Spodoptera litura Fabricius (Lepidoptera: Noctuidae) is a major pest of marigolds in Thailand. It causes severe damage to flower quantity and quality. The most common control method is synthetic ...insecticides, although this method struggles to control hidden larvae inside flowers or buds and underground pupae. Entomopathogenic nematodes (EPNs) are widely used as biocontrol agents against many foliage and cryptic insects. The virulence of two commercial EPNs strains, Steinernema siamkayai (Stock, Somsook and Reid) and S. carpocapsae (Weiser), isolated in Thailand, were assessed against different developmental stages of S. litura. The younger larvae of first and third instar were more susceptible to S. siamkayai with the LD50 of 15.84 and 40.34 dauer juveniles (DJs) per host, whereas S. carpocapsae caused higher mortality in older larvae. Against the fifth instar larvae and pupae, the LD50 ranged between 54.37 and 607.97 DJs/host. Field experiments compared the effect of EPNs with farmer's practice (insecticides). EPNs treatments with S. siamkayai and S. carpocapsae significantly reduced the percentage of damaged flowers to 2.53 ± 1.2% and 2.97 ± 1.5%, respectively, compared with insecticide treatment at 4.21 ± 2.1% in the first trial. In trial 2, the insecticide treatment had the highest percentage of damaged flowers of 5.02 ± 5.7%. The benefit–cost ratio (BCR) of EPNs treatments ranged from 2.42 to 2.53 in the first trial and 1.24–1.50 in the second trial and was superior to insecticide treatments with 2.21 and 1.16 in the two trials.
Plum sawflies are among the most damaging pests of European plum. Current control strategy implies insecticide application. Three species of entomopathogenic nematodes (EPN), Steinernema feltiae ...Filipjev, S. carpocapsae Weiser and Heterorhabditis bacteriophora Poinar were tested under laboratory and field conditions to assess effectiveness against larval and adult stages. Laboratory tests resulted in up to 100% mortality of last instar larvae before construction of a cocoon. However, the nematodes were not able to penetrate the cocoon. Foliar application did not result in plum sawflies larvae infestation by EPNs. Under field conditions, the nematodes reduced the number of emerging adults by application against sawfly larvae in the previous year before migration into the soil for overwintering by 62%–92%. Application of the nematodes against adults just before their anticipated emergence resulted in reduction of fruit infestation up to 100%. Mean results of 5 trials using caged trees were 47.8% with S. feltiae, 56.3% with S. carpocapsae and 62.9% with H. bacteriophora. In open field trails, control of adults obtained with S. feltiae at 0.5 million nematodes/m2 was 98.2 and 67.8% and at 0.25 million m−2 41.7 and 41.2%. Forecasting adult emergence and optimal soil moisture conditions are essential for success of the nematode application.
Entomopathogenic nematodes are effective biocontrol agents against arthropod pests. However, their efficacy is limited due to sensitivity to environmental extremes. The objective of the present study ...was to establish a foundation of genetic-based selection tools for beneficial traits of heat and desiccation tolerance in entomopathogenic nematodes. Screening of natural populations enabled us to create a diverse genetic and phenotypic pool. Gene expression patterns and genomic variation were studied in natural isolates. Heterorhabditis isolates were phenotyped by heat- and desiccation-stress bioassays to determine their survival rates compared to a commercial line. Transcriptomic study was carried out for the commercial line, a high heat-tolerant strain, and for the natural, low heat-tolerant isolate. The results revealed a higher number of upregulated vs. downregulated transcripts in both isolates vs. their respective controls. Functional annotation of the differentially expressed transcripts revealed several known stress-related genes and pathways uniquely expressed. Genome sequencing of isolates with varied degrees of stress tolerance indicated variation among the isolates regardless of their phenotypic characterization. The obtained data lays the groundwork for future studies aimed at identifying genes and molecular markers as genetic selection tools for enhancement of entomopathogenic nematodes ability to withstand environmental stress conditions.
The entomopathogenic nematode (EPN)
Heterorhabditis bacteriophora
is an effective biological-control agent of insect pests. The dauer juveniles (DJs) seek for, infect insects, and release cells of ...the carried symbiotic bacterium of the genus
Photorhabdus
. Inside the host, the DJs perceive signals from the insect’s haemolymph that trigger the exit from the arrested stage and the further development to mature adults. This developmental step is called DJ recovery. In commercial production, a high and synchronous DJ recovery determines the success of liquid-culture mass production. To enhance the understanding about genetic components regulating DJ recovery, more than 160 mutant- and 25 wild type inbred lines (WT ILs) were characterized for DJ recovery induced by cell-free bacterial supernatant. The mutant lines exhibited a broader DJ recovery range than WT ILs (4.6–67.2% vs 1.6–35.7%). A subset of mutant lines presented high variability of virulence against mealworm (
Tenebrio molitor
) (from 22 to 78% mortality) and mean time survival under oxidative stress (70 mM H
2
O
2
; from 10 to 151 h). Genotyping by sequencing of 96 mutant lines resulted in more than 150 single nucleotide polymorphisms (SNPs), of which four results are strongly associated with the DJ recovery trait. The present results are the basis for future approaches in improving DJ recovery by breeding under in vitro liquid-culture mass production in
H. bacteriophora
. This generated platform of EMS-mutants is as well a versatile tool for the investigation of many further traits of interest in EPNs.
