Clonal genomic population structure of Beauveria brongniartii and Beauveria pseudobassiana: Pathogens of the common European cockchafer (Melolontha melolontha L.).

Beauveria brongniartii is a fungal pathogen that infects the beetle Melolontha melolontha, a significant agricultural pest in Europe. While research has primarily focused on the use of B. brongniartii for controlling M. melolontha, the genomic structure of the B. brongniartii population remains unknown. This includes whether its structure is influenced by its interaction with M. melolontha, the timing of beetle-swarming flights, geographical factors, or reproductive mode. To address this, we analysed genome-wide SNPs to infer the population genomics of Beauveria spp., which were isolated from infected M. melolontha adults in an Alpine region. Surprisingly, only one-third of the isolates were identified as B. brongniartii, while two-thirds were distributed among cryptic taxa within B. pseudobassiana, a fungal species not previously recognized as a pathogen of M. melolontha. Given the prevalence of B. pseudobassiana, we conducted analyses on both species. We found no spatial or temporal genomic patterns within either species and no correlation with the population structure of M. melolontha, suggesting that the dispersal of the fungi is independent of the beetle. Both species exhibited clonal population structures, with B. brongniartii fixed for one mating type and B. pseudobassiana displaying both mating types. This implies that factors other than mating compatibility limit sexual reproduction. We conclude that the population genomic structure of Beauveria spp. is primarily influenced by predominant asexual reproduction and dispersal.

Spatial and temporal patterns in the population genomics of the European cockchafer Melolontha melolontha in the Alpine region.

The European cockchafer Melolontha melolontha is an agricultural pest in many European countries. Populations have a synchronized 3 or 4 years life cycle, leading to temporally isolated populations. Despite the economic importance and availability of comprehensive historical as well as current records on cockchafer occurrence, population genomic analyses of M. melolontha are missing. For example, the effects of geographic separation caused by the mountainous terrain of the Alps and of temporal isolation on the genomic structure of M. melolontha still remain unknown. To address this gap, we genotyped 475 M. melolontha adults collected during 3 years from 35 sites in a central Alpine region. Subsequent population structure analyses discriminated two main genetic clusters, i.e., the South Tyrol cluster including collections located southeast of the Alpine mountain range, and a northwestern alpine cluster with all the other collections, reflecting distinct evolutionary history and geographic barriers. The "passo di Resia" linking South and North Tyrol represented a regional contact zone of the two genetic clusters, highlighting genomic differentiation between the collections from the northern and southern regions. Although the collections from northwestern Italy were assigned to the northwestern alpine genetic cluster, they displayed evidence of admixture with the South Tyrolean genetic cluster, suggesting shared ancestry. A linear mixed model confirmed that both geographic distance and, to a lower extent, also temporal isolation had a significant effect on the genetic distance among M. melolontha populations. These effects may be attributed to limited dispersal capacity and reproductive isolation resulting from synchronized and non-synchronized swarming flights, respectively. This study contributes to the understanding of the phylogeography of an organism that is recognized as an agricultural problem and provides significant information on the population genomics of insect species with prolonged temporally shifted and locally synchronized life cycles.

Light in the box-photobiological examination chamber with light trap ventilation system for studying fungal surface cultures illustrated with Metarhizium brunneum and Beauveria brongniartii.

Due to their versatile way of life as saprophytes, endophytes, and entomopathogens, fungi of the genera Metarhizium and Beauveria are exposed to varying illumination conditions in their natural habitats, which makes a thorough adaptation to light very likely. While the few available studies for these genera support this assumption, research in this field is still in its infancy and the data material restricted to only a few fungal species. Thus, the aim of this work was to explore how light influences growth, conidial production and secondary metabolite formation of two industrial relevant strains of M. brunneum (MA 43, formerly M. anisopliae var. anisopliae BIPESCO 5/F52) and B. brongniartii (BIPESCO 2). To achieve this, we constructed an easily adjustable illumination device for highly standardized photophysiological studies of fungi on Petri dishes, the so-called LIGHT BOX. With the aid of this device, M. brunneum and B. brongniartii were grown on S4G or S2G agar at 25 °C for 14 days either in complete darkness or under constant illumination with red light (λpeak = 635 nm), green light (λpeak = 519 nm) or blue light (λpeak = 452 nm). In addition, for each wavelength the effect of different illumination intensities was tested, i.e., intensities of red light ranging from 22.1 ± 0.1 to 136.5 ± 0.3 µW cm-2, green light from 16.5 ± 0.1 to 96.2 ± 0.1 µW cm-2, and blue light from 56.1 ± 0.2 to 188.9 ± 0.6 µW cm-2. Both fungi strongly responded in terms of growth, conidial production, pigmentation and morphology to changes in the wavelength and irradiation intensity. The wavelength-dependent production of the well-known secondary metabolite oosporein which is secreted by the genus Beauveria in particular, was also increased under green and blue light exposure. The established LIGHT BOX system allows not only to optimize conidial production yields with these biotechnologically relevant fungi, but also allows the photobiological exploration of other fungi.

