Disentangling the mixed effects of soil management on microbial diversity and soil functions: A case study in vineyards.

Promoting soil functioning by maintaining soil microbial diversity and activity is central for sustainable agriculture. In viticulture, soil management often includes tillage, which poses a multifaceted disturbance to the soil environment and has direct and indirect effects on soil microbial diversity and soil functioning. However, the challenge of disentangling the effects of different soil management practices on soil microbial diversity and functioning has rarely been addressed. In this study, we investigated the effects of soil management on soil bacterial and fungal diversity as well as soil functions (soil respiration and decomposition) using a balanced experimental design with four soil management types in nine vineyards in Germany. Application of structural equation modelling enabled us to investigate the causal relationships of soil disturbance, vegetation cover, and plant richness on soil properties, microbial diversity, and soil functions. We could show that soil disturbance by tillage increased bacterial diversity but decreased fungal diversity. We identified a positive effect of plant diversity on bacterial diversity. Soil respiration showed a positive response to soil disturbance, while decomposition was negatively affected in highly disturbed soils via mediated effects of vegetation removal. Our results contribute to the understanding of direct and indirect effects of vineyard soil management on soil life and aids designing targeted recommendations for agricultural soil management.

Characterization of a Disease-Suppressive Isolate of Lysobacter enzymogenes with Broad Antagonistic Activity against Bacterial, Oomycetal and Fungal Pathogens in Different Crops.

Although synthetic pesticides play a major role in plant protection, their application needs to be reduced because of their negative impact on the environment. This applies also to copper preparations, which are used in organic farming. For this reason, alternatives with less impact on the environment are urgently needed. In this context, we evaluated eight isolates of the genus Lysobacter (mainly Lysobacter enzymogenes) for their activity against plant pathogens. In vitro, the investigated Lysobacter isolates showed broad antagonistic activity against several phytopathogenic fungi, oomycetes and bacteria. Enzyme assays revealed diverse activities for the tested isolates. The most promising L. enzymogenes isolate (LEC) was used for further detailed analyses of its efficacy and effective working concentrations. The experiments included in vitro spore and sporangia germination tests and leaf disc assays as well as ad planta growth chamber trials against Alternaria solani and Phytophthora infestans on tomato plants, Pseudoperonospora cubensis on cucumbers and Venturia inaequalis on young potted apple trees. When applied on leaves, dilutions of a culture suspension of LEC had a concentration-dependent, protective effect against the tested pathogens. In all pathosystems tested, the effective concentrations were in the range of 2.5-5% and similarly efficacious to common plant protection agents containing copper hydroxide, wettable sulphur or fenhexamid. Thus, the isolate of L. enzymogenes identified in this study exhibits a broad activity against common plant pathogens and is therefore a promising candidate for the development of a microbial biocontrol agent.

Altered volatile emission of pear trees under elevated atmospheric CO2 levels has no relevance to pear psyllid host choice.

The impact of climate change drivers on cultivated plants and pest insects has come into research focus. One of the most significant drivers is atmospheric carbon dioxide, which is converted into primary plant metabolites by photosynthesis. Increased atmospheric CO2 concentrations therefore affect plant chemistry. The chemical composition of non-volatile and volatile organic compounds of plants is used by insects to locate and identify suitable host plants for feeding and reproduction. We investigated whether elevated CO2 concentrations in the atmosphere affect the plant-pest interaction in a fruit crop of high economic importance in Europe. Therefore, potted pear trees were cultivated under specified CO2 conditions in a Free-Air Carbon dioxide Enrichment (FACE) facility at Geisenheim University in Germany for up to 14 weeks, beginning from bud swelling. We compared emitted volatiles from these pear trees cultivated for 7 and 14 weeks under two different CO2 levels (ambient: ca. 400 ppm and elevated: ca. 450 ppm CO2) and their impact on pest insect behavior. In total, we detected and analyzed 76 VOCs from pear trees. While we did not detect an overall change in VOC compositions, the relative release of single compounds changed in response to CO2 increase. Differences in VOC release were inconsistent over time (phenology stages) and between study years, indicating interactions with other climate parameters, such as temperature. Even though insect-plant interaction can rely on specific volatile compounds and specific mixtures of compounds, respectively, the changes of VOC patterns in our field study did not impact the host choice behavior of C. pyri females. In olfactometer trials, 64% and 60% of the females preferred the odor of pear trees cultivated under elevated CO2 for 7 and 14 weeks, respectively, over the odor from pear trees cultivated under ambient CO2. In binary-choice oviposition assays, C. pyri females laid most eggs on pears during April 2020; on average, 51.9 (± 51.3) eggs were laid on pears cultivated under eCO2 and 60.3 (± 48.7) eggs on aCO2.

