Pesticides are considered one of the main drivers of insect decline, and complex pesticide mixtures are frequently detected in our landscapes. However, the risk posed by mixtures of active substances is not systematically assessed. In this study using acute contact dose-response testing, we show that binary and triple combinations of relevant surrogates for major pesticide classes can lead to synergistic toxic effects on the non-target insect Oncopeltus fasciatus . First, we tested the individual active substances acetamiprid (insecticide), fluopyram (fungicide) and terbuthylazine (herbicide), detecting acute toxic effects only for acetamiprid. However, the combination of acetamiprid and terbuthylazine significantly increased toxicity compared to acetamiprid alone and the combination of acetamiprid and fluopyram with the effective dose 50 being 2.7 and 2.3 times lower respectively. Strikingly, a triple mixture of all active substances exceeded the toxicity of any other treatment. While the exact mechanisms remain unclear, we speculate that increased physiological stress contributes to the observed synergistic effects, as terbuthylazine and fluopyram have been shown to interfere, though to varying extents, with cellular energy metabolism, oxidative balance, detoxification and neurotransmission in several non-target organisms. These results are concerning and highlight the ecotoxicological uncertainty posed by traces of complex pesticide mixtures present in the air, soil, water and plants across landscapes. We therefore propose a systematic assessment of realistic exposure scenarios reflecting both agricultural practices and observed environmental mixtures. ### Competing Interest Statement The authors have declared no competing interest. University of Hohenheim, Graduate Academy, Baden-Württemberg Graduate Support Programme

Although pesticides have been proposed as one of the main causes of insect decline, there are still few studies assessing their effects on non-target species under field conditions. Here we investigated the effects of the neonicotinoid insecticide Mospilan (R) SG (active ingredient acetamiprid) on plant bugs (Heteroptera: Miridae), a dominant group of European grassland insect communities. In a controlled field study, the abundance of three focal species was reduced by up to 92% two days after field exposure at concentrations expected at field margins, with mortality varying among species. Follow-up feeding assays with insecticide-treated host plants in the greenhouse and controlled dose-response laboratory assays confirmed the strong negative effects on non-target species. Strikingly, when comparing the lethal dose 50 derived from Mospilan (R) SG with a value reported for honeybees using another acetamiprid formulation, the insecticide was over 11,000 times more toxic to plant bugs than to honeybees. In addition, males were 20 times more sensitive than females in the two tested species. Thus, continuous exposure to this neonicotinoid may reduce plant bug populations and promote insecticide-tolerant species, altering community composition. We suggest that sex-specific sensitivity be considered in risk assessment and conclude that the true risk to non-target insects is currently greatly underestimated.

The adaptation of monarch butterflies (Danaus plexippus) to milkweed plants and their ability to sequester toxic cardenolides is a model system for plant-herbivore coevolution. However, the physiological mechanisms underlying cardenolide sequestration and its temporal dynamics remain largely unknown. Here, we show that the polar cardenolide ouabain passes through the isolated midgut epithelium of D. plexippus in vitro and is also absorbed into the body cavity of monarch caterpillars. Remarkably, the same pattern was observed in caterpillars of the related, but non-sequestering milkweed butterfly Euploea core, and even in the non-adapted Solanaceae specialist Manduca sexta, although uptake across gut epithelia occurred at a lower rate. Furthermore, we demonstrated that cardenolides begin to cross the epithelium in the anterior part of the intestine and can be detected in body tissues as soon as one minute after ingestion. Finally, we show that not all cardenolides are translocated into butterfly tissues during metamorphosis, and that the most apolar cardenolides are removed with the last caterpillar exuviae. As a result, adult butterflies contain no cardenolides less polar than the milkweed cardenolide calactin. We conclude that uptake by the intestinal epithelium is a very rapid process and that quantitative differences in cardenolide sequestration among lepidopteran caterpillars are only partially mediated by the gut epithelium, likely involving additional mechanisms such as metabolism or excretion. In addition, the translocation of cardenolides from the caterpillar is a selective process which may be due to the limited mobility of highly apolar cardenolides.

Although pesticides are seen as one of the main drivers of insect decline, there are still only few studies that assess their effects on non-target species under field conditions. Here we investigated the effects of the neonicotinoid insecticide Mospilan®SG (active ingredient acetamiprid) on plant bugs (Heteroptera: Miridae), a dominant group of European grassland insect communities. Abundance of three focal mirid species was reduced by up to 78% two days after field exposure at concentrations expected at field margins, with mortality varying considerably among species. Follow-up feeding assays with insecticide-treated host plants in the greenhouse and controlled dose-response assays in the laboratory confirmed the strong negative effects on non-target species. Strikingly, the neonicotinoid was nearly 10,000 times more toxic to plant bugs than to honeybees. In addition, male bugs were 20 times more sensitive than females in two of the three tested species. Thus, continuous exposure to neonicotinoids in the field may reduce the plant bug populations and promote a shift towards more insecticide-tolerant species, altering community composition. We suggest that sex-specific sensitivity should be considered in risk assessment and conclude that the real risk to non-target insects is currently greatly underestimated. ### Competing Interest Statement The authors have declared no competing interest.

