Characterizing entomopathogenic nematode (EPN) biogeography with a goal of augmentation and conservation biological control requires fine-scale taxonomic resolution, because closely related EPN species can exhibit divergent phenotypes for key properties such as habitat adaptation and insect host specificity. Consequently, we employed high throughput genome sequencing (HTS) to identify and compare EPNs and natural enemies of EPNs in 58 citrus orchards in 2 ecoregions in Egypt (El Beheira and Al Qalyubia governorates). We designed improved primers targeting the ITS2 rDNA to discriminate EPN species and used pre-reported primers targeting D2-D3 region for soil microarthropods. Five EPN species ( Heterorhabditis bacteriophora , H. indica , H. taysearae , Steinernema glaseri , and S. scapterisci ) and one steinernematid not represented in Genbank databases were detected. This is the first report of S. scapterisci and possibly the unknown (perhaps undescribed) species in Egypt. Only heterorhabditid species, dominated by H. indica , were detected in the reclaimed, sandy desert soils of El Beheira governorate. In the fine textured, ancient farming lands of the Nile delta all six species were detected, but at lower frequency and abundance. Microarthropod family richness ( P = 0.01) and abundance ( P = 0.001) was higher in the reclaimed lands than in the Nile Delta. Soil clay content, pH and elevation explained significant variation in the mite community structure. Population density of H. indica , the only EPN found consistently and at high abundance in El-Beheira, was inversely related to abundance of species in the nematophagous mite family Rhodacaridae.

The pupal soil cell of the pecan weevil, Curculio caryae (Coleoptera: Curculionidae), was reported previously to exhibit antibiosis to an entomopathogenic fungus, Beauveria bassiana. The objectives of this study were to examine 1) if the antimicrobial effect occurs in other insects that form pupal cells, 2) whether the effect extends to plant pathogenic fungi, and 3) identify the source of antibiosis in pupal soil cells of C. caryae. Antibiosis of pupal cells against B. bassiana was confirmed in-vitro in three additional curculionids, Diaprepes abbreviatus, Conotrachelus nenuphar, and Pissodes nemorensis, all of which had fewer fungal colonies relative to controls. Pupal soil cells were found to suppress phytopathogenic fungi in-vitro, including suppression of Alternaria solani by D. abbreviatus pupal cell, and that of Monilinia fructicola by C. caryae. The detection of antibiosis of soil cells formed by surface-sterilized insects using sterile soil implies the antimicrobial effect stemmed from inside the insect. Further, a novel biotic mechanism was identified: a bacterium related to Serratia nematodiphila was isolated from C. caryae pupal soil cells and was found to be associated with antibiosis. The bacterial cultures with or without autoclave had similar effects but were not as potent as pupal soil cells for suppressing B. bassiana. Also, autoclaved soil cells and autoclaved bacterial culture suppressed M. fructicola but were not as inhibitory as non-autoclaved soil cells. This indicates that antibiosis may be due to bacterial metabolites, although other factors may also be involved. Our findings suggest potential to develop the antibiotic compounds as novel bio-fungicides to control plant diseases.

Entomopathogenic nematodes (EPNs) can provide a substantial control, especially of the root feeding larval pests. The objective of the present study was to examine beneficial traits of an indigenous entomopathogenic nematode species, Heterorhabditis bacteriophora Poinar (Hb-EG strain), and to identify superior biocontrol candidates for suppression of the scarab beetle Temnorhynchus baal (Reiche & Saulcy) (Coleoptera: Scarabaeidae: Dynastinae) larvae as serious pests of strawberry in Egypt. The nematodes were applied to infect the greater wax moth, Galleria mellonella L. cadavers, to represent natural emergence from the host in laboratory and under field conditions. Its average yield was 49604 infective juveniles (IJs) per G. mellonella larva. Overall averages of nematode-induced mortality in G . mellonella larvae were (4.63, 3.12, and 1.92) at 4, 8, and 12 weeks, respectively, after continuous weekly baiting with 5 cadavers. Nematode capability for infection and reproduction ranged from 72 to 26% one to five months, respectively, after field inoculation of the infected larvae into the strawberry rhizosphere. The number of IJs, moved to the North of the rhizosphere, was significantly ( P ≤ 0.05) less than that moved to the South or the East as reflected by the numbers of infected insects. The factors that may have a marked influence on EPN foraging behavior, persistence, and movement direction with implications for harnessing them as biological pest control agents were discussed.

