes as soil dried in summer. Root-feeding insects have only recently been recognized as a force in communities, and the regulation of these important herbivores is still largely an ecological terra incognita. All evidence indicates that entomopathogenic nematodes are found throughout terrestril ecosystems, and we propose that trophic chains similar to those described in this report should not be uncommon.     

Effects of Paenibacillus nematophilus on the entomopathogenic nematode Heterorhabditis megidis

The insect parasitic nematodes Heterorhabditis spp. are mutualistically associated with entomopathogenic bacteria, Photorhabdus spp. A novel association has been detected between H. megidis isolate EU17 and the endospore-forming bacterium Paenibacillus nematophilus. P. nematophilus sporangia adhere to infective juveniles (IJs) of H. megidis and develop in insect hosts along with the nematodes and their symbiont. We tested the effects of P. nematophilus on H. megidis. The yield and quality (size, energy reserves, and storage survival) of IJs were not affected by co-culture in insects with P. nematophilus. Dispersal of IJs in sand and on agar was inhibited by adhering P. nematophilus sporangia: fewer than 2% of IJs with P. nematophilus sporangia reached the bottom of a sand column, compared to 30% of the control treatment. Sporangia significantly reduced infectivity of H. megidis for wax moth larvae in sand, but not in a close contact (filter paper) assay. The results suggest that P. nematophilus may reduce the transmission potential of H. megidis through impeding the motility of IJs.     

噻虫嗪对昆虫病原线虫侵染韭菜迟眼蕈蚊能力的影响

【目的】为提高昆虫病原线虫对韭菜迟眼蕈蚊Bradysia odoriphaga Yang et Zhang幼虫的防治效果,将昆虫病原线虫与环境友好型化学杀虫剂混用是一条有效途径。【方法】测定了噻虫嗪与6个昆虫病原线虫品系混用对韭菜迟眼蕈蚊的作用效果,以及温度和土壤含水量对作用效果的影响,并进行了田间验证。【结果】田间推荐浓度噻虫嗪(100 mg·L~(-1))对6种供试线虫存活无显著影响。处理后3 d,低浓度噻虫嗪(15 mg·L~(-1))分别与6品系线虫混合后处理韭菜迟眼蕈蚊幼虫,其死亡率明显高于线虫和噻虫嗪单用处理。小卷蛾斯氏线虫SF-SN+噻虫嗪、印度异小杆线虫LN2+噻虫嗪和小卷蛾斯氏线虫All+噻虫嗪3种组合发挥杀虫作用的最适温度范围分别为20~25℃、25~30℃和25~30℃,显著高于其他温度;最适土壤含水量范围为10%~18%,也显著高于其他湿度。大田条件下,施用后7 d,单用噻虫嗪、线虫+噻虫嗪组合处理对韭菜迟眼蕈蚊的防治效果显著高于单线虫,且以芫菁夜蛾斯氏线虫SF-SN+噻虫嗪的防治效果最高,达到93%以上。【结论】芫菁夜蛾斯氏线虫SF-SN品系与噻虫嗪组合联合防治韭菜迟眼蕈蚊效果最好。     

In vitro production of the biological control agent Heterorhabditis indica SL0708 in different agar media

ABSTRACT

Heterorhabditis indica SL0708 is an entomopathogenic nematode isolated from Valle del Cauca-Colombia, whose bacterial symbiont, Photorhabdus luminescens subsp. akhurstii SL0708, has potential to control pests of economic importance in Colombia. Since in vivo production does not supply its demand, this investigation evaluated H. indica SL0708 production on different agar media. Five culture media (I, II, III, IV and V) were evaluated for productivity and pathogenicity of infective juveniles (IJs). IJs emerged between 11 and 16 days after inoculation in all media, with a total of 2.7?×?10⁴ and 4.7?×?10⁶ IJs produced during 15 days after IJs emergence, with maximum productivity at day five and high variability. Pathogenicity to Galleria mellonella larvae was not significantly different between in vitro and in vivo produced IJs on all media tested. Media IV and V were selected for their higher productivity. Subsequently, nematode inoculum size was evaluated in selected media at 2000, 4000 and 6000?IJs ml^?1, but significant differences were not observed in productivity and pathogenicity. Lastly, lipid source influence was evaluated in medium IV comparing canola, olive and soy oils. None of the plant-based oils had a significant effect on IJs production and pathogenicity. A medium was selected for H. indica SL0708 IJs production which was suitable in terms of productivity, culture time and pathogenicity of IJs produced. The medium and parameters selected in this study could be applied as an alternative for mass production of this entomopathogenic nematodes.     