Attempts have been made to determine the in vitro and in planta suppressive potential of particular Trichoderma strains (T16 and T23) and their secondary metabolites (SMs) against Asian soybean rust (ASR) incited by Phakopsora pachyrhizi. Aside from the previously identified SMs 6-pentyl-α-pyrone (6PAP) and viridiofungin A (VFA), the chemical structures of harzianic acid (HA), iso-harzianic acid (iso-HA), and harzianolide (HZL) were characterized in this study. Our results indicate that exposure of urediospores to 200 ppm 6PAP completely inhibits germination. A slightly higher dosage (250 ppm) of HZL and VFA reduces germination by 53.7% and 44%, respectively. Germ tube elongation seems more sensitive to 6PAP than urediospore germination. On detached leaves, application of conidia of T16 and T23 results in 81.4% and 74.3% protection, respectively. Likewise, 200 ppm 6PAP recorded the highest ASR suppression (98%), followed by HZL (78%) and HA (69%). Treatment of undetached leaves with 6PAP, HA, or HZL reduces ASR severity by 84.2%, 65.8%, and 50.4%, respectively. Disease reduction on the next, untreated trifoliate by T23 (53%), T16 (41%), HZL (42%), and 6PAP (32%) suggests a translocation or systemic activity of the SMs and their producers. To our knowledge, this study provides the first proof for controlling ASR using antifungal SMs of Trichoderma. Our findings strongly recommend the integration of these innovative metabolites, particularly 6PAP and/or their producers in ASR management strategies.

Crop production is increasingly affected by water limitation even in temperate climates due to a rising frequency of drought periods, related with global change. The stress-protective nutrients, discussed as a mitigation strategy, were investigated for their potential drought-protective effects of nutrient seed treatments, based on Ca, K, Fe, Zn, Mn on early growth of oilseed rape (OSR). Responses were observed in five OSR hybrids under greenhouse conditions on two soils with contrasting properties (sandy-loam pH 5.6 vs. silty-loam pH 6.9) in two independent pot experiments. A 7-days drought period with reduced soil moisture level (40% soil water-holding capacity. WHC) inhibited shoot and root growth and caused irreversible wilting and leaf necrosis (27-46% of total leaf area) particularly on the sandy-loam with lower WHC, depending on the investigated genotype. Nutrient seed treatment increased shoot (10-15%) and particularly root growth (14-23%) as well as nutrient accumulation, but also reduced the proportion of irreversibly damaged leaves to 17-21%, with the largest effect in strongly drought-affected genotypes under the challenging conditions on the sandy-loam soil. Analysis of physiological stress indicators revealed increased accumulation of phenolics (23-28%), antioxidants (14-47%) and higher activities of ascorbate peroxidase (APX) (23-87%) in the leaf tissue, counteracting drought-induced oxidative stress. Moreover, APX activity was positively related with root length (R20.8953), suggesting a protective effect on drought-induced oxidative IAA degradation with inhibitory effects on root growth. Increased levels of absisic, jasmonic and salicylic acids during drought stress recovery point to stress priming effects, strengthening the natural adaptive responses to water limitation.

Soil-borne pathogens can severely limit plant productivity. Induced defense responses are plant strategies to counteract pathogen-related damage and yield loss. In this study, we hypothesized that benzoic acid and lettucenin A are involved as defense compounds against Rhizoctonia solani and Olpidium virulentus in lettuce. To address this hypothesis, we conducted growth chamber experiments using hydroponics, peat culture substrate and soil culture in pots and minirhizotrons. Benzoic acid was identified as root exudate released from lettuce plants upon pathogen infection, with pre-accumulation of benzoic acid esters in the root tissue. The amounts were sufficient to inhibit hyphal growth of R. solani in vitro (30%), to mitigate growth retardation (51%) and damage of fine roots (130%) in lettuce plants caused by R. solani, but were not able to overcome plant growth suppression induced by Olpidium infection. Additionally, lettucenin A was identified as major phytoalexin, with local accumulation in affected plant tissues upon infection with pathogens or chemical elicitation (CuSO4) and detected in trace amounts in root exudates. The results suggest a two-stage defense mechanism with pathogen-induced benzoic acid exudation initially located in the rhizosphere followed by accumulation of lettucenin A locally restricted to affected root and leaf tissues.