Integrated Biological Control of the Sugar Beet Weevil Asproparthenis punctiventris with the Fungus Metarhizium brunneum: New Application Approaches.

The mass occurrence of the sugar beet weevil (Asproparthenis punctiventris, previously Bothynoderes punctiventris) has been endangering sugar beet cultivation in Austria for centuries. Exacerbated by climatic and political changes (warmer, drier spring and limited access to chemical pesticides), new approaches are needed to counter the problem. The aim of our work was to test whether the bioinsecticide Metarhizium brunneum Ma 43 (formerly M. anisopliae var. anisopliae BIPESCO 5/F52) can be used as a sustainable plant protection product against the sugar beet weevil. Our goal was to control the pest in all its development stages through multiple applications. Therefore, GranMetTM-P, a granular formulation of M. brunneum Ma 43, was applied in spring to establish the fungus in the soil, whereas GranMetTM-WP, a liquid formulation of the production strain, was used in early summer on trap ditches and leaves to target the adult weevils. Soil and plant samples as well as weevils were collected during the planting season from the trial sites to evaluate the development of the fungus and the mycosis of the treated weevils. In addition, data on hibernating weevils and their emigration from untreated field sites was collected. In all field sites, the Metarhizium spp. abundance increased above the background level (<1000 CFU g−1 soil dry weight) after application of the product. With an increasing number of treatments per plot, and thus an increased contact possibility between pest and the fungus, a rise in the mycosis rate was observed. In conclusion, the various Metarhizium application strategies, which are already available or in testing, must be implemented to ensure control in both old and new sugar beet fields. Metarhizium is a further asset in the successful control of this sugar beet pest.

Development of a SNP-based tool for the identification and discrimination of Melolontha melolontha and Melolontha hippocastani.

The European (Melolontha melolontha L.) and Forest (M. hippocastani F.) cockchafer are widespread pests throughout Central Europe. Both species exhibit a 3-5-year life cycle and occur in temporally shifted populations, which have been monitored and documented for more than 100 years. Visual identification of adults and larvae belonging to these morphologically similar species requires expertise and, particularly in the case of larvae, is challenging and equivocal. The goal of the study was the development of an efficient and fast molecular genetic tool for the identification and discrimination of M. melolontha and M. hippocastani. We established a collection of both species from Switzerland, Austria and Northern Italy in 2016, 2017 and 2018. An approximately 1550 bp long fragment of the cytochrome c oxidase subunit 1 (CO1) mitochondrial gene was amplified and sequenced in 13 M. melolontha and 13 M. hippocastani beetles. Alignment of the new sequences with reference sequences (NCBI GenBank and BOLDSYSTEMS databases) and subsequent phylogenetic analysis revealed consistent clustering of the two species. After the identification of M. melolontha and M. hippocastani species-specific single nucleotide polymorphisms (SNPs) in the CO1 alignment, we developed an effective SNP tool based on the ABI PRISM® SNaPshot™ Multiplex Kit for the rapid and accurate species discrimination of adults and larvae.

Analytical Methods for Secondary Metabolite Detection.

The entomopathogenic fungi Metarhizium brunneum, Beauveria bassiana, and B. brongniartii are widely applied as biological pest control agent in OECD countries. Consequently, their use has to be flanked by a risk management approach, which includes the need to monitor the fate of their relevant toxic metabolites. There are still data gaps claimed by regulatory authorities pending on their identification and quantification of relevant toxins or secondary metabolites. In this chapter, analytical methods are presented allowing the qualitative and quantitative analysis of the relevant toxic B. brongniartii metabolite oosporein and the three M. brunneum relevant destruxin (dtx) derivatives dtx A, dtx B, and dtx E.

Supercritical Fluid Chromatography as an Alternative Tool for the Qualitative and Quantitative Analysis of Metarhizium brunneum Metabolites from Culture Broth.