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.     

Laboratory Evaluation of Seven Pakistani Strains of Entomopathogenic Nematodes Against a Stored Grain Insect Pest, Pulse beetle Callosobruchus chinensis (L.)

Seven Pakistani strains of entomopathogenic nematodes belonging to the genera Steinernema and Heterorhabditis were tested against last instar and adult stages of the pulse beetle, Callosobruchus chinensis (L.). These nematodes included Steinernema pakistanense Shahina, Anis, Reid and Maqbool (Ham 10 strain); S. asiaticum Anis, Shahina, Reid and Rowe (211 strain); S. abbasi Elawad, Ahmad and Reid (507 strain); S. siamkayai Stock, Somsook and Reid (157 strain); S. feltiae Filipjev (A05 strains); Heterorhabditis bacteriophora Poinar (1743 strain); and H. indica Poinar, Karunakar and David (HAM-64 strain). Activity of all strains was determined at four different nematode densities in Petri dishes and in concrete containers. A significant nematode density effect was detected for all nematode species tested. Overall, Heterorhabditis bacteriophora, S. siamkayai, and S. pakistanense were among those that showed the highest virulence to pulse beetle larvae and adults. For all nematode species, the last larval stage of the pulse beetle seems to be more susceptible than the adult. LC(50) values in Petri dish and concrete containers were 14-340 IJs/larvae and 41-441 IJs/larvae, respectively, and 59-1376 IJs/adult and 170-684/adult, respectively.     

Phylogenetic and cophylogenetic relationships of entomopathogenic nematodes (Heterorhabditis: Rhabditida) and their symbiotic bacteria (Photorhabdus: Enterobacteriaceae)

Mutualistic association between entomopathogenic Photorhabdus bacteria and Heterorhabditis nematodes represents one of the emerging model systems in symbiosis studies, yet little is known about this partnership from a coevolutionary perspective. Herein, we investigated phylogenetic and cophylogenetic relationships of Heterorhabditis and Photorhabdus strains using molecular markers Internal Transcribed Spacer and gyrase B gene sequences, respectively. The phylogenies presented consistent, well supported, monophyletic groups in the parsimonious and likelihood analyses for both the nematode and bacterial strains and supported the placement of currently recognized taxa, from which a potentially new Heterorhabditis species represented by a Thailand strain MP68 was identified. While the nematode strains with distant geographic distributions showed no detectable phylogenetic divergence within H. bacteriophora or H. georgiana monophyletic groups, their respective symbiotic bacteria speciated into two Photorhabdus species: P. luminescens and P. temperata, indicating the occurrence of duplication. Although such evolutionary process reduces the phylogenetic congruence between Heterorhabditis nematodes and Photorhabdus bacteria, global cophylogenetic tests using ParaFit detected a highly significant correlation between the two phylogenies (ParaFitGlobal = 0.001). Further, the associations between H. zealandica, H. indica and H. megidis strains and their symbiotic bacteria exhibited significant contribution to the overall cophylogenetic structure. Overall, this study reveals evidence of coevolution between Photorhabdus bacteria and Heterorhabditis nematodes and provides a framework for further examination of the evolution of these associations.     