The scarab beetle, Temnorhynchus baal (Reiche & Saulcy) (Coleoptera: Scarabaeidae), becomes a key pest of strawberry, especially after increasing its cultivated area and economic importance in Egypt. Few entomopathogenic nematode (EPN) species/strains were tested against this pest, where only a foreign species had good effect comparable to the native populations tested previously. Thirty-eight indigenous Heterorhabditis indica populations were tested against the most damaging, third instar larvae of H. baal in two soil types, where strawberry cultivation prevails. The corrected mortality induced by indigenous H. indica population in T. baal larvae was (99.52 and 98.57%) for 15 populations in the sandy soil and 23 in loamy sand soil, respectively. Overall average of infective juveniles (IJs) per T. baal larva was about (41,000). The average of emerged nematode-IJs from the infected grubs for the 15 EPN populations in sandy soil, (46,960 IJs/grub), was significantly (P < 0.001) higher than that (36,502 IJs/grub) of 23 EPN populations in loamy sand soil. The insignificant difference was detected in the reproductive capacity among nematode populations in T. baal larvae in sandy or loamy sand soil. A highly significant difference was found among total IJ numbers collected 10, 18, 26, and 30 days post-inoculation in sandy or loamy sand soil. The persistence of the H. indica populations in the soil varied greatly. Obtained results suggest further use of at least ten populations of such indigenous nematodes under field conditions.

Abiotic and biotic factors in soil modulate the efficiency of entomopathogenic nematodes (EPNs) as biological control agents of insect pests. We tested the hypothesis that EPNs associate spatially with a saprophytic fungus Fusarium solani in nature and contribute to both the fungal and nematode population growth, as found in previous laboratory-based studies that measured interactions at a fine spatial scale. Spatial Analysis by Distance Indices (SADIE) revealed highly significant spatial associations between F. solani and EPN communities comprising up to three species (Steinernema diaprepesi, Heterorhabditis indica, and Heterorhabditis zealandica), all of which were measured using quantitative real-time PCR during a 6-month citrus orchard survey. During three successive winter months, the EPNs aggregated in patches where F. solani aggregated during the previous month, although this behavior was not detected during the spring. The F. solani abundance in all plots was also related to that of total EPNs the previous month (one-month time lag; r = 0.17, P = 0.02) and may have resulted, at least in part, from the influence of soil moisture on both organisms. This study is the first report of quantitative relationships between F. solani and EPNs in the field. The results showed that a mutualistic relationship between this saprophytic fungus and EPNs reported previously, may have had measurable, but relatively minor effect on the spatial patterns of either organism at the landscape scale measured in this orchard.

The abundance of three entomopathogenic nematode (EPN) species, seven species of nematophagous fungi, free living nematode competitors of EPNs and a bacterial ectoparasite of EPNs were monitored during approximately two years in four Florida citrus orchards. The objective of the surveys was to identify natural enemies that potentially regulate the temporal abundance of naturally occurring EPNs by using molecular tools. Nematodes were extracted from two soil depths at monthly intervals, DNA extracted, and target organisms were measured using qPCR. Potentially causal relationships between the temporal patterns of EPNs and their natural enemies were assessed primarily with stepwise regression of variables at various time lags. Drechslerella dactyloides was the only nematophagous fungus species exhibiting evidence of top-down regulation, being inversely related to population change of Heterorhabditis indica at two of three sites. Acrobeloides spp. (EPN competitors) were associated with reduced population growth of Steinernema diaprepesi at two sites and H. indica was associated with reduced abundance of H. zealandica and S. diaprepesi at one site each. A Paenibacillus sp. exhibited significant phase-space, predator-prey dynamics at two of three sites with abundant S. diaprepesi, a species-specific host of the bacterium. The initial abundance of both S. diaprepesi and Paenibacillus sp. explained significant variability in population changes of S. diaprepesi in a multiple regression model at a lag of three months. Controlled experiments showed that pH is directly related to adherence of the bacterial endospores to the S. diaprepesi cuticle. Soil pH in these surveys was directly related to the infestation rate (encumbrance) by Paenibacillus and inversely related to the abundance of S. diaprepesi. Vertical distribution patterns reflected potential routes for avoiding competition and predation between species. Nematode-trapping fungi tended to inhabit shallower soil horizons than did several other nematophagous fungal species and S. diaprepesi and H. indica. The nematophagous fungus Purpureocillium lilacinum was especially abundant in deeper soil horizons and in the flatwoods sites. Depth distribution among EPNs also differed significantly with H. indica > S. diaprepesi > than H. zealandica. These results support soil pH management to conserve the demonstrated services of S. diaprepesi. They also suggest a need to better understand potential non-target effects of practices favorable to nematophagous fungi and bactivorous nematode competitors of EPNs.

