Protein complexes from edible mushrooms as a sustainable potato protection against coleopteran pests.

Protein complexes from edible oyster mushrooms (Pleurotus sp.) composed of pleurotolysin A2 (PlyA2) and pleurotolysin B (PlyB) exert toxicity in feeding tests against Colorado potato beetle (CPB) larvae, acting through the interaction with insect-specific membrane sphingolipid. Here we present a new strategy for crop protection, based on in planta production of PlyA2/PlyB protein complexes, and we exemplify this strategy in construction of transgenic potato plants of cv Désirée. The transgenics in which PlyA2 was directed to the vacuole and PlyB to the endoplasmic reticulum are effectively protected from infestation by CPB larvae without impacting plant performance. These transgenic plants showed a pronounced effect on larval feeding rate, the larvae feeding on transgenic plants being on average five to six folds lighter than larvae feeding on controls. Further, only a fraction (11%-37%) of the larvae that fed on transgenic potato plants completed their life cycle and developed into adult beetles. Moreover, gene expression analysis of CPB larvae exposed to PlyA2/PlyB complexes revealed the response indicative of a general stress status of larvae and no evidence of possibility of developing resistance due to the functional inactivation of PlyA2/PlyB sphingolipid receptors.

A single point mutation expands the applicability of ostreolysin A6 in biomedicine.

An aegerolysin protein ostreolysin A6 (OlyA6) binds to cholesterol-complexed sphingomyelin and can be used for specific labelling of lipid rafts. In addition, OlyA6 interacts with even higher affinity with ceramide phosphoethanolamine (CPE), a sphingolipid that dominates in invertebrate cell membranes. In the presence of pleurotolysin B, a protein bearing the membrane-attack complex/perforin domain, OlyA6 forms pores in insect midgut cell membranes and acts as a potent bioinsecticide. It has been shown that a point mutation of glutamate 69 to alanine (E69A) allows OlyA6 to bind to cholesterol-free sphingomyelin. Using artificial lipid membranes and mammalian MDCK cells, we show that this mutation significantly enhances the interaction of OlyA6 with sphingomyelin and CPE, and allows recognition of these sphingolipids even in the absence of cholesterol. Our results suggest that OlyA6 mutant E69A could serve as complementary tool to detect and study cholesterol-associated and free sphingomyelin or CPE in membranes. However, the mutation does not improve the membrane-permeabilizing activity after addition of pleurotolysin B, which was confirmed in toxicity tests on insect and mammalian cell lines, and on Colorado potato beetle larvae.

Influence of Wireworm Diet on its Susceptibility to and Control With the Entomopathogenic Fungus Metarhizium brunneum (Hypocreales: Clavicipitaceae) in Laboratory and Field Settings.

Entomopathogenic fungi (EPF) represent promising control agents against wireworms but success in field experiments is inconsistent. The physiological condition of the targeted insect is crucial for its ability to withstand fungal infection. In particular, nutritional status is among the most important determinants of the insects' immune defense. In this study, we investigated the effects of diet on the development of the wireworm Agriotes obscurus (L.) (Coleoptera: Elateridae) and its subsequent susceptibility to the fungal pathogen Metarhizium brunneum (Petch) (Hypocreales: Clavicipitaceae) in a pot experiment. After being reared on one of five plant diets for eight weeks, wireworms were exposed to an environment inoculated with the EPF and monitored for their susceptibility to fungal infection. We then performed a field experiment in which three plant diets (clover, radish, and a cover crop mix), selected according to the insects' performance in the laboratory experiment, were grown as a cover crop with EPF application. Plant diet influenced growth and development of larvae, but there were no strong differences in susceptibility toward fungal infection in the laboratory experiment. Damage levels in EPF-treated plots in the field varied depending on the cover crop. Damage was highest in plots planted with a mix of cover crop species, whereas damage was lowest in plots with clover or radish alone. This agrees with the laboratory results where insect performance was inferior when fed on clover or radish. Cover crop effects on wireworm damage in the subsequent cash crop may thus vary depending on the cover crop species selected.

Testing Virulence of Different Species of Insect Associated Fungi against Yellow Mealworm (Coleoptera: Tenebrionidae) and Their Potential Growth Stimulation to Maize.