Mutualism and pathogenesis in Xenorhabdus and Photorhabdus: two roads to the same destination

Photorhabdus and Xenorhabdus bacteria colonize the intestines of the infective soil-dwelling stage of entomophagous nematodes, Heterorhabditis and Steinernema, respectively. These nematodes infect susceptible insect larvae and release the bacteria into the insect blood. The bacteria kill the insect larvae and convert the cadaver into a food source suitable for nematode growth and development. After several rounds of reproduction the nematodes are recolonized by the bacteria before emerging from the insect cadaver into the soil to search for a new host. Photorhabdus and Xenorhabdus bacteria therefore engage in both pathogenic and mutualistic interactions with different invertebrate hosts as obligate components of their life cycle. In this review we aim to describe current knowledge of the molecular mechanisms utilized by Photorhabdus and Xenorhabdus to control their host-dependent interactions. Recent work has established that there is a trade-off between pathogenicity and mutualism in both these species of bacteria suggesting that the transition between these interactions must be under regulatory control. Despite the superficial similarity between the life cycles of these bacteria, it is now apparent that the molecular components of the regulatory networks controlling pathogenicity and mutualism in Photorhabdus and Xenorhabdus are very different.     

Effect of Soil Texture and Moisture on the Activity of Entomopathogenic Nematodes Against Female Boophilus annulatus Ticks

Soil texture, chemistry and moisture have a profound effect upon the activity and persistence of entomopathogenic nematodes (EPNs). Whereas nematodes’ natural habitat is within the soil, ticks and other arthropod pests prefer to stay on the soil surface and under stones or leaf litter; they spend much of their life cycle in the humid environment of the soil upper layer, therefore consideration of the effect of the soil environment on nematode activity is a pre-requisite for the sucessful use of EPNs against arthropod pests. In the present study we investigated the effects of soil type, and humidity on various nematode strains and on their effectiveness against ticks. Many infective juveniles (IJs) of Steinernema carpocapsae and S. riobrave were found in the uppermost soil layer whereas the heterorhabditid strains were almost absent from the upper 6 cm of the soil profile. The IJs of S. feltiae, and the S. carpocapsae strain S-20, exhibited an intermediate behavior. It was found that the activity of IJs of S. carpocapsae in the soil upper layer (1 cm depth) was strongly affected by soil type: the greatest number of IJs were recorded from sandy loam soil; less were found in the lighter soils – ‘Marine sand’ and ‘Calcareous sandstone’ – and only very few were recovered from heavy soils. Strikingly, even when the soil moisture was low and the number of nematodes found in the upper layer correspondingly low, tick mortality remained high. The results demonstrate: (a) the possible use of the nematodes as an anti-tick agent; (b) the importance of knowing the exact interaction of nematodes with the immediate environment of the pest, in order to optimize the pest-control activity of the nematode.     

A survival-reproduction trade-off in entomopathogenic nematodes mediated by their bacterial symbionts

In this work, we investigate the investment of entomopathogenic Steinernema nematodes (Rhabditidae) in their symbiotic association with Xenorhabdus bacteria (Enterobacteriaceae). Their life cycle comprises two phases: (1) a free stage in the soil, where infective juveniles (IJs) of the nematode carry bacteria in a digestive vesicle and search for insect hosts, and (2) a parasitic stage into the insect where bacterial multiplication, nematode reproduction, and production of new IJs occur. Previous studies clearly showed benefits to the association for the nematode during the parasitic stage, but preliminary data suggest the existence of costs to the association for the nematode in free stage. IJs deprived from their bacteria indeed survive longer than symbiotic ones. Here we show that those bacteria-linked costs and benefits lead to a trade-off between fitness traits of the symbiotic nematodes. Indeed IJs mortality positively correlates with their parasitic success in the insect host for symbiotic IJs and not for aposymbiotic ones. Moreover mortality and parasitic success both positively correlate with the number of bacteria carried per IJ, indicating that the trade-off is induced by symbiosis. Finally, the trade-off intensity depends on parental effects and, more generally, is greater under restrictive environmental conditions.     

