Photorhabdus aballayi sp. nov. and Photorhabdus luminescens subsp. venezuelensis subsp. nov., isolated from Heterorhabditis amazonensis entomopathogenic nematodes.

Six Gram-negative, rod-shaped bacterial strains isolated from Heterorhabditis amazonensis entomopathogenic nematodes were characterized to determine their taxonomic position. 16S rRNA and gyrB gene sequences indicate that they belong to the class Gammaproteobacteria, family Morganellaceae and genus Photorhabdus, and that some of them are conspecifics. Two of them, APURET and JART, were selected for further molecular characterization using whole genome- and whole-proteome-based phylogenetic reconstructions and sequence comparisons. Phylogenetic reconstructions using whole genome and whole proteome sequences show that strains APURET and JART are closely related to Photorhabdus luminescens subsp. luminescens ATCC 29999T and to P. luminescens subsp. mexicana MEX47-22T. Moreover, digital DNA-DNA hybridization (dDDH) values between APURET and P. luminescens subsp. luminescens ATCC 29999T, APURET and P. luminescens subsp. mexicana MEX47-22T, and APURET and JART are 61.6, 61.2 and 64.1 %, respectively. These values are below the 70 % divergence threshold that delimits prokaryotic species. dDDH scores between JART and P. luminescens subsp. luminescens ATCC 29999T and between JART and P. luminescens subsp. mexicana MEX47-22T are 71.9 and 74.8 %, respectively. These values are within the 70 and 79 % divergence thresholds that delimit prokaryotic subspecies. Based on these genomic divergence values, APURET and JART represent two different taxa, for which we propose the names: Photorhabdus aballayi sp. nov. with APURET (=CCM 9236T =CCOS 2019T) as type strain and Photorhabdus luminescens subsp. venezuelensis subsp. nov. with JART (=CCM 9235T =CCOS 2021T) as type strain. Our study contributes to a better understanding of the biodiversity of an important bacterial group with enormous biotechnological and agricultural potential.

Photorhabdus antumapuensis sp. nov., a novel symbiotic bacterial species associated with Heterorhabditis atacamensis entomopathogenic nematodes.

One motile, Gram-negative, non-spore-forming and rod-shaped symbiotic bacterium, strain UCH-936T, was isolated from Heterorhabditis atacamensis nematodes. Results of biochemical, physiological, molecular and genomic analyses suggest that it represents a new species, which we propose to name Photorhabdus antumapuensis sp. nov. Digital DNA-DNA hybridization shows that strain UCH-936T is more closely related to Photorhabdus kleinii DSM 23513T, but shares solely 50.5 % similarity, which is below the 70% cut-off value that delimits species boundaries in bacteria. Phylogenetic reconstructions using whole-genome sequences show that strain UCH-936T forms a unique clade, suggesting its novel and distinct taxonomic status again. Similarly, comparative genomic analyses shows that the virulence factor flagella-related gene fleR, the type IV pili-related gene pilL and the vibriobactin-related gene vibE are present in the genome of strain UCH-936T but absent in the genomes of its closest relatives. Biochemically and physiologically, UCH-936T differs also from all closely related Photorhabdus species. Therefore, Photorhabdus antumapuensis sp. nov. is proposed as a new species with the type strain UCH-936T (CCCT 21.06T=CCM 9188T=CCOS 1991T).

Xenorhabdus lircayensis sp. nov., the symbiotic bacterium associated with the entomopathogenic nematode Steinernema unicornum.

Xenorhabdus is a symbiotic group of bacteria associated with entomopathogenic nematodes of the family Steinernematidae. Although the described Steirnernema species list is extensive, not all their symbiotic bacteria have been identified. One single motile, Gram-negative and non-spore-forming rod-shaped symbiotic bacterium, strain VLST, was isolated from the entomopathogenic nematode Steinernema unicornum. Analyses of the 16S rRNA gene determined that the VLST isolate belongs to the genus Xenorhabdus, and its closest related species is Xenorhabdus szentirmaii DSM 16338T (98.2 %). Deeper analyses using the whole genome for phylogenetic reconstruction indicate that VLST exhibits a unique clade in the genus. Genomic comparisons considering digital DNA-DNA hybridization (dDDH) values confirms this result, showing that the VLST values are distant enough from the 70 % threshold suggested for new species, sharing 30.7, 30.5 and 30.3 % dDDH with Xenorhabdus khoisanae MCB, Xenorhabdus koppenhoeferi DSM 18168T and Xenorhabdus miraniensis DSM 18168T, respectively, as the closest species. Detailed physiological, biochemical and chemotaxonomic tests of the VLST isolate reveal consistent differences from previously described Xenorhabdus species. Phylogenetic, physiological, biochemical and chemotaxonomic approaches show that VLST represents a new species of the genus Xenorhabdus, for which the name Xenorhabdus lircayensis sp. nov. (type strain VLST=CCCT 20.04T=DSM 111583T) is proposed.

