Phyllosphere-associated microbiota in built environment: Do they have the potential to antagonize human pathogens?

INTRODUCTION: The plant microbiota is known to protect its host against invasion by plant pathogens. Recent studies have indicated that the microbiota of indoor plants is transmitted to the local built environment where it might fulfill yet unexplored functions. A better understanding of the interplay of such microbial communities with human pathogens might provide novel cues related to natural inhibition of them. OBJECTIVE: We studied the plant microbiota of two model indoor plants, Musa acuminata and Chlorophytum comosum, and their effect on human pathogens. The main objective was to identify mechanisms by which the microbiota of indoor plants inhibits human-pathogenic bacteria. METHODS: Microbial communities and functioning were investigated using a comprehensive set of experiments and methods combining amplicon and shotgun metagenomic analyses with results from interaction assays. RESULTS: A diverse microbial community was found to be present on Musa and Chlorophytum grown in different indoor environments; the datasets comprised 1066 bacterial, 1261 fungal, and 358 archaeal ASVs. Bacterial communities were specific for each plant species, whereas fungal and archaeal communities were primarily shaped by the built environment. Sphingomonas and Bacillus were found to be prevalent components of a ubiquitous core microbiome in the two model plants; they are well-known for antagonistic activity towards plant pathogens. Interaction assays indicated that they can also antagonize opportunistic human pathogens. Moreover, the native plant microbiomes harbored a broad spectrum of biosynthetic gene clusters, and in parallel, a variety of antimicrobial resistance genes. By conducting comparative metagenomic analyses between plants and abiotic surfaces, we found that the phyllosphere microbiota harbors features that are clearly distinguishable from the surrounding abiotic surfaces. CONCLUSIONS: Naturally occurring phyllosphere bacteria can potentially act as a protective shield against opportunistic human pathogens. This knowledge and the underlying mechanisms can provide an important basis to establish a healthy microbiome in built environments.

Impact of Cultivation and Origin on the Fruit Microbiome of Apples and Blueberries and Implications for the Exposome.

Vegetables and fruits are a crucial part of the planetary health diet, directly affecting human health and the gut microbiome. The objective of our study was to understand the variability of the fruit (apple and blueberry) microbiome in the frame of the exposome concept. The study covered two fruit-bearing woody species, apple and blueberry, two countries of origin (Austria and Finland), and two fruit production methods (naturally grown and horticultural). Microbial abundance, diversity, and community structures were significantly different for apples and blueberries and strongly influenced by the growing system (naturally grown or horticultural) and country of origin (Austria or Finland). Our results indicated that bacterial communities are more responsive towards these factors than fungal communities. We found that fruits grown in the wild and within home gardens generally carry a higher microbial diversity, while commercial horticulture homogenized the microbiome independent of the country of origin. This can be explained by horticultural management, including pesticide use and post-harvest treatments. Specific taxonomic indicators were identified for each group, i.e., for horticultural apples: Pseudomonas, Ralstonia, and Stenotrophomonas. Interestingly, Ralstonia was also found to be enriched in horticultural blueberries in comparison to such that were home and wildly grown. Our study showed that the origin of fruits can strongly influence the diversity and composition of their microbiome, which means that we are exposed to different microorganisms by eating fruits from different origins. Thus, the fruit microbiome needs to be considered an important but relatively unexplored external exposomic factor.

Function-Based Rhizosphere Assembly along a Gradient of Desiccation in the Former Aral Sea.