Fertilization management can affect plant performance and soil microbiota, involving still poorly understood rhizosphere interactions. We hypothesized that fertilization practice exerts specific effects on rhizodeposition with consequences for recruitment of rhizosphere microbiota and plant performance. To address this hypothesis, we conducted a minirhizotron experiment using lettuce as model plant and field soils with contrasting properties from two long-term field experiments (HUB-LTE: loamy sand, DOK-LTE: silty loam) with organic and mineral fertilization history. Increased relative abundance of plant-beneficial arbuscular mycorrhizal fungi and fungal pathotrophs were characteristic of the rhizospheres in the organically managed soils (HU-org; BIODYN2). Accordingly, defense-related genes were systemically expressed in shoot tissues of the respective plants. As a site-specific effect, high relative occurrence of the fungal lettuce pathogen Olpidium sp. (76–90%) was recorded in the rhizosphere, both under long-term organic and mineral fertilization at the DOK-LTE site, likely supporting Olpidium infection due to a lower water drainage potential compared to the sandy HUB-LTE soils. However, plant growth depressions and Olpidium infection were exclusively recorded in the BIODYN2 soil with organic fertilization history. This was associated with a drastic (87–97%) reduction in rhizosphere abundance of potentially plant-beneficial microbiota (Pseudomonadaceae, Mortierella elongata) and reduced concentrations of the antifungal root exudate benzoate, known to be increased in presence of Pseudomonas spp. In contrast, high relative abundance of Pseudomonadaceae (Gammaproteobacteria) in the rhizosphere of plants grown in soils with long-term mineral fertilization (61–74%) coincided with high rhizosphere concentrations of chemotactic dicarboxylates (succinate, malate) and a high C (sugar)/N (amino acid) ratio, known to support the growth of Gammaproteobacteria. This was related with generally lower systemic expression of plant defense genes as compared with organic fertilization history. Our results suggest a complex network of belowground interactions among root exudates, site-specific factors and rhizosphere microbiota, modulating the impact of fertilization management with consequences for plant health and performance.

Biological Nitrification Inhibition (BNI) of Brachiaria humidicola has been mainly attributed to the root-exuded fusicoccane-type diterpene brachialactone. We hypothesized, however, that according to the high diversity of fusicoccanes described for plants and microorganisms, BNI of B. humidicola is caused by an assemblage of bioactive fusicoccanes. B. humidicola root exudates were collected hydroponically and compounds isolated by semi-preparative HPLC. Chemical structures were revealed by spectroscopic techniques, including HRMS as well as 1D and 2D NMR. Nitrification inhibiting (NI) potential of isolated compounds was evaluated by a Nitrosomonas europaea based bioassay. Besides the previously described brachialactone (1), root exudates contained 3-epi-brachialactone (2), the C3-epimer of 1 (m/z 334), as well as 16-hydroxy-3-epi-brachialactone (3) with an additional hydroxyl group at C16 (m/z 350) and 3,18-epoxy-9-hydroxy-4,7-seco-brachialactone (4), which is a ring opened brachialactone derivative with a 3,18 epoxide ring and a hydroxyl group at C9 (m/z 332). The 3-epi-brachialactone (2) showed highest NI activity (ED50 similar to 20 mu g mL(-1), ED80 similar to 40 mu g mL(-1)), followed by compound 4 with intermediate (ED50 similar to 40 mu g mL(-1)) brachialactone (1) with low and compound 3 without activity. In coherence with previous reports on fusicoccanes, stereochemistry at C3 was of high relevance for the biological activity (NI potential) of brachialactones.