In this study, molecular (ribosomal sequence data), morphological and cross-hybridization properties were used to identify a new Steinernema sp. from Florida, USA. Molecular and morphological data provided evidence for placing the novel species into Clade V, or the 'glaseri-group' of Steinernema spp. Within this clade, analysis of sequence data of the rDNA genes, 28S and internal transcribed spacer (ITS), depicted the novel species as a distinctive entity and closely related to S. glaseri and S. cubanum. Additionally, cross-hybridization assays showed that the new species is unable to interbreed with either of the latter two species, reinforcing its uniqueness from a biological species concept standpoint. Key morphological diagnostic characters for S. khuongi n. sp. include the mean morphometric features of the third-stage infective juveniles: total body length (average: 1066 μm), tail length (average: 65 μm), location of the excretory pore (average: 80.5 μm) and the values of c (average: 16.4), D% (average: 60.5), E% (average: 126) and H% (average: 46.6). Additionally, males can be differentiated from S. glaseri and S. cubanum by the values of several ratios: D% (average: 68), E% (average: 323) and SW% (average: 120). The natural distribution of this species in Florida encompasses both natural areas and citrus groves, primarily in shallow groundwater ecoregions designated as 'flatwoods'. The morphological, molecular, phylogenetic and ecological data associated with this nematode support its identity as a new species in the S. glaseri-group.

Caribbean fruit fly, also known as Caribfly or Anastrepha suspensa , is a major tephritid pest of guavas. A virulent entomopathogenic nematode (EPN) species was investigated to suppress the fruit-to-soil stages of Caribflies, which are also attacked by the koinobiont parasitoid Diachasmimorpha longicaudata in south Florida. The main objective was to develop a feasible and cost-effective EPN-application method for integrated pest management (IPM) of Caribfly to improve guava production. Naturally infested guavas were treated with increasing Heterorhabditis bacteriophora infective juvenile (IJ) concentration or rate (0, 25, 50, …, 1,600 IJs cm -2 ) in field trials to measure the optimum IJ rate and then examine sensitivity of producing guavas to inclusion of Heterorhabditis bacteriophora in Caribfly IPM plans. Relative survival of Caribfly in treatments significantly decreased with increasing IJ rate from 0 to 100 IJs cm -2 . Similarly, probability of observing large numbers of parasitoid wasps ( Diachasmimorpha longicaudata ) in EPN treatments significantly declined with increasing IJ rate (0-100 IJs cm -2 ), even though the non-target effects of Heterorhabditis bacteriophora on relative survival of Diachasmimorpha longicaudata could not be determined because of few emerging parasitoid wasps. Optimum suppression (⩾ 60%) of Caribfly was consistently achieved at 100 IJs cm -2 or 17,500 IJs fruit -1 . Profitability analysis showed that Heterorhabditis bacteriophora can be included in Caribfly IPM tactics to produce guavas. Costs of EPNs in Caribfly IPM are minimized if Heterorhabditis bacteriophora is strategically applied by spot treatment of fruit. Repayment of costs of EPN-augmentation by spot treatments appears achievable by recovering 5.71% of the annual yield losses (⩾1,963 kg ha -1 ≈ US$ 8,650 ha -1 ), which are largely due to Caribfly infestation. Hectare-wide EPN-augmentation (or broadcasting) method requires more fruit recovery than the total annual yield losses to repay its high costs. Profitability of guava production in south Florida will not be very sensitive to marginal costs of the spot treatment method, when compared to the field-wide broadcasting of Heterorhabditis bacteriophora .