This paper investigates 71 isolates of two genera of entomopathogens, Metarhizium and Beauveria, and a biostimulative genus Trichoderma, for their ability to infect yellow mealworms (Tenebrio molitor) and to stimulate maize (Zea mays) growth. Fungal origin, host, and isolation methods were taken into account in virulence analysis as well. Isolates Metarhizium brunneum (1154) and Beauveria bassiana (2121) showed the highest mortality (100%) against T. molitor. High virulence seems to be associated with fungi isolated from wild adult mycosed insects, meadow habitats, and Lepidopteran hosts, but due to uneven sample distribution, we cannot draw firm conclusions. Trichoderma atroviride (2882) and Trichoderma gamsii (2883) increased shoot length, three Metarhizium robertsii isolates (2691, 2693, and 2688) increased root length and two M. robertsii isolates (2146 and 2794) increased plant dry weight. Considering both criteria, the isolate M. robertsii (2693) was the best as it caused the death of 73% T. molitor larvae and also significantly increased maize root length by 24.4%. The results warrant further studies with this isolate in a tri-trophic system.

Lipid-Binding Aegerolysin from Biocontrol Fungus Beauveria bassiana.

Fungi are the most common pathogens of insects and thus important regulators of their populations. Lipid-binding aegerolysin proteins, which are commonly found in the fungal kingdom, may be involved in several biologically relevant processes including attack and defense against other organisms. Aegerolysins act alone or together with membrane-attack-complex/perforin (MACPF)-like proteins to form transmembrane pores that lead to cell lysis. We performed an in-depth bioinformatics analysis of aegerolysins in entomopathogenic fungi and selected a candidate aegerolysin, beauveriolysin A (BlyA) from Beauveria bassiana. BlyA was expressed as a recombinant protein in Escherichia coli, and purified to further determine its functional and structural properties, including lipid-binding ability. Aegerolysins were found to be encoded in genomes of entomopathogenic fungi, such as Beauveria, Cordyceps, Metarhizium and Ophiocordyceps. Detailed bioinformatics analysis revealed that they are linked to MACPF-like genes in most genomes. We also show that BlyA interacts with an insect-specific membrane lipid. These results were placed in the context of other fungal and bacterial aegerolysins and their partner proteins. We believe that aegerolysins play a role in promoting the entomopathogenic and antagonistic activity of B. bassiana, which is an active ingredient of bioinsecticides.

Dissecting Out the Molecular Mechanism of Insecticidal Activity of Ostreolysin A6/Pleurotolysin B Complexes on Western Corn Rootworm.

Ostreolysin A6 (OlyA6) is a protein produced by the oyster mushroom (Pleurotus ostreatus). It binds to membrane sphingomyelin/cholesterol domains, and together with its protein partner, pleurotolysin B (PlyB), it forms 13-meric transmembrane pore complexes. Further, OlyA6 binds 1000 times more strongly to the insect-specific membrane sphingolipid, ceramide phosphoethanolamine (CPE). In concert with PlyB, OlyA6 has potent and selective insecticidal activity against the western corn rootworm. We analysed the histological alterations of the midgut wall columnar epithelium of western corn rootworm larvae fed with OlyA6/PlyB, which showed vacuolisation of the cell cytoplasm, swelling of the apical cell surface into the gut lumen, and delamination of the basal lamina underlying the epithelium. Additionally, cryo-electron microscopy was used to explore the membrane interactions of the OlyA6/PlyB complex using lipid vesicles composed of artificial lipids containing CPE, and western corn rootworm brush border membrane vesicles. Multimeric transmembrane pores were formed in both vesicle preparations, similar to those described for sphingomyelin/cholesterol membranes. These results strongly suggest that the molecular mechanism of insecticidal action of OlyA6/PlyB arises from specific interactions of OlyA6 with CPE, and the consequent formation of transmembrane pores in the insect midgut.

Evaluation of Sown Cover Crops and Spontaneous Weed Flora as a Potential Reservoir of Black-Foot Pathogens in Organic Viticulture.