Local conditions matter: Minimal and variable effects of soil disturbance on microbial communities and functions in European vineyards.

Soil tillage or herbicide applications are commonly used in agriculture for weed control. These measures may also represent a disturbance for soil microbial communities and their functions. However, the generality of response patterns of microbial communities and functions to disturbance have rarely been studied at large geographical scales. We investigated how a soil disturbance gradient (low, intermediate, high), realized by either tillage or herbicide application, affects diversity and composition of soil bacterial and fungal communities as well as soil functions in vineyards across five European countries. Microbial alpha-diversity metrics responded to soil disturbance sporadically, but inconsistently across countries. Increasing soil disturbance changed soil microbial community composition at the European level. However, the effects of soil disturbance on the variation of microbial communities were smaller compared to the effects of location and soil covariates. Microbial respiration was consistently impaired by soil disturbance, while effects on decomposition of organic substrates were inconsistent and showed positive and negative responses depending on the respective country. Therefore, we conclude that it is difficult to extrapolate results from one locality to others because microbial communities and environmental conditions vary strongly over larger geographical scales.

Three Methods Assessing the Association of the Endophytic Entomopathogenic Fungus Metarhizium robertsii with Non-Grafted Grapevine Vitis vinifera.

Characterizing the association of endophytic insect pathogenic fungi (EIPF) with plants is an important step in order to understand their ecology before using them in biological control programs. Since several methods are available, it is challenging to identify the most appropriate for such investigations. Here, we used two strains of Metarhizium robertsii: EF3.5(2) native to the French vineyard environment and ARSEF-2575-GFP a laboratory strain expressing a green fluorescent protein, to compare their potential of association with non-grafted grapevine Vitis vinifera. Three methods were used to evaluate the kinetics of rhizosphere and grapevine endospheric colonization: (i) Droplet Digital (ddPCR), a sensitive molecular method of M. robertsii DNA quantification in different plant parts, (ii) culture-based method to detect the live fungal propagules from plant tissues that grew on the medium, (iii) confocal imaging to observe roots segments. Both strains showed evidence of establishment in the rhizosphere of grapevines according to the culture-based and ddPCR methods, with a significantly higher establishment of strain EF3.5(2) (40% positive plants and quantified median of exp(4.61) c/µL) compared to strain ARSEF-2575-GFP (13% positive plants and quantified median of exp(2.25) c/µL) at 96-98 days post-inoculation. A low incidence of association of both strains in the grapevine root endosphere was found with no significant differences between strains and evaluation methods (15% positive plants inoculated with strain EF3.5(2) and 5% with strain ARSEF-2575-GFP according to culture-based method). ddPCR should be used more extensively to investigate the association between plants and EIPF but always accompanied with at least one method such as culture-based method or confocal microscopy.

Insights in genetic diversity of German and Italian grape berry moth (Eupoecilia ambiguella) populations using novel microsatellite markers.

The grape berry moth Eupoecilia ambiguella (Lepidoptera: Tortricidae) is causing significant damage to grape berries, however, little is known on population genetics of this lepidopteran pest insect, hindered so far by the lack of suitable molecular markers. Here we report on the development of ten microsatellite markers of which six were used to characterise 21 E. ambiguella populations obtained from two viticultural regions in Germany and Italy. Moths were sampled during two subsequent generations (flights) in the same vineyard as well as in vineyards surrounded by different landscape types. German and Italian populations were genetically differentiated and a significant isolation by distance was evident. No significant divergence was observed among the populations from first or second flight moths, however, inbreeding was higher in first than in second flight populations. Moreover, inbreeding was influenced by habitat composition and complexity of landscape around vineyards, being positively associated with the percentage of area covered by grapevine. Population genetics of E. ambiguella could thus be affected by the presence of alternative host plants in viticultural landscapes, which is important in the light of both insecticide resistance management and sustainable pest management.

Mutual and Opposing Responses of Carabid Beetles and Predatory Wasps to Local and Landscape Factors in Vineyards.