A fast and selective ultrahigh-performance supercritical fluid chromatography photodiode array detector method was established for the qualitative and quantitative analysis of destruxins, cyclic hexadepsipeptides, from fungal culture broth samples. Prior to analysis, sample purification was carried out using an off-line solid-phase extraction protocol on a reversed-phase material in order to remove unwanted matrix constituents. For separation, detection, and identification, an ultrahigh-performance supercritical fluid chromatography photodiode array detector system hyphenated to a triple quadrupole mass spectrometer was utilized. Analyses were performed on an Acquity ethylene bridged hybrid 2-ethylpyridine sub 2 µm particle size column with CO2 and an acidified (0.02% trifluor acetic acid) modifier mixture of methanol/acetonitrile (8/2 v/v) serving as mobile phase. For the optimal separation of destruxins, the amount of the modifier was increased in a 10 min linear gradient from 2% to 20%, and the column outlet pressure and temperature was set at 140 bars and 60 °C, respectively. Seventeen analytes were separated within an elution window of 4 minutes. Five destruxin congeners (destruxin A, destruxin B, destruxin D, destruxin E, and destruxin E-diol) were identified using reference material. Additionally, eight analytes were tentatively assigned as known destruxins by the evaluation of mass spectrometry data performed as multiple reaction monitoring experiments in the positive electrospray ionization mode.

Quantitative Assessment of Destruxins from Strawberry and Maize in the Lower Parts per Billion Range: Combination of a QuEChERS-Based Extraction Protocol with a Fast and Selective UHPLC-QTOF-MS Assay.

The entomopathogenic fungus Metarhizium brunneum is widely applied as a biological pest control agent. Consequently, its use has to be accompanied by a risk management approach, which includes the need to monitor the fate of its bioactive metabolites in the environment, for example, in treated crops. A fast and selective UHPLC-QTOF-MS method was developed to monitor the presence of secreted destruxins in two model food plants for the application of this fungal biocontrol agent, namely, strawberry and maize. The liquid chromatography-mass spectrometric assay for destruxin trace analysis is combined with a novel QuEChERS-based extraction protocol. The whole assay was optimized for the application in these crops, and it allows quantitative analysis of the major M. brunneum metabolites destruxin A, 1, destruxin B, 2, and destruxin E, 3, down to the parts per billion range. In strawberry, limits of quantitation (LOQs) were found to be <2.0 ppb for all analytes; in maize LOQs were found to be <3.2 ppb for destruxin A and destruxin B. Destruxin E showed a distinctive loss of recovery in maize and was excluded from further quantitative analysis in this crop. For both crops assay linearities ranged from the LOQs to 100 ppb, interassay repeatabilities (RSD) were found to be better than 16.4%, and accuracies ranged from 83.5 to 105.3% (assessed at four spiking levels between 5 and 75 ppb).

Development of a fast and selective UHPLC-DAD-QTOF-MS/MS method for the qualitative and quantitative assessment of destruxin profiles.

A fast and selective ultrahigh-performance liquid chromatography diode array detector (UHPLC-DAD) method combined with an off-line solid phase extraction (SPE) protocol was established to monitor destruxins (dtxs), a secondary metabolite class of highly bioactive cyclic depsipeptides. Sample purification via SPE was tailored to remove both more polar and apolar matrix constituents by applying analyte class-selective washing and elution conditions. To separate and detect destruxin congeners an UHPLC-DAD system hyphenated to a quadrupole-time-of-flight (Q-TOF) hybrid mass spectrometer was utilized. Analyses were performed on a sub-2-µm-particle-size RP-18 column with an acidified (0.02% acetic acid) 12 min water/acetonitrile solvent gradient. In the dtx congener elution zone 22 chromatographic peaks were separated. Four of these were identified by comparison with reference materials as dtx A, dtx B, dtx E, and dtx E-diol; 16 were tentatively assigned as known or novel dtx congeners by the analysis of high resolution UHPLC-DAD-QTOF-MS/MS data recorded in the positive electrospray ionization (ESI) mode. The applicability of the UHPLC-DAD assay to investigate biological materials in a qualitative and quantitative manner was proven by the application of the platform to monitor the dtx production profile of three Metarhizium brunneum strain fungal culture broths.

Formation of exudate droplets by Metarhizium anisopliae and the presence of destruxins.