(1) Background. An extensive survey of grapevine-sown cover crops and spontaneous weed flora was conducted from 2019 to 2020 in organic vineyards in six European countries (France, Italy, Romania, Slovenia, Spain, Switzerland). Our main objective was to detect and identify the presence of Cylindrocarpon-like asexual morphs species associated with black-foot disease on their roots. (2) Methods. Fungal isolations from root fragments were performed on culture media. Cylindrocarpon-like asexual morph species were identified by analyzing the DNA sequence data of the histone H3 (his3) gene region. In all, 685 plants belonging to different botanical families and genera were analyzed. Cylindrocarpon-like asexual morphs were recovered from 68 plants (9.9% of the total) and approximately 0.97% of the plated root fragments. (3) Results. Three fungal species (Dactylonectria alcacerensis, Dactylonectria torresensis, Ilyonectria robusta) were identified. Dactylonectria torresensis was the most frequent, and was isolated from many cover crop species in all six countries. A principal component analysis with the vineyard variables showed that seasonal temperatures and organic matter soil content correlated positively with Cylindrocarpon-like asexual morphs incidence. (4) Conclusions. The presence of Cylindrocarpon-like asexual morphs on roots of cover crops suggests that they can potentially act as alternative hosts for long-term survival or to increase inoculum levels in vineyard soils.

Stressor-Dependant Changes in Immune Parameters in the Terrestrial Isopod Crustacean, Porcellio scaber: A Focus on Nanomaterials.

We compared the changes of selected immune parameters of Porcellio scaber to different stressors. The animals were either fed for two weeks with Au nanoparticles (NPs), CeO2 NPs, or Au ions or body-injected with Au NPs, CeO2 NPs, or lipopolysaccharide endotoxin. Contrary to expectations, the feeding experiment showed that both NPs caused a significant increase in the total haemocyte count (THC). In contrast, the ion-positive control resulted in a significantly decreased THC. Additionally, changes in phenoloxidase (PO)-like activity, haemocyte viability, and nitric oxide (NO) levels seemed to depend on the stressor. Injection experiments also showed stressor-dependant changes in measured parameters, such as CeO2 NPs and lipopolysaccharide endotoxin (LPS), caused more significant responses than Au NPs. These results show that feeding and injection of NPs caused an immune response and that the response differed significantly, depending on the exposure route. We did not expect the response to ingested NPs, due to the low exposure concentrations (100 µg/g dry weight food) and a firm gut epithelia, along with a lack of phagocytosis in the digestive system, which would theoretically prevent NPs from crossing the biological barrier. It remains a challenge for future research to reveal what the physiological and ecological significance is for the organism to sense and respond, via the immune system, to ingested foreign material.

Aegerolysins from the fungal genus Pleurotus - Bioinsecticidal proteins with multiple potential applications.

The aegerolysin proteins ostreolysin A6, pleurotolysin A2 and erylysin A are produced by mushrooms of the genus Pleurotus. These aegerolysins can interact specifically with sphingolipid-enriched membranes. In particular, they strongly bind insect cells and to artificial lipid membranes that contain physiologically relevant concentrations of the main invertebrate-specific sphingolipid, ceramide phosphoethanolamine. Moreover, the aegerolysins permeabilise these membranes when combined with their protein partner pleurotolysin B, which contains a membrane-attack-complex/perforin domain. These aegerolysin/ pleurotolysin B complexes show strong and selective toxicity towards western corn rootworm larvae and adults and Colorado potato beetle larvae. Their insecticidal activities arise through aegerolysin binding to ceramide phosphoethanolamine in the insect midgut. This mode of membrane binding is different from those described for similar aegerolysin-based complexes of bacterial origin (e.g., Cry34Ab1/Cry35Ab1), or other Bacillus thuringiensis proteinaceous crystal toxins, which associate with protein receptors. The ability of Pleurotus aegerolysins to specifically interact with sphingolipid-enriched domains in mammalian cells can be further exploited to visualize lipid rafts in living cells, and to treat certain types of tumours and metabolic disorders. Finally, these proteins can strongly enhance fruiting initiation of P. ostreatus even when applied externally. In this review, we summarise the current knowledge of the potential biotechnological and biomedical applications of the Pleurotus aegerolysins, either alone or when complexed with pleurotolysin B, with special emphasis on their bioinsecticidal effects.

Bioaugmentation of Entomopathogenic Fungi for Sustainable Agriotes Larvae (Wireworms) Management in Maize.