Interactions of two idiobiont parasitoids (Hymenoptera: Ichneumonidae) of codling moth (Lepidoptera: Tortricidae) with the entomopathogenic nematode Steinernema carpocapsae (Rhabditida: Steinernematidae)

Simultaneous use of parasitoids and entomopathogenic nematodes for codling moth (CM) control could produce an antagonistic interaction between the two groups resulting in death of the parasitoid larvae. Two ectoparasitic ichneumonid species, Mastrus ridibundus and Liotryphon caudatus, imported for classical biological control of cocooned CM larvae were studied regarding their interactions with Steinernema carpocapsae. Exposure of M. ridibundus and L. caudatus developing larvae to infective juveniles (IJs) of S. carpocapsae (10 IJs/cm2; approximately LC(80-90) for CM larvae) within CM cocoons resulted in 70.7 and 85.2% mortality, respectively. However, diapausing full grown parasitoid larvae were almost completely protected from nematode penetration within their own tightly woven cocoons. M. ridibundus and L. caudatus females were able to detect and avoid ovipositing on nematode-infected cocooned CM moth larvae as early as 12h after treatment of the host with IJs. When given the choice between cardboard substrates containing untreated cocooned CM larvae and those treated with an approximate LC95 of S. carpocapsae IJs (25 IJs/cm2) 12, 24, or 48h earlier, ovipositing parasitoids demonstrated a significant preference for untreated larvae. The ability of these parasitoids to avoid nematode-treated larvae and to seek out and kill cocooned CM larvae that survive nematode treatments enhances the complementarity of entomopathogenic nematodes and M. ridibundus and L. caudatus.     

Nematodes, bacteria, and flies: a tripartite model for nematode parasitism

More than a quarter of the world's population is infected with nematode parasites, and more than a hundred species of nematodes are parasites of humans [1-3]. Despite extensive morbidity and mortality caused by nematode parasites, the biological mechanisms of host-parasite interactions are poorly understood, largely because of the lack of genetically tractable model systems. We have demonstrated that the insect parasitic nematode Heterorhabditis bacteriophora, its bacterial symbiont Photorhabdus luminescens, and the fruit fly Drosophila melanogaster constitute a tripartite model for nematode parasitism and parasitic infection. We find that infective juveniles (IJs) of Heterorhabditis, which contain Photorhabdus in their gut, can infect and kill Drosophila larvae. We show that infection activates an immune response in Drosophila that results in the temporally dynamic expression of a subset of antimicrobial peptide (AMP) genes, and that this immune response is induced specifically by Photorhabdus. We also investigated the cellular and molecular mechanisms underlying IJ recovery, the developmental process that occurs in parasitic nematodes upon host invasion and that is necessary for successful parasitism. We find that the chemosensory neurons and signaling pathways that control dauer recovery in Caenorhabditis elegans also control IJ recovery in Heterorhabditis, suggesting conservation of these developmental processes across free-living and parasitic nematodes.     

Comparison of Bioassays to Measure Virulence of Different Entomopathogenic Nematodes

Five bioassays were compared for their usefulness to determine the virulence of four nematode strains. The objective of this study was to develop standard assays for particular nematode species. In all assays, the nematodes Steinernema feltiae (strain UK), S. riobravis, S. scapterisci Argentina and Heterorhabditis bacteriophora HP88 were exposed to Galleria mellonella larvae. All bioassays except the sand column assay were conducted in multi-well plastic dishes. In the penetration rate assay, the number of individual nematodes invading the insect was determined after a 48-h exposure to 200 infective juveniles (IJs). In the one-on-one assay, each larva was exposed to an individual nematode for 72 h before insect mortality was recorded. In the exposure time assay, insect mortality was recorded after exposure to 200 IJs for variable time periods. The dose-response assay involved exposing larvae to different nematode concentrations over the range 1-200 IJs/insect and recording mortality every 24 h for a 96-h period. In the sand columns assay, insects were placed in the bottom of a plastic cylinder filled with sand. Nematodes were applied on top of the sand and insect mortality was determined after IJs had migrated through the cylinder. The highest mortality level in the sand column assay was obtained with IJs of S. feltiae followed by H. bacteriophora; treatments with S. riobravis and S. scapterisci produced low levels of insect mortality. In the other four assays, S riobravis was the most virulent, followed by S. feltiae, H. bacteriophora and S. scapterisci. In the exposure time assay, rapid mortality was achieved when the insects were exposed to S. feltiae and S. riobravis. For these nematode species, a gradual increase in the number of individuals which penetrated into cadavers was recorded. Conversely, the number of nematodes in the cadavers of insects infected by H. bacteriophora and S. scapterisci remained low during the entire exposure period. In this assay, exposing the insects to these nematodes resulted in a gradual increase in mortality. In the dose-response assay, complete separation among nematode species was obtained only after 48 h of incubation at a concentration of 15 IJs/insect. LD and LD values were calculated from 50 90 dose-response assay data. However, these values did not indicate differences among the different nematode species. The present study demonstrated the variation in entomopathogenic nematode performance in different bioassays and supports the notion that one common bioassay cannot be used as a universal measure of virulence for all species and strains because nematodes differ in their behavior. Furthermore, particular assays should be used for different purposes. To select a specific population for use against a particular insect, assays that are more laborious but which simulate natural environmental conditions (e.g. the sand column assay) or invasion by the nematode (e.g. the penetration rate assay) should be considered. In cases where commercial production batches of the same nematode strains are compared, simple and fast assays are needed (e.g. the one-on-one and exposure time assays). Further studies are needed to determine the relationships between data obtained in each assay and nematode efficacy in the field.     