AimsLow soil temperature in spring is a major constraint for the cultivation of tropical crops in temperate climates. This study aims at the exploitation of synergistic interactions of micronutrients, consortia of plant growth-promoting microorganisms and N forms as cold-stress protectants.MethodsMaize seedlings were exposed for two weeks to low root zone temperatures at 8–14°C under controlled conditions on a silty clay-loam soil (pH 6.9) collected from a maize field cultivation site. A pre-selection trial with fungal and bacterial PGPM strains revealed superior cold-protective performance for a microbial consortium of Trichoderma harzianum OMG16 and Bacillus spp. with Zn/Mn supplementation (CombiA+), particularly in combination with N-ammonium as a starting point for the characterization of the underlying physiological and molecular mechanisms.ResultsIn nitrate-treated plants, the cold stress treatment increased oxidative leaf damage by 133% and reduced the shoot biomass by 25%, related with reduced acquisition of phosphate (P), zinc (Zn) and manganese (Mn). The supplying of N as ammonium improved the Zn and Mn nutritional status and increased the ABA shoot concentration by 33%, as well as moderately increased detoxification of reactive oxygen species (ROS). Moreover, use of N as ammonium also increased the root auxin (IAA) concentration (+76%), with increased expression of auxin-responsive genes, involved in IAA synthesis (ZmTSA), transport (ZmPIN1a), and perception (ZmARF12). Additional inoculation with the microbial consortium promoted root colonization with the inoculant strain T. harzianum OMG16 in combination with ammonium fertilization (+140%). An increased ABA/cytokinin ratio and increased concentrations of jasmonic (JA) and salicylic acids (SA) were related to a further increase in enzymatic and non-enzymatic ROS detoxification. Additional supplementation with Zn and Mn further increased shoot IAA, root length and total antioxidants, resulting in the highest shoot biomass production and the lowest leaf damage by oxidative chemical species.ConclusionOur results suggest the mitigation of cold stress and reduction of stress priming effects on maize plants due to improved ROS detoxification and induction of hormonal stress adaptations relying on the strategic combination of stress-protective nutrients with selected microbial inoculants.

The form of nitrogen (N) supply has a significant impact on rhizosphere chemistry and root growth responses of higher plants. The respective effects are also employed as management options to improve nutrient acquisition and to minimize nutrient losses in cropping systems. However, surprisingly little is known concerning the interactions with rhizosphere biota. In this study, we investigated the effects of selected bacterial and fungal inoculants with proven plant growth-promoting and phosphate (P)-solubilizing potential (plant growth-promoting microorganisms, PGPM) in maize with nitrate or stabilized ammonium supply, on soils with limited P availability and sparingly soluble rock phosphate (Rock-P) applied as P fertilizer. The combination of the bacterial inoculants Pseudomonas sp. DSMZ 13134 (Proradix) and Bacillus amyloliquefaciens FZB42 with ammonium sulphate fertilization, stabilized with the nitrification inhibitor 3,4-dimethylpyrazole-phosphate (DMPP), resulted in a superior shoot biomass production (79-111%) and shoot P accumulation (109-235%) as compared with nitrate supply. This effect could be partially attributed to (1) ammonium-induced rhizosphere acidification via increased root extrusion of protons, (2) promotion of root hair elongation, and (3) increased shoot concentrations of hormonal growth regulators (indole-3-acetic acid, zeatin, gibberellic acid). The effects, induced by the microbial inoculants were mainly related to increased root length development (43-44%), associated with a 60% increase in auxin production potential. No inoculant effects were detected on root hair elongation or on chemical modifications of the rhizosphere involved in P solubilisation, such as rhizosphere acidification, release of carboxylates or secretory phosphohydrolases. However, the ammonium-induced stimulation of root hair elongation increased preferential sites for root colonization by the selected inoculants, which may explain the increase in rhizosphere abundance of PGPMs, exemplarily recorded for the fungal inoculant Trichoderma harzianum OMG16 (210%). The presented data suggest a network of positive interactions between stabilized ammonium fertilization and plant growth-promoting functions of various bacterial and fungal PGPM inoculants. This offers perspectives to increase the efficiency and the reproducibility of PGPM-assisted fertilization strategies.

Low soil temperature in spring is a major constraint for the cultivation of tropical and subtropical crops in temperate climates, associated with inhibition of root growth and activity, affecting early growth and frequently plant performance and final yield. This study was initiated to investigate the physiological base of cold-protective effects induced by supplementation with silicon (Si), widely recommended as a stress-protective mineral nutrient. Maize was used as a cold-sensitive model plant, exposed to chilling stress and low root-zone temperature (RZT) during early growth in a lab to field approach. In a pot experiment, 2–weeks exposure of maize seedlings to low RZT of 12–14°C, induced leaf chlorosis and necrosis, inhibition of shoot and root growth and micronutrient limitation (particularly Zn and Mn). These phenotypes were mitigated by seed treatments with the respective micronutrients, but surprisingly, also by Si application. Both, silicon and micronutrient treatments were associated with increased activity of superoxide dismutase in shoot and roots (as a key enzyme for detoxification of reactive oxygen species, depending on Zn and Mn as cofactors), increased tissue concentrations of phenolics, proline, and antioxidants, but reduced levels of H2O2. These findings suggest that mitigation of oxidative stress is a major effect of Zn, Mn, and Si applied as cold stress protectants. In a soil–free culture system without external nutrient supply, Si significantly reduced large leaching losses of Zn and Mn from germinating seeds exposed to low-temperature stress. Silicon also increased the translocation of micronutrient seed reserves to the growing seedling, especially the Zn shoot translocation. In later stages of seedling development (10 days after sowing), cold stress reduced the root and shoot contents of important hormonal growth regulators (indole acetic acid, gibberellic acid, zeatin). Silicon restored the hormonal balances to a level comparable with non-stressed plants and stimulated the production of hormones involved in stress adaptation (abscisic, salicylic, and jasmonic acids). Beneficial effects of Si seed treatments on seedling establishment and the nutritional status of Zn and Mn were also measured for a field-grown silage maize, exposed to chilling stress by early sowing. This translated into increased final biomass yield.