Preserving agro-biodiversity is one of the main means at the moment to counteract the global biodiversity crisis. Vineyard inter-rows offer vegetation covers which could function as foraging grounds for arthropods. Furthermore, organic management and enhanced landscape complexity often support biodiversity. Here, species richness and abundance of two groups of arthropod predators in vineyards were studied. Fifteen pairs of organically and conventionally managed vineyards were chosen along a gradient of landscape complexity in Rhine-Hesse, Germany. Carabid beetles were sampled using pitfall traps and cavity-nesting wasps with trap nests, respectively. Proportions of different land-use types surrounding the vineyards were calculated and inter-row vegetation cover was characterized. Species richness and abundances of both predator groups were not significantly affected by the management system. Likewise, increased cover of semi-natural habitats in the surrounding landscape did not promote their diversity or abundance. Instead, the increasing cover of annual crops diminished both groups. Cavity-nesting wasps profited from dense inter-row vegetation cover, while carabids were disadvantaged. The results indicate that distinct taxa within the same trophic group can respond oppositely to vineyard management. Thus, inter-row vegetation management with densely and sparsely vegetated elements might be best to support predator diversity. Overall, our results suggest that organic viticulture alone is insufficient to assist the studied insect groups, and that other local and landscape management options are needed for their protection.

Correction to: Evaluation of microbial communities in peels of Brazilian tropical fruits by amplicon sequence analysis.

The original version of this article unfortunately contained two mistakes in the "Materials and methods" section, subsection "DNA extraction and PCR" of the article. The correct information is given below.

Occurrence and diversity of entomopathogenic fungi (Beauveria spp. and Metarhizium spp.) in Australian vineyard soils.

Entomopathogenic Ascomycetes: Hypocreales fungi occur worldwide in the soil; however, the abundance and distribution of these fungi in a vineyard environment is unknown. A survey of Australian vineyards was carried out in order to isolate and identify entomopathogenic fungi. A total of 240 soil samples were taken from eight vineyards in two states (New South Wales and Victoria). Insect baiting (using Tenebrio molitor) and soil dilution methods were used to isolate Beauveria spp. and Metarhizium spp. from all soil samples. Of the 240 soil samples, 60% contained either Beauveria spp. (26%) or Metarhizium spp. (33%). Species of Beauveria and Metarhizium were identified by sequencing the B locus nuclear intergenic region (Bloc) and elongation factor-1 alpha (EFT1) regions, respectively. Three Beauveria species (B. bassiana, B. australis and B. pseudobassiana) and six Metarhizium species (M. guizhouense, M. robertsii, M. brunneum, M. flavoviride var. pemphigi, M. pingshaense and M. majus) were identified. A new sister clade made up of six isolates was identified within B. australis. Two potentially new phylogenetic species (six isolates each) were found within the B. bassiana clade. This study revealed a diverse community of entomopathogenic fungi in sampled Australian vineyard soils.

Evaluation of microbial communities in peels of Brazilian tropical fruits by amplicon sequence analysis.

Elucidation of the distinctive microbial taxonomic profiles of tropical fruit peels is the indispensable component of investigations aimed at the detection of microorganisms responsible for the post-harvest loss. The objective of the present work was to dissect the bacterial and fungal community of five tropical fruit peels (banana, guava, mango, papaya, and passion fruit) in wild (non-cultivated) and conventionally produced samples from Brazil. To that end, 16S rRNA-encoding gene and ITS rDNA amplicon analysis of the five tropical fruit peels were performed to discriminate the bacterial and fungal communities, respectively. The result showed that bacterial communities of the five types of fruit peels were by far more diversified than that of fungal communities, independent of the type of production system involved. Among the investigated fruits, non-cultivated papaya peels hosted the most diversified bacterial community while the least bacterial community diversity was found in the conventionally produced papaya fruit peels. The gene amplicon analysis clearly discriminated the bacterial community into their respective classes, while fungal communities were better classified in their phyla, yet with clearer component discrimination of fungal community based on the type of cultivation system practiced. Conventionally produced banana and non-cultivated passion fruit peels were characteristically dominated by fungal and bacterial groups, respectively. Overall, in conventionally produced fruit peels, bacterial community was mainly composed of Proteobacteria, Actinobacteria, and Bacilli. The result provided a broad microbial diversity profile that could be used as an important input for seeking alternative fruit spoilage control and post-harvest treatments.

Elimination of Phytoplasmas in Rubus Mother Plants by Tissue Culture Coupled with Heat Therapy.