Keypoints
• Exposure to bacterial supernatants of Photorhabdus laumondii induces the recovery of Heterorhabditis bacteriophora dauer juveniles (DJs). Both, the bacteria and the nematode partner, influence this response. However, the complete identity of its regulators is not known.
• We dissected the genetic component of DJ recovery regulation in H. bacteriophora nematodes by generating a large array of EMS mutant lines and characterizing their recovery pheno- and genotypes.
• We determined sets of mutants with contrasting DJ recovery and genotyped a subset of the EMS-mutant lines via genotyping by sequencing (GBS) and identified SNPs with significant correlation to the recovery trait.
Although the plant microbiome is crucial for plant health, little is known about the significance of the seed microbiome. Here, we studied indigenous bacterial communities associated with the seeds ...in different cultivars of oilseed rape and their interactions with symbiotic and pathogenic microorganisms.
We found a high bacterial diversity expressed by tight bacterial co-occurrence networks within the rape seed microbiome, as identified by llumina MiSeq amplicon sequencing. In total, 8362 operational taxonomic units (OTUs) of 40 bacterial phyla with a predominance of Proteobacteria (56%) were found. The three cultivars that were analyzed shared only one third of the OTUs. The shared core of OTUs consisted mainly of Alphaproteobacteria (33%). Each cultivar was characterized by having its own unique bacterial structure, diversity, and proportion of unique microorganisms (25%). The cultivar with the lowest bacterial abundance, diversity, and the highest predicted bacterial metabolic activity rate contained the highest abundance of potential pathogens within the seed. This data corresponded with the observation that seedlings belonging to this cultivar responded more strongly to the seed treatments with bacterial inoculants than other cultivars. Cultivars containing higher indigenous diversity were characterized as having a higher colonization resistance against beneficial and pathogenic microorganisms. Our results were confirmed by microscopic images of the seed microbiota.
The structure of the seed microbiome is an important factor in the development of colonization resistance against pathogens. It also has a strong influence on the response of seedlings to biological seed treatments. These novel insights into seed microbiome structure will enable the development of next generation strategies combining both biocontrol and breeding approaches to address world agricultural challenges.
The entomopathogenic nematode
Heterorhabditis bacteriophora
, symbiotically associated with enterobacteria of the genus
Photorhabdus
, is a biological control agent against many insect pests. Dauer ...Juveniles (DJ) of this nematode are produced in industrial-scale bioreactors up to 100 m
3
in liquid culture processes lasting approximately 11 days. A high DJ yield (> 200,000 DJ·mL
−1
) determines the success of the process. To start the mass production, a DJ inoculum proceeding from a previous monoxenic culture is added to pre-cultured (24 h)
Photorhabdus
bacteria. Within minutes after contact with the bacteria, DJ are expected to perceive signals that trigger their further development (DJ recovery) to reproductive hermaphrodites. A rapid, synchronized, and high DJ recovery is a key factor for an efficient culture process. In case of low percentage of DJ recovery, the final DJ yield is drastically reduced, and the amount of non-desired stages (males and non-fertilized females) hinders the DJ harvest. In a preliminary work, a huge DJ recovery phenotypic variability in
H. bacteriophora
ethyl methanesulphonate (EMS) mutants was determined. In the present study, two EMS-mutant lines (M31 and M88) with high and low recovery phenotypes were analyzed concerning their differences in gene expression during the first hours of contact with
Photorhabdus
supernatant containing food signals triggering recovery. A snapshot (RNA-seq analysis) of their transcriptome was captured at 0.5, 1, 3 and 6 h after exposure. Transcripts (3060) with significant regulation changes were identified in the two lines. To analyze the RNA-seq data over time, we (1) divided the expression profiles into clusters of similar regulation, (2) identified over and under-represented gene ontology categories for each cluster, (3) identified
Caenorhabditis elegans
homologous genes with recovery-related function, and (4) combined the information with available single nucleotide polymorphism (SNP) data. We observed that the expression dynamics of the contrasting mutants (M31 and M88) differ the most within the first 3 h after
Photorhabdus
supernatant exposure, and during this time, genes related to changes in the DJ cuticle and molting are more active in the high-recovery line (M31). Comparing the gene expression of DJ exposed to the insect food signal in the haemolymph, genes related to host immunosuppressive factors were not found in DJ upon bacterial supernatant exposure. No link between the position of SNPs associated with high recovery and changes in gene expression was determined for genes with high differential expression. Concerning specific transcripts, nine
H. bacteriophora
gene models with differential expression are provided as candidate genes for further studies.