The desiccation of the Aral Sea represents one of the largest human-made environmental regional disasters. The salt- and toxin-enriched dried-out basin provides a natural laboratory for studying ecosystem functioning and rhizosphere assembly under extreme anthropogenic conditions. Here, we investigated the prokaryotic rhizosphere communities of the native pioneer plant Suaeda acuminata (C.A.Mey.) Moq. in comparison to bulk soil across a gradient of desiccation (5, 10, and 40 years) by metagenome and amplicon sequencing combined with quantitative PCR (qPCR) analyses. The rhizosphere effect was evident due to significantly higher bacterial abundances but less diversity in the rhizosphere compared to bulk soil. Interestingly, in the highest salinity (5 years of desiccation), rhizosphere functions were mainly provided by archaeal communities. Along the desiccation gradient, we observed a significant change in the rhizosphere microbiota, which was reflected by (i) a decreasing archaeon-bacterium ratio, (ii) replacement of halophilic archaea by specific plant-associated bacteria, i.e., Alphaproteobacteria and Actinobacteria, and (iii) an adaptation of specific, potentially plant-beneficial biosynthetic pathways. In general, both bacteria and archaea were found to be involved in carbon cycling and fixation, as well as methane and nitrogen metabolism. Analysis of metagenome-assembled genomes (MAGs) showed specific signatures for production of osmoprotectants, assimilatory nitrate reduction, and transport system induction. Our results provide evidence that rhizosphere assembly by cofiltering specific taxa with distinct traits is a mechanism which allows plants to thrive under extreme conditions. Overall, our findings highlight a function-based rhizosphere assembly, the importance of plant-microbe interactions in salinated soils, and their exploitation potential for ecosystem restoration approaches. IMPORTANCE The desertification of the Aral Sea basin in Uzbekistan and Kazakhstan represents one of the most serious anthropogenic environmental disasters of the last century. Since the 1960s, the world's fourth-largest inland body of water has been constantly shrinking, which has resulted in an extreme increase of salinity accompanied by accumulation of many hazardous and carcinogenic substances, as well as heavy metals, in the dried-out basin. Here, we investigated bacterial and archaeal communities in the rhizosphere of pioneer plants by combining classic molecular methods with amplicon sequencing as well as metagenomics for functional insights. By implementing a desiccation gradient, we observed (i) remarkable differences in the archaeon-bacterium ratio of plant rhizosphere samples, (ii) replacement of archaeal indicator taxa during succession, and (iii) the presence of specific, potentially plant-beneficial biosynthetic pathways in archaea present during the early stages. In addition, our results provide hitherto-undescribed insights into the functional redundancy between plant-associated archaea and bacteria.

Modulation of the food microbiome by apple fruit processing.

During the early life, introduction to external exposures such as consumption of solid foods contribute to the development of the gut microbiota. Among solid foods, fruit and vegetables are normally consumed during early childhood making them key components of a healthy human diet. The role of the indigenous microbiota of fruits as a source for beneficial gut microbes, especially during food processing, is largely unknown. Therefore, we investigated the apple fruit microbiota before and after processing using functional assays, advanced microscopic as well as sequencing technologies. Apple fruits carried a high absolute bacterial abundance (1.8 × 105 16S rRNA copies per g of apple pulp) and diversity of bacteria (Shannon diversity index = 2.5). We found that heat and mechanical treatment substantially affected the fruit's microbiota following a declining gradient of absolute bacterial abundance and bacterial diversity from shredded > boiled > pureed > preserved > dried apples. Betaproteobacteriales and Enterobacteriales were the two dominant bacterial orders (51.3%, 20.4% of the total 16S rRNA sequence reads) in the unprocessed apple. Boiling and air drying reduced the microbial load, but an unexpected, substantial fraction of 1/3 of the microbiota survived. Boiling and air drying shifted the microbiota leading to a relative increase in low abundant taxa such as Pseudomonas and Ralstonia (>2 log2 fold change), while others such as Bacillus decreased. Bacillus spp., frequently found in raw fruits, were shown to have specific traits, i.e. antagonist activity against opportunistic pathogens, biosurfactant production, and bile salt resistance indicating a probiotic potential. Our findings provide novel insights into food microbial changes during processing and demonstrate that food microbiome studies need a combined methodological approach. Food inhabiting microbes, currently considered being a risk factor for food safety, are a potential resource for the infant gut microbiome.

The Brassica napus seed microbiota is cultivar-specific and transmitted via paternal breeding lines.

Seed microbiota influence germination and plant health and have the potential to improve crop performance, but the factors that determine their structure and functions are still not fully understood. Here, we analysed the impact of plant-related and external factors on seed endophyte communities of 10 different oilseed rape (Brassica napus L.) cultivars from 26 field sites across Europe. All seed lots harboured a high abundance and diversity of endophytes, which were dominated by six genera: Ralstonia, Serratia, Enterobacter, Pseudomonas, Pantoea, and Sphingomonas. The cultivar was the main factor explaining the variations in bacterial diversity, abundance and composition. In addition, the latter was significantly influenced by diverse biotic and abiotic factors, for example host germination rates and disease resistance against Plasmodiophora brassicae. A set of bacterial biomarkers was identified to discriminate between characteristics of the seeds, for example Sphingomonas for improved germination and Brevundimonas for disease resistance. Application of a Bayesian community approach suggested vertical transmission of seed endophytes, where the paternal parent plays a major role and might even determine the germination performance of the offspring. This study contributes to the understanding of seed microbiome assembly and underlines the potential of the microbiome to be implemented in crop breeding and biocontrol programmes.