Nutritional conditions causing droplet exudation by Metarhizium anisopliae var. anisopliae were studied. Exudation in droplets occurred only on media with more than one carbon source and was highly dependent on the ratio of a well metabolized sugar such as trehalose and a nonpreferred sugar, in particular arabinose. Exuded droplets contained destruxin A, B and E in concentrations similar to those on submerged culture on Czapek Dox medium with equivalent C:N ratios but was clearly less than previously reported on standard Czapek Dox or Sabouraud dextrose broth. Destruxins also were found in agar samples from directly below mycelium and from up to 2 cm from the colony edge. Exudates retrieved from different media were proven to have Pr1 protease-related enzyme activity. Additional HPLC analysis indicated that droplets from diverse media did not differ in their sugar and acid content. A hypothesis is presented regarding the trigger for guttation in Metarhizium during growth under these conditions.

Carbon utilization pattern as a potential quality control criterion for virulence of Beauveria brongniartii.

The registered entomopathogenic fungus Beauveria brongniartii (BIPESCO 2) was tested for its virulence after one, five and 10 times sub-culturing on four types of selective synthetic nutrient media. Bioassays with third instar Melolontha melolontha larvae showed that sub-culturing negatively affects the virulence of the fungus after 10 transfers. With the Biolog SF-P2 and Biolog SF-N2 microtiter plate systems the sub-cultivated B. brongniartii conidia were monitored for any change in the carbon utilization pattern of 128 carbon sources. With the help of Spearman's rank correlation, principal components analysis and canonical correspondence analysis, respectively, six carbon sources were identified as potential virulence indicators for BIPESCO 2 (pyruvic acid, maltose, glycyl-L-glutamic acid, malonic acid, glucuronamide and phenylethylamine). The Biolog microtiter plate system is suggested as a simple and inexpensive test-system for virulence determination of B. brongniartii strain BIPESCO 2 in routine quality control.

Destruxin production of Metarhizium anisopliae under carbon and nitrogen exhaustion.

Destruxins (dtx) A, B, and E, showing a variety of biological activities, are the main toxic secondary metabolites of the entomopathogenous ascomycete Metarhizium anisopliae Bipesco 5, a widely used biocontrol production strain. Dynamics of dtx biosynthesis were monitored during liquid fermentation in a chemically defined medium. During shake flask cultivation with excess carbon, nitrogen and phosphate, approximately 50, 20, and 100 mg l(-1) dtx A, B, and E were produced after 12 d. Destruxins were produced during exponential growth phase and in the stationary phase. Carbon exhaustion in the culture broth was demonstrated to affect destruxin production to a minor degree: Absolute dtx amounts in the liquid increased also after glucose exhaustion; dtx amounts referred to biomass increased further evidently in shake flasks or slightly in bioreactor experiments after carbon limitation occurred. Contrarily, nitrogen exhaustion resulted in an evident decline in dtx amounts referred to biomass. Absolute amounts in the culture broth, however, still increased slightly the following four days in bioreactor experiments. From this we conclude that dtx production is highly influenced by nitrogen availability. Generally, dtx production in bioreactors with controlled aeration (1 vvm) was significantly lower than in shake flasks.

Apolar chromatography on Sephadex LH-20 combined with high-speed counter-current chromatography. High yield strategy for structurally closely related analytes-Destruxin derivatives from Metarhizium anisopliae as a case study.

A novel high yield isolation procedure for lipophilic cyclic peptide derivatives is presented. Destruxin (dtx) A, B, D, E, and E-diol retrieval from Metarhizium anisopliae culture broth was achieved with a three-step purification protocol. After liquid-liquid extraction column chromatography over Sephadex LH-20 served as enrichment step. High-speed counter-current chromatography (HSCCC) was used for the final purification. Within the first chromatographic step dtx D and dtx E-diol were separated in purities exceeding 90%. The separation of dtx A, B, and E was achieved from an enriched Sephadex LH-20 fraction by a HSCCC protocol using light petroleum-ethyl acetate-methanol-water = 2:5:2:5 (v/v) as eluent system. These derivatives were obtained in purities above 98% and total yields exceeding 40%.

High-performance liquid chromatography-diode array detection assay for the detection and quantification of the Beauveria metabolite oosporein from potato tubers.