Fusarium graminearum (teleomorph: Gibberella zeae) is the causal agent of several destructive diseases in cereal crops worldwide. In the present study we have evaluated the potential of two strains of Trichoderma sp. (T23, and T16), a strain of Paecilomyces sp. (PS1), and their secondary metabolites (SMs) in suppressing F. graminearum. Results from dual culture experiments show that in the presence of either Trichoderma sp., or Paecilomyces sp. mycelial growth of F. graminearum is considerably inhibited. Strain T23 causes the greatest inhibition (83.8%), followed by strain T16 (72.2%), and strain PS1 (61.9%). Likewise, mycelial growth of the pathogen is completely inhibited (≥ 98%) when grown under exposure to volatile metabolites excreted from Trichoderma cultures. Bioautographic analyses using culture filtrates revealed that several antifungal SMs are excreted. Among five metabolites tested, 6-pentyl-alpha-pyrone (6PAP) from strain T23, and PF3 from strain PS1 exhibit pronounced antifungal activity against F. graminearum. A new method for mass production of perithecia of F. graminearum which is simple and more effective than traditional methods was developed, which allows an increase in perithecial formation of more than 5-fold. Using this method, we found, that in the presence of SMs perithecial formation was negatively affected. Perithecial production was suppressed by 81.4% and 76.6% using 200 μg ml−1 of either 6PAP or PF3, respectively. Moreover, ascospore discharge was significantly suppressed (67.0%) when perithecia were exposed to the metabolite F116 produced by T16. Including 6PAP or PF3 in conidial suspensions impeded germination of conidia completely. Similarly, both metabolites strongly inhibited ascospore germination (˃ 90%).

Antibiosis is assumed to be an essential mechanism exerted by potential biocontrol agents (BCAs) of Trichoderma spp. Therefore, in the present study, we report for the first time on the elucidation and production of viridiofungin A (VFA) from T. harzianum isolate T23 cultures and investigate the antifungal potential of VFA and some other secondary metabolites purified from T. harzianum cultures against Fusarium moniliforme. The bioautography assay revealed that T. harzianum isolates T16 and T23 excreted several secondary metabolites with antifungal activity. Following isolation and purification of the antifungal zones, three fractions (F223, F323 and F423) from extracts of isolate T23 and two fractions (F416 and F516) from extracts of isolate T16 exhibited pronounced fungitoxic activity in the bioautography and antibiotic disk assays against Cladosporium spp. and F. moniliforme, respectively. The structure of the antifungal metabolite in fraction F323 was identified as viridiofungin A (VFA), the first report of production of VFA by isolate T23 of T. harzianum. Following cultivation of isolate T23 in PDB medium for 9 days, 94.6 mg l−1 of VFA were determined. VFA and fraction F516 retarded the mycelial growth of F. moniliforme in the non-volatile phase assay by >90% for each 250 μg ml−1 7 days post-inoculation (dpi). While VFA and fraction F416 showed both volatile and non-volatile effects, fraction F516 seemed to exhibit mainly non-volatile activity. Microscopic examination revealed that hyphae of F. moniliforme grown on VFA-amended medium were less branched and appeared thicker than untreated hyphae. Furthermore, in the presence of VFA, formation of chlamydospores by F. moniliforme was increased. Finally, the antifungal spectrum of VFA towards various important plant pathogens was evaluated. Germination of propagules of a variety of fungal pathogens in vitro was differentially inhibited by VFA. While in the presence of 100 μg ml−1 VFA conidial germination of V. dahliae was completely inhibited, a slightly higher concentration (150 μg ml−1) of the inhibitor was required to suppress germination of Phytophthora infestans sporangia or sclerotia of Sclerotinia sclerotiorum. Contrary to several reports in the literature, VFA proved to be fungistatic rather than fungicidal. However, neither VFA nor the other Trichoderma metabolites, such as 6PAP, F416 and F516, exhibited any antibacterial activity against Gram-positive and Gram-negative bacteria.