Soil microorganisms influence biotic and abiotic stress tolerance of crops. Most interactions between plant symbiotic and non-symbiotic soil microorganisms and plants occur in the rhizosphere and are sustained through plant exudation/rhizodeposition. Bioaugmentation, i.e., the introduction or amplification of certain plant beneficial microbes (e.g., entomopathogenic fungi) into the rhizosphere, could contribute to controlling insect crop pests and replacing chemical, environmentally unfriendly insecticides. Wireworms, the soil-burrowing larval stages of click beetles (Coleoptera: Elateridae), are major pests of crops including maize, wheat and potatoes, worldwide. Alternative strategies for controlling wireworms are needed because several chemical pesticides used successfully in the past are being phased out because of their ecotoxicity. Therefore, virulence to Agriotes lineatus L. wireworms and plant beneficial traits of entomopathogenic fungi were investigated in a series of laboratory experiments. Tested taxa included environmentally retrieved Metarhizium brunneum Petch. (two strains), M. robertsii Bisch., Rehner & Humber (Hypocreales: Clavicipitaceae), and Beauveria brongniartii (Sacc.) Petch. and commercially formulated B. bassiana (Bals.-Criv.) Vuill. (Cordycipitaceae) and Bacillus thuringiensis Berliner 1915 var. kurstaki. In-house reared larvae were dipped in conidial suspension, and maize and wheat seeds were coated with fungal conidia. Metarhizium brunneum strains 1154 and 1868 significantly increased wireworm mortality. Fungi were significantly more often re-isolated from maize than wheat rhizoplanes in laboratory assays. The strains tested were rarely isolated as endophytes. Metarhizium brunneum strain 1154 stimulated wheat growth, while M. robertsii 1880 stimulated maize growth, whereas M. brunneum 1868 and others did not affect root or shoot length or plant biomass significantly in laboratory settings. Metarhizium brunneum strain 1868, re-isolated most often from maize rhizoplane, caused the highest wireworm mortality. It was further evaluated whether M. brunneum 1868 can protect maize varieties FeroXXY, LG 34.90 and Chapalu from wireworm damage and promote plant growth at field conditions. Plants of all three varieties stemming from seeds treated with conidia of M. brunneum 1868 showed significantly less wireworm damage 3 to 4 weeks after sowing (5- to 6-leaf stage) resulting in a significantly higher initial maize stand. However, only in the variety LG 34.90 a significant increase of the maize stand was observed at harvest time.

Validating the Potential of Double-Stranded RNA Targeting Colorado Potato Beetle Mesh Gene in Laboratory and Field Trials.

Colorado potato beetle (CPB) is an agricultural pest of solanaceous crops, notorious for its rapid resistance development to chemical pesticides. Foliar spraying of dsRNA formulations is a promising innovative technology providing highly specific and environmentally acceptable option for CPB management. We designed dsRNA to silence CPB mesh gene (dsMESH) and performed laboratory feeding trials to assess impacts on beetle survival and development. We compared the effectiveness of in vivo and in vitro produced dsRNA in a series of laboratory experiments. We additionally performed a field trial in which the efficacy of dsRNA sprayed onto potato foliage was compared to a spinosad-based insecticide. We showed that dsMESH ingestion consistently and significantly impaired larval growth and decreased larval survival in laboratory feeding experiments. In vivo produced dsRNA performed similarly as in vitro synthesized dsRNA in laboratory settings. In the field trial, dsMESH was as effective in controlling CPB larvae as a commercial spinosad insecticide, its activity was however slower. We discuss limitations and benefits of a potential dsMESH-based CPB management strategy and list some important RNAi based CPB research topics, which will have to be addressed in future.

An update of the Worldwide Integrated Assessment (WIA) on systemic pesticides. Part 4: Alternatives in major cropping systems.

We present a synthetic review and expert consultation that assesses the actual risks posed by arthropod pests in four major crops, identifies targets for integrated pest management (IPM) in terms of cultivated land needing pest control and gauges the implementation "readiness" of non-chemical alternatives. Our assessment focuses on the world's primary target pests for neonicotinoid-based management: western corn rootworm (WCR, Diabrotica virgifera virgifera) in maize; wireworms (Agriotes spp.) in maize and winter wheat; bird cherry-oat aphid (Rhopalosiphum padi) in winter wheat; brown planthopper (BPH, Nilaparvata lugens) in rice; cotton aphid (Aphis gossypii) and silver-leaf whitefly (SLW, Bemisia tabaci) in cotton. First, we queried scientific literature databases and consulted experts from different countries in Europe, North America, and Asia about available IPM tools for each crop-pest system. Next, using an online survey, we quantitatively assessed the economic relevance of target pests by compiling country-level records of crop damage, yield impacts, extent of insecticide usage, and "readiness" status of various pest management alternatives (i.e., research, plot-scale validation, grower-uptake). Biological control received considerable scientific attention, while agronomic strategies (e.g., crop rotation), insurance schemes, decision support systems (DSS), and innovative pesticide application modes were listed as key alternatives. Our study identifies opportunities to advance applied research, IPM technology validation, and grower education to halt or drastically reduce our over-reliance on systemic insecticides globally.