Infection parameters of Heterorhabditis amazonensis (Nematoda: Heterorhabditidae) in different stages of Hibiscus pink mealybug.

The pink hibiscus mealybug Maconellicoccus hirsutus (Hemiptera: Pseudococcidae) is an invasive pest of an enormous variety of crops and has become a concern in many parts of the world. Early attempts to control M. hirsutus with chemical insecticides and cultural methods have failed due to the cryptic habit of the insect. We assessed the entomopathogenic nematode Heterorhabditis amazonensis as a biological agent against different insect stages. Comparing different concentrations of the nematode, insect females were very susceptible, with more than 90% of the insects killed. In second and third nymphal stages mortality rates varied from 20 to 60% depending on the nematode concentration. The first nymphal stage as much less susceptible to nematodes due to their small size. The number of nematodes capable of invading the insect host did not vary between the different concentrations. However, the LC50 for females (35.2 IJ/insect), second and third nymphal stages (83.9 IJ/insect) demonstrated that H. amazonensis should be considered as a potential biocontrol agent of the pink hibiscus mealybug.The pink hibiscus mealybug Maconellicoccus hirsutus (Hemiptera: Pseudococcidae) is an invasive pest of an enormous variety of crops and has become a concern in many parts of the world. Early attempts to control M. hirsutus with chemical insecticides and cultural methods have failed due to the cryptic habit of the insect. We assessed the entomopathogenic nematode Heterorhabditis amazonensis as a biological agent against different insect stages. Comparing different concentrations of the nematode, insect females were very susceptible, with more than 90% of the insects killed. In second and third nymphal stages mortality rates varied from 20 to 60% depending on the nematode concentration. The first nymphal stage as much less susceptible to nematodes due to their small size. The number of nematodes capable of invading the insect host did not vary between the different concentrations. However, the LC50 for females (35.2 IJ/insect), second and third nymphal stages (83.9 IJ/insect) demonstrated that H. amazonensis should be considered as a potential biocontrol agent of the pink hibiscus mealybug.

Soil texture, infective juvenile concentration, and soil organic matter influence the efficacy of Steinernema feltiae isolate Lican Ray.

The influence of infective juveniles (IJs) concentration, soil texture, IJ-host distance and organic matter (OM) content, at different decomposition degree, on the activity of the nematode Steinernema feltiae isolate Lican Ray (LR) was examined using Galleria mellonella larvae. Bioassays were conducted in tubes of varied length, filled with soil of different textures, placed either vertically or horizontally. In the concentration assay, highest IJ concentrations caused maximum larval mortality in all soil types (440, 2,200 and 4,400 IJs in clay, loam and sandy loam). In the second assay, soil texture (loam, clay or sandy loam) interacted significantly with IJ-host distance (10, 20, 30, 40 cm, horizontally; 30, 50, 70 cm, vertically), and distances of 30 cm or more affected IJ effectiveness on the control of G. mellonella. The effect was stronger in clay and sandy loam than in loam soils, where IJ moved up to 40 cm horizontally and 70 cm vertically. In the third assay, OM content (0, 2, 4, 6 and 8%) and its decomposition degree (initial, medium and advanced) did not interact to influence IJ movement in all treatments that contained any percentage of OM (2-8%). Only in the soil with no OM, IJ did not cause death of larvae at all. These results show the potential of S. feltiae LR to be used in different soil textures, as long as the content of soil OM allows its dispersal and host infection, in order to optimize the pest-control activity of the nematode.The influence of infective juveniles (IJs) concentration, soil texture, IJ-host distance and organic matter (OM) content, at different decomposition degree, on the activity of the nematode Steinernema feltiae isolate Lican Ray (LR) was examined using Galleria mellonella larvae. Bioassays were conducted in tubes of varied length, filled with soil of different textures, placed either vertically or horizontally. In the concentration assay, highest IJ concentrations caused maximum larval mortality in all soil types (440, 2,200 and 4,400 IJs in clay, loam and sandy loam). In the second assay, soil texture (loam, clay or sandy loam) interacted significantly with IJ-host distance (10, 20, 30, 40 cm, horizontally; 30, 50, 70 cm, vertically), and distances of 30 cm or more affected IJ effectiveness on the control of G. mellonella. The effect was stronger in clay and sandy loam than in loam soils, where IJ moved up to 40 cm horizontally and 70 cm vertically. In the third assay, OM content (0, 2, 4, 6 and 8%) and its decomposition degree (initial, medium and advanced) did not interact to influence IJ movement in all treatments that contained any percentage of OM (2­8%). Only in the soil with no OM, IJ did not cause death of larvae at all. These results show the potential of S. feltiae LR to be used in different soil textures, as long as the content of soil OM allows its dispersal and host infection, in order to optimize the pest-control activity of the nematode.