Nematicidal prenylated flavanones from Phyllanthus niruri

Two prenylated flavanones have been isolated from the hexane extract of Phyllanthus niruri plant. The structure of these flavanones were established as 8-(3-Methyl-but-2-enyl)-2-phenyl chroman-4-one (1) and 2-(4-hydroxyphenyl)-8-(3-methyl-but-2-enyl)-chroman-4-one (2) on the basis of spectral analysis. These were evaluated for nematicidal activity against root-knot, Meloidogyne incognita, and reniform, Rotylenchulus reniformis, nematodes. Compound 2 exhibited nematicidal activity at par with the standard carbofuran (LC50 3.3 and 3.1ppm, respectively) when tested against reniform nematode. The LC50 value against root-knot nematode was found to be 14.5ppm. Compound 1 however, showed moderate activity against both the test nematodes.     

Host cadavers protect entomopathogenic nematodes during freezing

The entomopathogenic nematodes Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema glaseri, and Steinernema feltiae were exposed to freezing while inside their hosts. Survival was assessed by observing live and dead nematodes inside cadavers and by counting the infective juveniles (IJs) that emerged after freezing. We (1) measured the effects of 24h of freezing at different times throughout the course of an infection, (2) determined the duration of freezing entomopathogenic nematodes could survive, (3) determined species differences in freezing survival. Highest stage-specific survival was IJs for S. carpocapsae, and adults for H. bacteriophora. When cadavers were frozen two or three days after infection, few IJs emerged from them. Freezing between five and seven days after infection had no negative effect on IJ production. No decrease in IJ production was measured for H. bacteriophora after freezing. H. bacteriophora also showed improved survival inside versus outside their host when exposed to freezing.     

A Phosphopantetheinyl transferase homolog is essential for Photorhabdus luminescens to support growth and reproduction of the entomopathogenic nematode Heterorhabditis bacteriophora

The bacterium Photorhabdus luminescens is a symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora. The nematode requires the bacterium for infection of insect larvae and as a substrate for growth and reproduction. The nematodes do not grow and reproduce in insect hosts or on artificial media in the absence of viable P. luminescens cells. In an effort to identify bacterial factors that are required for nematode growth and reproduction, transposon-induced mutants of P. luminescens were screened for the loss of the ability to support growth and reproduction of H. bacteriophora nematodes. One mutant, NGR209, consistently failed to support nematode growth and reproduction. This mutant was also defective in the production of siderophore and antibiotic activities. The transposon was inserted into an open reading frame homologous to Escherichia coli EntD, a 4'-phosphopantetheinyl (Ppant) transferase, which is required for the biosynthesis of the catechol siderophore enterobactin. Ppant transferases catalyze the transfer of the Ppant moiety from coenzyme A to a holo-acyl, -aryl, or -peptidyl carrier protein(s) required for the biosynthesis of fatty acids, polyketides, or nonribosomal peptides. Possible roles of a Ppant transferase in the ability of P. luminescens to support nematode growth and reproduction are discussed.