From Genome to Field-Observation of the Multimodal Nematicidal and Plant Growth-Promoting Effects of Bacillus firmus I-1582 on Tomatoes Using Hyperspectral Remote Sensing.

Root-knot nematodes are considered the most important group of plant-parasitic nematodes due to their wide range of plant hosts and subsequent role in yield losses in agricultural production systems. Chemical nematicides are the primary control method, but ecotoxicity issues with some compounds has led to their phasing-out and consequential development of new control strategies, including biological control. We evaluated the nematicidal activity of Bacillus firmus I-1582 in pot and microplot experiments against Meloidogyne luci. I-1582 reduced nematode counts by 51% and 53% compared to the untreated control in pot and microplot experiments, respectively. I-1582 presence in the rhizosphere had concurrent nematicidal and plant growth-promoting effects, measured using plant morphology, relative chlorophyll content, elemental composition and hyperspectral imaging. Hyperspectral imaging in the 400-2500 nm spectral range and supervised classification using partial least squares support vector machines successfully differentiated B. firmus-treated and untreated plants, with 97.4% and 96.3% accuracy in pot and microplot experiments, respectively. Visible and shortwave infrared spectral regions associated with chlorophyll, N-H and C-N stretches in proteins were most relevant for treatment discrimination. This study shows the ability of hyperspectral imaging to rapidly assess the success of biological measures for pest control.

Evaluation of the Field Efficacy of Heterorhabditis Bacteriophora Poinar (Rhabditida: Heterorhabditidae) and Synthetic Insecticides for the Control of Western Corn Rootworm Larvae.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera, Chrysomelidae), is an important insect pest of maize in North America and Central and Eastern Europe. In Central Europe, the larvae emerge in May and its three instars feed intensively on maize roots in June, causing plant lodging that leads to a loss of economic yield. A three-year field experiment (2016-2018) was conducted to compare the effectiveness i) of soil-applied granular insecticide based on the active ingredient tefluthrin, ii) of maize seeds dressed with thiacloprid, and iii) entomopathogenic nematodes Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae, product Dianem) against WCR larvae. An additional treatment with alcohol ethoxylate (i.e., soil conditioner) mixed with entomopathogenic nematodes was performed in 2017 and 2018 to check for any increase of entomopathogenic nematodes' effectiveness. Field tests were carried out in two fields infested naturally with a WCR pest population, one in Bucecovci (Eastern Slovenia) and the other in Smartno pri Cerkljah (northern Slovenia), exhibiting dissimilar pedo-climatic conditions and soil pest densities. The treatments were performed in five replicates per experiment in each year. The efficacy of the treatments was very similar at both locations, despite the approximately five-fold lower WCR soil pest densities in northern than in eastern Slovenia, as well as being constant over time. The largest number of WCR beetles was observed in the negative control, followed by that of beetles subjected to thiacloprid treatment (insignificant decrease taking into account the entire three-year dataset). Treatments with tefluthrin (44.1 ± 11.7%), H. bacteriophora (46.2 ± 7.4%), and H. bacteriophora + alcohol ethoxylate (49.2 ± 1.8%) significantly decreased the numbers of emerging beetles. Treatments of thiacloprid, H. bacteriophora, and H. bacteriophora + alcohol ethoxylate additionally led to significantly increased maize plant weights. Furthermore, entomopathogenic nematodes were able to persist in maize fields for almost five months at both experimental locations in silty and sandy loam soils. It was concluded that the control of WCR larvae in maize using the entomopathogenic nematode H. bacteriophora is as effective as a tefluthrin treatment, and could thus offer a sustainable Diabrotica v. virgifera biological control management option in Europe.

Pipeline for imaging, extraction, pre-processing, and processing of time-series hyperspectral data for discriminating drought stress origin in tomatoes.