A high-performance liquid chromatography-diode array detection (HPLC-DAD) assay is described for the detection and quantification of the secreted Beauveria brongniartii metabolite oosporein from potato tubers. Analyte recovery was achieved with a Britton-Robinson buffer system at pH 5.5 diluted with methanol 3:7 (v/v) (BR5.5-MeOH). An internal standard protocol using 2-iodobenzoic acid was established to minimize analytical error. The resulting assay, using a binary solvent gradient with acidic modifiers and detecting the metabolite at 287 nm, showed a limit of detection (LOD) of 2.4 mg oosporein/kg potato tubers. The oosporein content of potato tuber samples obtained from a field trial using the biological pest control B. brongniartii formulation Melocont-Pilzgerste in up to five-fold higher doses (250 kg Melocont-Pilzgerste/ha) as recommended per year was found to be below the established LOD.

Development of a sensitive high-performance liquid chromatography-diode array detection assay for the detection and quantification of the beauveria metabolite oosporein from submerged culture broth and bio-control formulations.

A sensitive high-performance liquid chromatography-diode array detection (HPLC-DAD) assay is described for the detection and quantification of the Beauveria metabolite oosporein from fungal culture broth and two biocontrol agent formulations. In all cases, analyte recovery was achieved with a Britton-Robinson buffer system at pH 5.5 diluted with methanol 3:7 (v/v) (BR5.5-MeOH). The HPLC-DAD assay, using a binary solvent gradient with acidic modifiers and detecting the metabolite at 287 nm, showed linearity over 3 orders of magnitude and a limit of detection (LOD) of 6.0 +/- 2.3 microg of oosporein/L of BR5.5-MeOH. The oosporein content of the representative fungal culture broth samples and two Beauveria formulations (Melocont-Pilzgerste and Melocont-WP) was found to be 504.7 +/-13.6 mg of oosporein/L of culture filtrate, 7.4 +/- 0.6 mg of oosporein/kg of Melocont-Pilzgerste, and 38.2 +/- 1.3 mg of oosporein/kg of Melocont-WP with recovery rates of 93 +/- 2, 99 +/- 8, and 92 +/- 3%, respectively.

Accurate determination of oosporein in fungal culture broth by differential pulse polarography.

A simple and accurate differential pulse polarographic method has been developed for the determination of oosporein in the culture broth of the fungus Beauveria brongniartii. This hydroxybenzoquinone derivative is the only major secondary metabolite secreted by this entomopathogenic fungus, which is used as biological pest control agent (BCA) against Melolontha melolontha larvae. It can be found in the host organism as well as in the formulated product. The polarographic behavior of oosporein was examined in various buffer systems over the pH range 3-10. In Britton-Robinson buffer/methanol solution (3:7 v/v, pH 5.5) the differential pulse polarograms exhibited reproducible peaks at E(p) = -0.18 V vs silver/silver chloride/potassium chloride (3 M). Under these conditions, a plot of peak height vs concentration of oosporein was found to be linear over the range 5.9 x 10(-)(7) to 2.5 x 10(-)(5) M (0.18-7.74 microg mL(-)(1); r = 0.9998). The detection limit was calculated to be 54 ng mL(-)(1). To evaluate the concentration of oosporein, the standard addition method was applied. The analysis of oosporein in the culture broth led to a mean value of 524.9 microg mL(-)(1) broth with a relative standard deviation (S(rel)) of +/-2.6%. The proposed polarographic method is accurate, not time-consuming, and it is of low cost because no separation steps are necessary.

Combination of a new sample preparation strategy with an accelerated high-performance liquid chromatography assay with photodiode array and mass spectrometric detection for the determination of destruxins from Metarhizium anisopliae culture broth.

A method is presented allowing the qualitative and quantitative analysis of destruxins (dtxs) in fungal culture broth. Sample preparation was carried out by ultrafiltration over a commercially available acetylated cellulose (CTA) membrane with a Mr 10000 cut-off. The developed high-performance liquid chromatography assay with diode array detection (HPLC-DAD) cuts down the analysis time by 50% compared to most of the currently applied methods (retention times: dtx A = 8.3 min, dtx B = 8.9 min, dtx E= 7.5 min) and enables dtx detection down to sub-ppm range (limits of detection: dtx A = 0.19 mg/l, dtx B = 0.41 mg/l, dtx E = 0.10 mg/l). Stability of dtx E in filtrated culture broth was found to be much lower than anticipated (half-life time = 64.5 +/- 1.7 h). Thus, the detoxification of this metabolite is an abiotic process. Coupling of the HPLC-DAD system to an ion trap mass spectrometer with an electrospray ionization source operating in the positive mode allowed identification of most dtxs encountered by utilizing multiple stage MS-MS experiments and retention time rules.