Trabajo presentado en el 33rd European Society of Nematologist meeting, celebrado en Ghent (Belgica), del 9 al 13 de septiembre de 2018

Entomopathogenic nematode (EPN) species richness merits studies towards making rational decisions for effective management of Caribfly, Anastrepha suspensa (Loew) in southern Florida. Competition for Caribfly and efficacies of EPN biodiversity were examined under laboratory conditions. Similar EPN species treatments to Caribfly-infested fruits, periodically obtained from the ground in groves which were also infested by the parasitoid Diachasmimorpha longicaudata Ashmead (Braconidae), were studied in a series of field trials. Treatments with individual EPN species and their mixtures caused similar mortalities of Caribfly larvae, though the various EPN species competed for larvae in multiple-species treatments. Laboratory trials showed that mortalities of EPN-treated Caribfly pupae were mostly inversely related to EPN diversity. In the field, population densities of emerging adult Caribfly increased with increasing number of EPN species combined in treatments. Thus, single-EPN species treatments proved to be more effective for the management of fruit-to-soil stages of Caribfly. Relative to controls, the proportions of surviving adult Caribfly observed in EPN treatments with Heterorhabditis bacteriophora (exotic in Florida), Steinernema feltiae (exotic EPN) and Heterorhabditis indica (the endemic species) in field plots were 22.5 ± 6, 45 ± 13 and 47 ± 13%, respectively. Number of emerging D. longicaudata in each of EPN species treatments was similar to that observed in control, suggesting that none of the EPN species significantly affected the emergence of D. longicaudata, a parasitoid of Caribfly. Heterorhabditis bacteriophora will be more promising, with insignificant side effects on D. longicaudata in Caribfly-integrated pest management.

In two field surveys, high proportions of Galleria mellonella L. (Lepidoptera: Pyralidae) sentinel larval cadavers were infected by Fusarium solani without evidence of concomitant entomopathogenic nematode (EPN) or entomopathogenic fungus (EPF) reproduction. Because F. solani is not considered entomopathogenic, the survey suggested the possibility that F. solani competes with EPNs. We tested the hypotheses that F. solani attracts the EPN, Steinernema diaprepesi, to facilitate infection of Diaprepes root weevils (Diaprepes abbreviatus L.) and thereafter competes with the nematode in the insect cadaver. In two-choice olfactometer assays where one side was treated with F. solani mycelia and conidia, juvenile S. diaprepesi were attracted to the fungus, in either raw soil, or in autoclaved soil in the presence or absence of insects. However, this attraction was attenuated as the habitat became more complex, by using raw soil in combination with insect larvae. Fusarium oxysporum did not recruit the nematode. When soil microcosms were tested with F. solani conidia and S. diaprepesi, the concomitant infection increased the mortality of the insect (P = 0.02) to 83%, compared to 58% and 0% mortality when nematodes or fungi were individually applied, respectively. Concomitant inoculation also increased the number of cadavers that supported nematode reproduction and increased the population density of fungus in soil. The number of IJs entering the host insect was not affected by F. solani. These results support the possibility that F. solani can facilitate the insecticidal efficiency of S. diaprepesi in order to exploit the resources in the cadaver.

Abstract This chapter covers subtropical and tropical fruit and nut tree crops, as well as vines, for many of which detailed information concerning nematode damage is relatively scarce. It includes 22 tree and vine crops that, by virtue of their production value on a world basis, their importance in world trade or their local significance at the present time, may largely be regarded in this context as major crops among the long list of tropical and subtropical fruit cultivated worldwide. These include eight fruit tree crops, three nut crops and eleven vine crops. Grapes are included in the vine crops section due to the cultivation increase of grapevines for wine, raisins and table grape production in subtropical areas. In all crops, those nematode pests for which some evidence of economic impact exists are emphasized.