Phytoplasmas are plant-pathogenic bacteria that cause a disease in Rubus species which is referred to as Rubus stunt. As phytoplasmas can be spread by vegetative propagation and latency periods of Rubus stunt can be up to one year, the use of pathogen-free Rubus propagation material in plant nurseries is important in order to stop the spread of this disease. Even though heat therapy has been commonly applied against viruses in many plants, its potential for phytoplasma eradication has been much less explored. Here, the efficacy of heat therapy with subsequent tissue culture to eliminate phytoplasmas from infected raspberry and blackberry plants is evaluated. Heat therapy was performed on 25 phytoplasma-infected raspberry and 33 infected blackberry plants, out of which 100 raspberry and 65 blackberry plants were regenerated via subsequent tissue culture. All plants were negative for the presence of phytoplasma DNA by qPCR at the end of cultivation periods of 481 to 565 days for the treated raspberry plants and 231 to 337 days for the treated blackberry plants. These results show the suitability of heat therapy combined with tissue culture as a routine tool to ensure the presence of phytoplasma-free Rubus mother plants in nurseries.

Elevated atmospheric CO2 concentrations alter grapevine (Vitis vinifera) systemic transcriptional response to European grapevine moth (Lobesia botrana) herbivory.

Atmospheric carbon dioxide (CO2) concentrations are among the chief factors shaping the mode and magnitude of interactions between plants and herbivorous insects. Here, we describe the first global analysis of systemic transcriptomic responses of grapevine Vitis vinifera plants to feeding of European grapevine moth Lobesia botrana larvae at future elevated CO2 concentrations. The study was conducted on mature, fruit-bearing grapevine plants under ambient and elevated CO2 concentrations in a grapevine free-air carbon dioxide enrichment (FACE) facility. Grapevine transcriptional response to herbivory was clearly dependent on phenological stage, with a higher number of differentially expressed genes identified at fruit development compared to berry ripening. At fruit development, more transcripts were differentially expressed as a response to herbivory under elevated compared to ambient CO2 concentrations. Classification of the respective transcripts revealed that in particular genes involved in metabolic pathways, biosynthesis of secondary metabolites and plant-pathogen interactions were significantly enriched. Most of these genes had similar expression patterns under both CO2 concentrations, with a higher fold-change under elevated CO2 concentrations. Differences in expression levels of a subset of herbivory responsive genes were further validated by RT-qPCR. Our study indicates that future elevated CO2 concentrations will affect interactions between grapevine plants and one of its key insect pests, with consequences for future relevance of L. botrana in worldwide viticulture.

A multiplex TaqMan qPCR assay for sensitive and rapid detection of phytoplasmas infecting Rubus species.

Rubus stunt is an economically important disease in the production of raspberries, blackberries, and loganberries. A fast, sensitive, and reliable diagnosis of phytoplasmas, the causal agent of the disease, is of prime importance to stop its spread by vegetative propagation and by insect vectors. Therefore, multiplex qPCR assays using TaqMan probes with different kinds of fluorophores in one reaction were developed, allowing the detection of phytoplasmas in general as well as a more specific detection of phytoplasmas belonging to group 16SrV and host DNA (either plant or insect). This assay now provides a practical tool for the screening of motherplants and monitoring the presence and distribution of phytoplasmas in Rubus plants of different geographic origins, cultivars, and cultivation systems, as well as in putative insect vectors like leafhoppers.

Laboratory Bioassays with Three Different Substrates to Test the Efficacy of Insecticides against Various Stages of Drosophila suzukii (Diptera: Drosophilidae).

Rapid worldwide spread and polyphagous nature of the spotted wing Drosophila Drosophila suzukii Matsumura (Diptera: Drosophilidae) calls for efficient and selective control strategies to prevent severe economic losses in various fruit crops. The use of insecticides is one option for management of this invasive pest insect. Efficacy of insecticides is usually assessed first in laboratory bioassays, which are compounded by the cryptic nature of D. suzukii larvae and the fact that fruits used in bioassays often start to rot and dissolve before larvae have reached the adult stage. Here, we report on laboratory bioassays using three different types of substrates allowing a thorough screening of insecticides for their potential effects against D. suzukii eggs, larvae and adults. Suitability of our bioassays was validated in an assessment of the efficacy of four bioinsecticides and one synthetic insecticide against various developmental stages of D. suzukii Water-apple juice agar used as a bioassay substrate allowed egg counting and observation of larval development due to its transparency, while apple-nutrition medium allowed complete metamorphosis. Use of grape berries in bioassays made it possible to assess effects of an insecticide present on a fruit's surface on oviposition and larval hatch from eggs. Insecticides tested in these three different bioassays with acetamiprid, spinosad or natural pyrethrins as active ingredients achieved a significant D. suzukii control if they were applied before egg deposition. Number of adult flies was significantly reduced if the bioassay medium was treated with an azadirachtin A containing insecticide both before or after egg deposition.

Are Epiphytic Microbial Communities in the Carposphere of Ripening Grape Clusters (Vitis vinifera L.) Different between Conventional, Organic, and Biodynamic Grapes?