Biological control agents can substitute hazardous pesticides and provide environmentally safe and sustainable control of pests, diseases and weeds. Often, exaggerating registration requirements ...prevent or delay market introduction of micro-organisms, semiochemicals and botanicals. This book is the result of the Europe-wide, two-year policy support action REBECA, a forum for representatives of academia, regulation authorities and biocontrol industry to discuss improved regulation rules. It provides a comprehensive review on existing data requirements and registration procedures. Risks and benefits of biocontrol in relation to trade-off effects resulting from unbalanced regulation are discussed. As a result, proposals for innovative and balanced regulation procedures are presented including recommendations how to accelerate the authorisation process. Considering concerns about possible non-target effects of beneficial insects, mites and nematodes this book proposes harmonisation of regulation rules and provides comprehensive material on the data requirements for authorisation, including guidelines for completion of possible application forms for macro-organisms. This book is a valuable source on state-of-the-art registration practice and possible policy steps to promote further introduction of biocontrol in agriculture practice.
The entomopathogenic nematode
Heterorhabditis bacteriophora
(Nematoda: Rhabditidae) is used in biological insect control. Their dauer juveniles (DJs) are free-living and developmentally arrested, ...invading host insects. They carry cells of their bacterial symbiont
Photorhabdus
spp. in the intestine. Once inside the insect´s hemolymph the DJs perceive a food signal, triggering them to exit the DJ stage and regurgitate the
Photorhabdus
cells into the insect’s haemocoel, which kill the host and later provide essential nutrients for nematode reproduction. The exit from the DJ stage is called “recovery”. For commercial pest control, nematodes are industrially produced in monoxenic liquid cultures. Artificial media are incubated with
Photorhabdus
before DJs are added. In absence of the insect’s food signal, DJs depend on unknown bacterial food signals to trigger exit of the DJ stage. A synchronized and high DJ recovery determines the success of the industrial in vitro production and can significantly vary between nematode strains, inbred lines and mutants. In this study, fourteen bacterial strains from
H. bacteriophora
were isolated and identified as
P. laumondii
,
P. kayaii
and
P. thracensis
. Although the influence of bacterial supernatants on the DJ recovery of three inbred lines and two mutants differed significantly, the bacterial impact on recovery has a subordinate role whereas nematode factors have a superior influence. Recovery of inbred lines decreased with age of the DJs. One mutant (M31) had very high recovery in bacterial supernatant and spontaneous recovery in Ringer solution. Another mutant (M88) was recovery defective.
Summary
Soil texture and moisture have significant effects on the infectivity and persistence of entomopathogenic nematodes (EPN). Both EPN and chili fruit flies (Bactrocera latifrons) spend most of ...their lifetime in soil, therefore making it possible to consider the successful use of EPN against chili fruit flies. This study aimed to investigate the effects of soil texture and moisture contents on chili fruit fly pupation and adult emergence, and on the infectivity of the EPN, Steinernema siamkayai, against this pest. The results revealed that the highest percentage of pupation occurred at a depth of 2 cm in all soil types. At a depth of 5 cm in the sandy clay loam only, no pupation of B. latifrons was observed. The adult emergence ranged from 51.9 to 82.5%, with significant differences between soil types. The horizontal movement of S. siamkayai showed that the concentration of EPN at a rate of 20 000 dauer juveniles pupa−1 yielded maximum pupae mortality (77.5-91.3%) in all soil types containing 15% moisture. In the vertical displacement test, S. siamkayai was capable of killing pupae up to a distance of 15 cm in all soil types. These results confirmed the potential of S. siamkayai to be used within pest control programmes of various soil textures, as long as the soil moisture contents allow its dispersal and host infection.
Commercial-scale field studies were conducted in citrus orchards to test the efficacy of entomopathogenic nematodes (EPNs) in controlling the soil-dwelling life stages of the key pest, Thaumatotibia ...leucotreta. Trials were conducted in three provinces of South Africa from 2011 to 2013. Two EPN species were tested: Heterorhabditis bacteriophora and Steinernema feltiae. Both EPN species were applied underneath citrus tree canopies, either with a spray machine or through the microsprinkler irrigation system. The concentration of nematodes and frequency of application varied. Efficacy of these treatments was measured by evaluation of EPN infection of sentinel T. leucotreta larvae in the soil at various intervals after application and by T. leucotreta infestation of fruit. Successful control was recorded in several trials, with EPN infection of larvae frequently reaching 80 %. However, in a few trials, this level of infection was assisted by naturally occurring EPNs. Fruit infestation by T. leucotreta larvae was reduced by up to 88 %. Efficacy appeared to be limited by suboptimal soil moisture and temperature. The former was influenced mainly by mode and relative thoroughness of irrigation before and after EPN application. This was best obtained through microsprinkler application. Nematode species and concentration also influenced efficacy. Both EPN species survived and persisted in the soil of the treated orchards for up to 4 months. Promising results support their potential for use in an integrated pest management programme in citrus in South Africa.
•Thaumatotibia leucotreta is an important pest of citrus in sub-Saharan Africa.•Heterorhabditis bacteriophora and Steinernema feltiae are virulent to T. leucotreta.•In field trials, more than 80 % of T. leucotreta larvae were infected with nematodes.•Fruit infestation with T. leucotreta larvae was reduced by up to 88 %.•Best results were achieved with monthly applications of EPNs for four months.
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