Relationships between entomopathogenic nematodes (EPNs), nematophagous fungi (NF) and soil physical and chemical properties were studied in a survey of 53 citrus orchards in central ridge and flatwoods ecoregions of Florida. Seven species of NF associated with nematodes were quantified directly using a real time qPCR assay. All nematophagous fungi studied except Arthrobotrys musiformis and Hirsutella rhossiliensis were frequently detected (24-56%) in both regions. Paecilomyces lilacinus and Gamsylella gephyropagum were encountered more frequently in the flatwoods (P = 0.03) and on the ridge (P = 0.02), respectively. Redundancy analysis revealed seven abiotic and biotic factors as significantly related to the NF occurrence. Multiple regression of fungi on these variables explained 78%, 66%, 48%, 36%, 23% and 4% of the variation in Catenaria sp., A. musiformis, A dactyloides, P. lilacinus, A. oligospora and G. gepharopagum, respectively. When the data from citrus were pooled with those reported previously from natural areas and subjected to principle component analysis, the first two principle components explained 43% of the variation in NF communities. The surveys (citrus vs natural areas) were discriminated by PC2 (P < 0.001) and the ecoregion by PC1 (P < 0.002), and all but one NF species were related (P < 0.01) to one or both components. NF communities tended to have more species and greater diversity in the flatwoods, where EPN richness and diversity were the least. However, the strength of associations between individual EPN and NF species as measured by SADIE reflected the associations between each species and ground water depth, suggesting that ecoregion preferences affected the species associations. Within each ecoregion, significant relationships between the individual NF and EPN species measured by stepwise regression tended to be positive. The results did not support the hypothesis that NF modulate the spatial patterns of EPN species between or within these two ecoregions. (C) 2017 Elsevier Inc. All rights reserved.

BACKGROUNDCaribbean fruit fly (Caribfly) is a serious economic insect pest because of development of larvae that hatch from eggs oviposited into fruits by female adults. This study assessed the virulence of twelve entomopathogenic nematode (EPN) isolates to Caribfly in laboratory bioassays as a starting point toward evaluation of management strategies for the fruit-to-soil-dwelling stages of A. suspensa in fields infested by Caribfly.RESULTSInoculation of A. suspensa with 1 mL of ca 200 IJs larva-1 killed Caribfly at either larval or pupal stage. Pupae were more resistant to EPN infections than larvae. Adult emergence from inoculated pupae in soil microcosms was significantly lower than that observed in filter paper assays. Longest or largest steinernematids suppressed emergence of more adult Caribfly from pupae in soils, whereas shorter heterorhabditids were more infectious to Caribfly larvae. The highest mortalities of A. suspensa were caused by exotic nematodes Steinernema feltiae and Heterorhabditis bacteriophora, followed by the native Heterorhabditis indica and the exotic Steinernema carpocapsae.CONCLUSIONEntomopathogenic nematodes reduced the development of Caribfly larvae and pupae to adult in our bioassays, suggesting that EPNs have potential for biological control of A. suspensa. Future work will assess management strategies, using the virulent EPNs, in orchards infested by A. suspensa. © 2016 Society of Chemical Industry.

Plant defense pathways play a critical role in mediating tritrophic interactions between plants, herbivores, and natural enemies. While the impact of plant defense pathway stimulation on natural enemies has been extensively explored aboveground, belowground ramifications of plant defense pathway stimulation are equally important in regulating subterranean pests and still require more attention. Here we investigate the effect of aboveground stimulation of the salicylic acid pathway through foliar application of the elicitor methyl salicylate on belowground recruitment of the entomopathogenic nematode, Steinernema diaprepesi. Also, we implicate a specific root-derived volatile that attracts S. diaprepesi belowground following aboveground plant stimulation by an elicitor. In four-choice olfactometer assays, citrus plants treated with foliar applications of methyl salicylate recruited S. diaprepesi in the absence of weevil feeding as compared with negative controls. Additionally, analysis of root volatile profiles of citrus plants receiving foliar application of methyl salicylate revealed production of d-limonene, which was absent in negative controls. The entomopathogenic nematode S. diaprepesi was recruited to d-limonene in two-choice olfactometer trials. These results reinforce the critical role of plant defense pathways in mediating tritrophic interactions, suggest a broad role for plant defense pathway signaling belowground, and hint at sophisticated plant responses to pest complexes.