Using barcoded pyrosequencing fungal and bacterial communities associated with grape berry clusters (Vitis vinifera L.) obtained from conventional, organic and biodynamic vineyard plots were investigated in two subsequent years at different stages during berry ripening. The four most abundant operational taxonomic units (OTUs) based on fungal ITS data were Botrytis cinerea, Cladosporium spp., Aureobasidium pullulans and Alternaria alternata which represented 57% and 47% of the total reads in 2010 and 2011, respectively. Members of the genera Sphingomonas, Gluconobacter, Pseudomonas, Erwinia, and Massilia constituted 67% of the total number of bacterial 16S DNA reads in 2010 samples and 78% in 2011 samples. Viticultural management system had no significant effect on abundance of fungi or bacteria in both years and at all three sampling dates. Exceptions were A. alternata and Pseudomonas spp. which were more abundant in the carposphere of conventional compared to biodynamic berries, as well as Sphingomonas spp. which was significantly less abundant on conventional compared to organic berries at an early ripening stage in 2011. In general, there were no significant differences in fungal and bacterial diversity indices or richness evident between management systems. No distinct fungal or bacterial communities were associated with the different maturation stages or management systems, respectively. An exception was the last stage of berry maturation in 2011, where the Simpson diversity index was significantly higher for fungal communities on biodynamic compared to conventional grapes. Our study highlights the existence of complex and dynamic microbial communities in the grape cluster carposphere including both phytopathogenic and potentially antagonistic microorganisms that can have a significant impact on grape production. Such knowledge is particularly relevant for development, selection and application of effective control measures against economically important pathogens present in the grape carposphere.

In vivo expression of genes in the entomopathogenic fungus Beauveria bassiana during infection of lepidopteran larvae.

The entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin is commercially available as a bio insecticide. The expression of three genes previously identified to have a role in pathogenicity in in vitro studies was validated in vivo in three lepidopteran insects infected with B. bassiana. Expression of all three genes was observed in all the tested insects starting from 48 or 72h to 10d post infection corroborating their role in pathogenicity. We suggest that it is essential to test the expression of putative pathogenicity genes both in vitro and in vivo to understand their role in different insect species.

Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein.

The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode-of-action of the two Bt Cry toxins.

Leaf microbiota of strawberries as affected by biological control agents.

The increasing use of biological control agents (BCAs) against Botrytis cinerea in strawberry raises the question of whether there are any undesirable impacts of foliar applications of BCAs on nontarget microorganisms in the phyllosphere. Therefore, our objective was to investigate this issue within a field study. Strawberry plants were repeatedly sprayed with three BCAs-namely, RhizoVital 42 fl. (Bacillus amyloliquefaciens FZB42), Trianum-P (Trichoderma harzianum T22), and Naturalis (Beauveria bassiana ATCC 74040)-to suppress Botrytis cinerea infections. Microbial communities of differentially treated leaves were analyzed using plate counts and pyrosequencing and compared with the microbial community of nontreated leaves. Plate count results indicate that the applied Bacillus and Trichoderma spp. survived in the strawberry phyllosphere throughout the strawberry season. However, no significant impacts on the leaf microbiota could be detected by this culture-dependent technique. Pyrosequencing of internal transcribed spacer ribosomal RNA and 16S RNA sequences revealed a change in fungal composition and diversity at class level after the introduction of T. harzianum T22 to the phyllosphere, whereas the bacterial composition and diversity was not affected by either this Trichoderma preparation or the other two BCAs. Our results suggest that pyrosequencing represents a useful method for studying microbial interactions in the phyllosphere.

Effects on diversity of soil fungal community and fate of an artificially applied Beauveria bassiana strain assessed through 454 pyrosequencing.

The entomopathogenic fungus Beauveria bassiana is widely used as a biological control agent (BCA) for insect pest control, with fungal propagules being either incorporated into the potting media or soil or sprayed directly onto the foliage or soil. To gain a better understanding of entomopathogenic fungal ecology when applied as a BCA to the soil environment, a case study using tag-encoded 454 pyrosequencing of fungal ITS sequences was performed to assess the fate and potential effect of an artificially applied B. bassiana strain on the diversity of soil fungal communities in an agricultural field in India. Results show that the overall fungal diversity was not influenced by application of B. bassiana during the 7 weeks of investigation. Strain-specific microsatellite markers indicated both an establishment of the applied B. bassiana strain in the treated plot and its spread to the neighboring nontreated control plot. These results might be important for proper risk assessment of entomopathogenic fungi-based BCAs.