Crop infestation with root-knot nematodes (RKN) and water deficiency lead to similar visible symptoms in the plant canopy. Identification of biotic or abiotic stress origin is therefore a problem, and currently the only reliable methods for determination of RKN infestation are invasive and applicable only for point-searches. In this study the applicability of hyperspectral remote sensing for early identification of drought stress and RKN infestations in tomato plants was tested. A four-stage image and data management pipeline was established: (1) image acquisition, (2) data extraction, (3) pre-processing, and (4) processing. •This pipeline reduces atmospheric impacts, facilitates data extraction (by using specially designed spectral libraries and supervised classification procedures), diminishes the impact of viewing geometry, and emphasized small spectral variations not apparent in the raw data.•By combining partial least squares - discriminant analysis and support vector machines with time series analysis, we achieved up to 100% classification success when determining watering regime and infestation, and their severity.•This pipeline could be at least partially automated, thus facilitating high throughput identification of stress origin in plants. Furthermore, the same pipeline could be applied to hyperspectral phenotyping procedures, which are gaining importance in breeding programs.

Pore-forming protein complexes from Pleurotus mushrooms kill western corn rootworm and Colorado potato beetle through targeting membrane ceramide phosphoethanolamine.

Aegerolysins ostreolysin A (OlyA) and pleurotolysin A (PlyA), and pleurotolysin B (PlyB) with the membrane-attack-complex/perforin domain are proteins from the mushroom genus Pleurotus. Upon binding to sphingomyelin/cholesterol-enriched membranes, OlyA and PlyA can recruit PlyB to form multimeric bi-component transmembrane pores. Recently, Pleurotus aegerolysins OlyA, PlyA2 and erylysin A (EryA) were demonstrated to preferentially bind to artificial lipid membranes containing 50 mol% ceramide phosphoethanolamine (CPE), the main sphingolipid in invertebrate cell membranes. In this study, we demonstrate that OlyA6, PlyA2 and EryA bind to insect cells and to artificial lipid membranes with physiologically relevant CPE concentrations. Moreover, these aegerolysins permeabilize these membranes when combined with PlyB. These aegerolysin/PlyB complexes show selective toxicity toward western corn rootworm larvae and adults and Colorado potato beetle larvae. These data strongly suggest that these aegerolysin/PlyB complexes recognize CPE as their receptor molecule in the insect midgut. This mode of binding is different from those described for similar aegerolysin-based bacterial complexes, or other Bacillus thuringiensis Cry toxins, which have protein receptors. Targeting of Pleurotus aegerolysins to CPE and formation of transmembrane pores in concert with PlyB suggest the use of aegerolysin/PlyB complexes as novel biopesticides for the control of western corn rootworm and Colorado potato beetle.

Laboratory Investigation of Cauliflower-Fungus-Insect Interactions for Cabbage Maggot Control.

The cabbage maggot (also known as cabbage root fly [CRF]; Delia radicum L.) is a serious pest of brassicas. The pest's soil-dwelling larvae are especially damaging to young brassica transplants. In light of toxic soil insecticide phase-out novel biocontrol management solutions are sought for. Our research is focused on the development of a biological control strategy involving cauliflower plantlet inoculation with insect pathogenic fungi. This article presents the results of a laboratory investigation of cauliflower × microbe × CRF interactions. Seven isolates of fungi (entomopathogenic and rhizosphere-competent fungi and soil saprotrophs) were tested for their pathogenicity to CRF and their effects on cauliflower plantlets. The laboratory experiments were performed in sterilized substrate. Several strains significantly increased CRF mortality, some at par with a commercial bioinsecticide based on B. bassiana (Balsamo-Crivelli) Vuillemin (Hypocreales: Cordycipitaceae). All strains colonized the rhizoplane, however to varying extent. Some isolates were also reisolated from within healthy plant tissues and thus identified as endophytes. The method of applying conidia had a significant effect on survival and weight of seedlings and rhizoplane and endophytic colonization rates. Two Metarhizium brunneum Petsch (Hypocreales: Clavicipitaceae) isolates exhibited plant growth promotion effects when ungerminated seeds were coated with conidia. The ecological implications of plant × microbe × pest interactions and options for improving the effectiveness of a fungal-based biological CRF management strategy are discussed.

Comparison of cauliflower-insect-fungus interactions and pesticides for cabbage root fly control.