The geospatial patterns of four species of native entomopathogenic nematodes in Florida were previously shown to be related to soil properties that affect soil water potential. Here we compared the responses to water potential of third stage, infective juvenile (IJ), Steinernema sp. (Sx), and Steinernema diaprepesi (Sd) in controlled conditions. The two species were selected because they are closely related (Steinernema glaseri-group), but tend to occupy different habitats. In columns of sandy soil with moisture gradients ranging from field capacity (6% w:w) to saturated (18%), Sx migrated toward wetter soil whereas Sd migrated toward drier soil. Survival of two isolates each of Sx and Sd for 7 days in the absence of food was greatest at 18 and 6% soil moisture, respectively. After three cycles of migration through soil to infect insect larvae 10 cm distant, Sd dominated EPN communities when soil columns were maintained at 6% moisture, whereas Sx was dominant in soil maintained at 18% moisture. When rehydrated after 24 h on filter paper at 90% RH, 50% of Sd survived compared to no Sx. Two isolates of Sd also survived better than two isolates of Sx during up to 24 h in a hypertonic solution (30% glycerol). The behavioral responses of both species to water potential and osmotic gradients were consistent with surveys in which Sx was recovered only from flatwoods ecoregions with shallow water tables and poorly drained soils, whereas Sd most frequently inhabited the central ridge ecoregion comprising well-drained soils and deeper water tables. Comparative proteomic analysis revealed differential expression of proteins involved in thermo-sensation (guanylyl cyclase and F13E6-4) and mechano-sensation and movement (paramyosin, Actin 3, LET-99, CCT-2), depending on whether Sd was in soil at 6 or 18% moisture. Proteins involved in metabolism, lectin detoxification, gene regulation, and cell division also differed between the two conditions. Our data suggest the plausibility of modifying soil moisture conditions in flatwoods orchards in ways that favor more desirable (effective) EPN species. Similarly, these particular behavioral traits are likely to be useful in guiding the selection or engineering of EPN species for use in different ecoregions.

In Florida citrus orchards, root weevil herbivory is tempered by four native entomopathogenic nematode (EPN) species whose spatial patterns are associated with soil properties related to water content. Manipulation of these variables could affect the efficacy and conservation of native EPNs for control of root weevils. The diversity of EPNs in non-agricultural areas it is unknown. We speculated that, whereas different species composition may occur in some natural areas by virtue of non-citrus niche adaptations, similar soil properties and environmental conditions should shape the communities in both systems. We characterized EPN food web assemblages during summer-fall 2011 in 91 sites comprising five naturally occurring botanical groups (oak, pine, palm and palmetto, mixed and other) and two ecoregions (central ridge and flatwoods). We used species-specific qPCR probes for 13 EPN species, two species of Paenibacillus (ectoparasitically associated with EPNs), seven species of nematophagous fungi (NF), an oomycete pathogen of citrus (Phytophthora nicotianae) and free-living bactivorous nematodes (Acrobeloides-group), some of which compete with EPNs. Seven EPN species were detected at frequencies about 60% of that reported from citrus. Additional species detected were Steinernema glaseri and Heterorhabditis floridensis, whereas citrus inhabitants Steinernema riobrave and Steinernema scapterisci, were absent, suggesting a possible niche adaptation. EPN recovery frequency did not differ between two ecoregions, except for Heterorhabditis indica which was detected at more sites in the flatwoods than on the central ridge and Heterorhabditis zealandica exhibiting the opposite pattern for detection frequency and abundance. Nor did the botanical habitats much affect EPNs other than H. indica which occurred with greatest frequency and abundance in abandoned citrus orchards. Soil moisture appeared to be important in modulating these subterranean communities, with groundwater depth, organic matter, soil clay and pH explaining significant EPN variability between sites. There was little evidence that regional or habitat differences in natural enemy occurrence affected the EPN spatial patterns. Indeed, numerous significant direct associations between species of nematodes and nematophagous fungi suggested that the local abundance of the r-selected EPN and Acrobeloides-group nematodes is an important resource modulating population growth of these trapping and endoparasitic fungi. Congruent relationships between EPN spatial patterns and soil properties that affect water potential in both natural areas and citrus orchards suggests that soil moisture drives these patterns and modulates the regulation of root herbivores by EPNs in this part of the Florida peninsula. Consequently, management of soil moisture by manipulating these properties has the greatest potential to enhance and conserve EPN services.