Entomopathogenic nematology in Latin America: A brief history, current research and future prospects.

Since the 1980s, research into entomopathogenic nematodes (EPNs) in Latin America has produced many remarkable discoveries. In fact, 16 out of the 117 recognized species of EPNs have been recovered and described in the subcontinent, with many more endemic species and/or strains remaining to be discovered and identified. In addition, from an applied perspective, numerous technological innovations have been accomplished in relation to their implementation in biocontrol. EPNs have been evaluated against over 170 species of agricultural and urban insects, mites, and plant-parasitic nematodes under laboratory and field conditions. While much success has been recorded, many accomplishments remain obscure, due to their publication in non-English journals, thesis dissertations, conference proceedings, and other non-readily available sources. The present review provides a brief history of EPNs in Latin America, including current findings and future perspectives.

First Report and Comparative Study of Steinernema surkhetense (Rhabditida: Steinernematidae) and its Symbiont Bacteria from Subcontinental India.

Two populations (CS19 and CS20) of entomopathogenic nematodes were isolated from the soils of vegetable fields from Bijnor district, India. Based on morphological, morphometrical, and molecular studies, the nematodes were identified as Steinernema surkhetense. This work represents the first report of this species in India. The infective juveniles (IJs) showed morphometrical and morphological differences, with the original description based on longer IJs size. The IJs of the Indian isolates possess six ridges in their lateral field instead of eight reported in the original description. The analysis of ITS-rDNA sequences revealed nucleotide differences at 345, 608, and 920 positions in aligned data. No difference was observed in D2-D3 domain. The S. surkhetense COI gene was studied for the first time as well as the molecular characterization of their Xenorhabdus symbiont using the sequences of recA and gyrB genes revealing Xenorhabdus stockiae as its symbiont. These data, together with the finding of X. stockiae, suggest that this bacterium is widespread among South Asian nematodes from the "carpocapsae" group. Virulence of both isolates was tested on Spodoptera litura. The strain CS19 was capable to kill the larvae with 31.78 IJs at 72 hr, whereas CS20 needed 67.7 IJs.

First Report of Heterorhabditis amazonensis from Venezuela and Characterization of Three Populations.

During a survey in western Venezuela in 2011, three new populations of Heterorhabditis amazonensis (LPV081, LPV156, and LPV498) were isolated. Some differences were found in terms of morphometry compared with the original description; however, the distance from the anterior end to the excretory pore is the most variable character; significantly shorter in all infective juveniles and in other developmental stages depending on the population. According to a Principal Component Analysis, LPV498 possesses more differences in morphometric characteristics and can be separated from the other two. Those intraspecific differences could be attributed to the geographic origin of the nematode. Molecular studies of ITS regions demonstrated that the sequences of the Venezuelan strains were identical to those of the type species originally isolated in the Brazilian Amazonian forest. This is an interesting fact because in several studies on heterorhabditids, intraspecific variability has been recorded. Herein, we present the first report of H. amazonensis in Venezuela and the characterization of three populations of this species.

Steinernema goweni n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Zulia State, Venezuela.

A nematode from the genus Steinernema was isolated in Zulia state (North-western, Venezuela). Morphological, morphometric and molecular studies indicated that this nematode belongs to the "bicornutum" group and is described herein as Steinernema goweni n. sp. Steinernema goweni n. sp. is characterised by morphometrics of its infective juveniles, with body length 640 (531-719) µm, pharynx length of 119 (109-126) µm, tail of 67 (59-89) µm, c ratio = 9 (6-11) and E% = 77 (48-94). First generation male specimens can be recognised by the shape and size of spicules (55 (50-57) µm long) and gubernaculum (35 (30-40) µm long), and %D value 42 (28-59), which is at the lower limit within the "bicornutum" group. The number of genital papillae in males is also distinctive for S. goweni n. sp. presenting up to 27 (13 pairs + 1) papillae in 15% of specimens. The phasmids can be seen in scanning electron microscopy in all stages, characters not previously reported within the "bicornutum" group. Phylogenetic analyses of the "bicornutum" group based on both ITS and D2D3 regions showed a clear separation of S. goweni n. sp. from the other species. In both analyses S. goweni n. sp. formed a strongly supported group of American species.

Size does matter: the life cycle of Steinernema spp. in micro-insect hosts.