Cabbage root fly (Delia radicum L.) control represents a major challenge in brassica production, therefore different management strategies for its control were tested in conventionally managed open field cauliflower production. Strategies included treatments with low-risk methods such as nitrogen lime, the insecticide spinosad and the Beauveria bassiana ATCC 74040-based biopesticide Naturalis. Their effects were compared with treatments based on nonformulated fungal species Metarhizium brunneum, B. bassiana, Clonostachys solani, Trichoderma atroviride, T. koningiopsis, and T. gamsii and commercial insecticides λ-cyhalothrin and thiamethoxam. Spinosad and thiamethoxam were pipetted to individual plants before transplanting; λ-cyhalothrin was sprayed after transplanting; nitrogen lime was applied at first hoeing. Nonformulated fungi were delivered onto cauliflower plantlets' roots as a single pretransplantation inoculation. The cabbage root fly population dynamics exhibited a strong spatiotemporal variation. The lowest number of cabbage root fly pupae recovered from cauliflower roots in the field experiments was recorded in plants treated with spinosad (significant reduction), followed by Naturalis and one of the tested M. brunneum strains (nonsignificant reduction). Significantly more pupae were counted in the nitrogen lime treatment. The field experiments showed that a single drench of cauliflower plantlets with spinosad offered consistent and enduring cabbage root fly control. Naturalis and nonformulated fungal isolates did not decrease cabbage root fly pressure significantly, apparently due to lack of statistical power. The implications of the substantial intra- and inter-annual pest pressure variation and the benefits of using single plant treatments are discussed, and recommendations for improvement of rhizosphere-competence utilizing biological control strategies provided.

On-farm evaluation of inundative biological control of Ostrinia nubilalis (Lepidoptera: Crambidae) by Trichogramma brassicae (Hymenoptera: Trichogrammatidae) in three European maize-producing regions.

BACKGROUND: A 2 year study was conducted to evaluate the efficacy of biological control with optimally timed Trichogramma brassicae releases as an integrated pest management tool against the European corn borer (ECB), Ostrinia nubilalis (Hübner), in on-farm experiments (i.e. real field conditions) in three European regions with dissimilar geoclimatic conditions and ECB pressure and conventional management (i.e. insecticide treated and untreated). RESULTS: Biological control with Trichogramma (1) provided ECB protection comparable with conventional management, (2) in all cases maintained mycotoxin levels below the EU threshold for maize raw materials destined for food products, (3) was economically sustainable in southern France and northern Italy, but not in Slovenia where it resulted in a significant decrease in gross margin, mainly owing to the cost of Trichogramma product, and (4) enabled avoidance of detrimental environmental effects of lambda-cyhalothrin use in northern Italy. CONCLUSION: Optimally timed mass release of T. brassicae could be considered a sustainable tool for IPM programmes against ECB in southern France and northern Italy. Better involvement of regional advisory services is needed for the successful dissemination and implementation of biological control. Subsidy schemes could also motivate farmers to adopt this IPM tool and compensate for high costs of Trichogramma product.

Evaluation of insect associated and plant growth promoting fungi in the control of cabbage root flies.

Delia radicum L. or cabbage maggot is an important pest for Brassicaceous crops. There are currently no registered chemical control agents for its control in Slovenia. Fungal control agents for cabbage maggot were therefore sought among nine rhizosphere-compatible and plant growth-promoting, soil-adapted, and entomopathogenic species to cabbage maggots and were assayed in in vitro and soil laboratory bioassays. In the in vitro tests, the conidial suspensions were applied directly to cabbage maggot eggs. The soil tests mimicked pathways of natural exposure of various insect life stages to the fungal strains. Conidial concentrations used in soil tests were comparable to economic rates for in-furrow application. The following fungi were tested: Trichoderma atroviride P. Karst. (2 isolates), Trichoderma koningiopsis Samuels, C. Suárez & H.C. Evans (1), Trichoderma gamsii Samuels & Druzhin. (3), Beauveria brongniartii (Saccardo) Petch (1), Beauveria bassiana (Balsamo-Crivelli) Vuillemin (2), Metarhizium robertsii J.F. Bisch., Rehner & Humber (1), Metarhizium anisopliae (Metschn.) Sorokin (4), Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel-Jones & Samson (2), and Clonostachys solani f. nigrovirens (J.F.H. Beyma) Schroers (2). Abbott's corrected mortality in the in vitro tests ranged from 0.0 +/- 18.9 to 47.6 +/- 9.0% and in the soil test from 2.4 +/- 13.0 to 68.2 +/- 21.5%. Seven isolates (B. bassiana [isolate 1174], C. solani [1828], M. anisopliae [1154 and 1868], T. atroviride [1872], T. koningiopsis [1874], and T. gamsii [1876]) caused significant cabbage maggot mortality in either in vitro or soil tests. The importance of fungal ecology as a criterion during the screening of potential biological control agents is discussed.