The life cycle of four Steinernema species was observed in 4 insect micro-insect host species (less than 5mm long). Several parameters were measured: sex ratio of invading nematodes, percentages of host infection and offspring production, penetration rate of infective juveniles per insect and number of new generation of infective juveniles. All parameters varied among nematode species, micro-host species and application rates. All Steinernema species were capable to invade micro-insect hosts, however, invasion decreased as insect size decreased and as nematode species size increased. None of the nematode species achieved 100% mortality in the micro-hosts. Due to size differences in the nematode species, Steinernema glaseri was less capable of completing its life cycle and unable to invade smaller hosts whereas S. riobrave completed its life cycle in smaller hosts more frequently. The number of invading nematodes and the number of offspring produced had the same levels regardless of the nematode application rates, those results showed a maximum top in the number of individuals per micro-insect host. The offspring production in thrips species was only possible by endotokia matricida in S. riobrave. The sex of the invader nematodes also impeded the life cycle of S. affine because males colonized the entire body of the micro-insect host leaving no room for female invasion. The size of the host plays an irrefutable role in limiting the development of nematodes and it appears improbable that an entomopathogenic nematode population can persist in the soil without the presence of bigger insects.

Ammonia concentration at emergence and its effects on the recovery of different species of entomopathogenic nematodes.

The life cycle of entomopathogenic nematodes (EPN) occurs inside an insect cadaver and an accumulation of ammonia initiates as a consequence of the nematodes defecation. This accumulation reduces the food resources quality and creates a detrimental environment for nematodes. When a given ammonia concentration is reached, the nematodes start their emergence process, searching for a new host. In the present work, this parameter, ammonia triggering point (ATP) was measured in 7 Steinernema species/strains. The effect of different ammonia concentrations on the recovery process and their consequences in the nematodes survival were also investigated. The results indicate that ATP varies among nematode species; Steinernema glaseri showed the highest ATP of the evaluated species (1.98±2.6 mg of NH4-N*g of Galleria mellonella(-1)); whereas Steinernema riobrave presented the lowest ATP (1.16±0.1 mg of NH4-N*g of G. mellonella(-1)). On the other hand, the nematode emergence could be a repulsive response when ATP is reached. As the ammonia concentration increased the recovery percentage of Steinernema feltiae (Chile strain) dropped gradually from 79.4±11.9% in the control treatment to 0% when 1mg of NH4-N*ml of bacterial broth(-1) was added. It is possible, that emergence process could be a repulsive response of the nematodes due to ammonia concentration when is reaching the ATP. The role of ammonia inside the insect cadavers, might suggests connections with some stages of the EPN life cycle.

Characterization of Xenorhabdus and Photorhabdus bacteria by Fourier transform mid-infrared spectroscopy with attenuated total reflection (FT-IR/ATR).

The use of Fourier transform mid-infrared spectroscopy with attenuated total reflection for characterizing entomopathogenic bacteria from genera Xenorhabdus and Photorhabdus is evaluated for the first time. The resulting spectra of Xenorhabdus poinarii and Photorhabdus luminiscens were compared with the spectrum of Escherichia coli samples. The absorption spectra generated by the bacteria samples, were very different at the region below 1400cm(-1) which represents the stretching vibrations of phosphate and carbohydrates. Star diagrams of the fingerprint section of nematodes spectra (between 1,350 and 1,650 cm(-1)) for separation between spectra was used and showed to be a useful tool for classification purposes.

Steinernema feltiae: ammonia triggers the emergence of their infective juveniles.

Entomopathogenic nematodes complete their life cycles inside dead insects. The emergence of new infective juveniles from the cadaver has been attributed (but never demonstrated) to food depletion or to the accumulation of metabolites from the breakdown of the host's tissues. Here we give evidence that emergence is triggered by ammonia, a product of nematode defecation. We found that the emergence of Steinernemafeltiae infective juveniles from Galleriamellonella cadavers was stimulated by a particular level of ammonia. Emergence was delayed when ammonia in the cadaver was decreased and was prompted when increased. These findings will further improve the understanding of the nematode life cycle. Here we speculate that production of infective juveniles can be mediated by ammonia and work in a manner analogous to that of the dauer recovery inhibiting factor (DRIF) in Caenorhabditiselegans.

Facultative scavenging as a survival strategy of entomopathogenic nematodes.

Entomopathogenic nematodes cannot be considered only as parasitic organisms. With dead Galleria mellonella larvae, we demonstrated that these nematodes use scavenging as an alternative survival strategy. We consider scavenging as the ability of entomopathogenic nematodes to penetrate, develop and produce offspring in insects which have been killed by causes other than the nematode-bacteria complex. Six Steinernema and two Heterorhabditis species scavenged but there were differences among them in terms of frequency of colonisation and in the time after death of G. mellonella larvae that cadavers were penetrated. The extremes of this behaviour were represented by Steinernema glaseri which was able to colonise cadavers which had been freeze-killed 240 h earlier and Heterorhabditis indica which only colonised cadavers which had been killed up to 72 h earlier. Also, using an olfactometer, we demonstrated that entomopathogenic nematodes were attracted to G. mellonella cadavers.