Insights into the microbiome assembly during different growth stages and storage of strawberry plants.

BACKGROUND: Microbiome assembly was identified as an important factor for plant growth and health, but this process is largely unknown, especially for the fruit microbiome. Therefore, we analyzed strawberry plants of two cultivars by focusing on microbiome tracking during the different growth stages and storage using amplicon sequencing, qPCR, and microscopic approaches. RESULTS: Strawberry plants carried a highly diverse microbiome, therein the bacterial families Sphingomonadaceae (25%), Pseudomonadaceae (17%), and Burkholderiaceae (11%); and the fungal family Mycosphaerella (45%) were most abundant. All compartments were colonized by high number of bacteria and fungi (107-1010 marker gene copies per g fresh weight), and were characterized by high microbial diversity (6049 and 1501 ASVs); both were higher for the belowground samples than in the phyllosphere. Compartment type was the main driver of microbial diversity, structure, and abundance (bacterial: 45%; fungal: 61%) when compared to the cultivar (1.6%; 2.2%). Microbiome assembly was strongly divided for belowground habitats and the phyllosphere; only a low proportion of the microbiome was transferred from soil via the rhizosphere to the phyllosphere. During fruit development, we observed the highest rates of microbial transfer from leaves and flowers to ripe fruits, where most of the bacteria occured inside the pulp. In postharvest fruits, microbial diversity decreased while the overall abundance increased. Developing postharvest decay caused by Botrytis cinerea decreased the diversity as well, and induced a reduction of potentially beneficial taxa. CONCLUSION: Our findings provide insights into microbiome assembly in strawberry plants and highlight the importance of microbe transfer during fruit development and storage with potential implications for food health and safety.

Microbiome Modulation-Toward a Better Understanding of Plant Microbiome Response to Microbial Inoculants.

Plant-associated microorganisms are involved in important functions related to growth, performance and health of their hosts. Understanding their modes of action is important for the design of promising microbial inoculants for sustainable agriculture. Plant-associated microorganisms are able to interact with their hosts and often exert specific functions toward potential pathogens; the underlying in vitro interactions are well studied. In contrast, in situ effects of inoculants, and especially their impact on the plant indigenous microbiome was mostly neglected so far. Recently, microbiome research has revolutionized our understanding of plants as coevolved holobionts but also of indigenous microbiome-inoculant interactions. Here we disentangle the effects of microbial inoculants on the indigenous plant microbiome and point out the following types of plant microbiome modulations: (i) transient microbiome shifts, (ii) stabilization or increase of microbial diversity, (iii) stabilization or increase of plant microbiome evenness, (iv) restoration of a dysbiosis/compensation or reduction of a pathogen-induced shift, (v) targeted shifts toward plant beneficial members of the indigenous microbiota, and (vi) suppression of potential pathogens. Therefore, we suggest microbiome modulations as novel and efficient mode of action for microbial inoculants that can also be mediated via the plant.

Microbiome-Assisted Breeding to Understand Cultivar-Dependent Assembly in Cucurbita pepo.

Recently, it was shown that long-term plant breeding does not only shape plant characteristics but also impacts plant-associated microbiota substantially. This requires a microbiome-integrative breeding approach, which was not yet shown. Here we investigate this for the Styrian oil pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by analyzing the microbiome of six genotypes (the complete pedigree of a three-way cross-hybrid, consisting of three inbred lines and one open pollinating cultivar) in the seed and rhizosphere as well as the progeny seeds. Using high-throughput amplicon sequencing targeting the 16S rRNA and the ITS1 genes, the bacterial and fungal microbiomes were accessed. Seeds were found to generally carry a significantly lower microbial diversity compared to the rhizosphere and soil as well as a different microbial composition, with an especially high fraction of Enterobacteriaceae (40-83%). Additionally, potential plant-beneficial bacterial taxa, including Bacillaceae, Burkholderiaceae, and Pseudomonadaceae, were found to be enriched in progeny seeds. Between genotypes, more substantial changes can be observed for seed microbiomes compared to the rhizosphere. Moreover, rhizosphere communities were assembled for the most part from soil. Interestingly, bacterial signatures are mainly linked from seed to seed, while fungal communities are shaped by the soil and rhizosphere. Our findings provide a deep look into the rhizosphere and seed microbiome assembly of pumpkin-associated communities and represent the first steps into microbiome-driven breeding for plant-beneficial microbes.

Antimicrobial-specific response from resistance gene carriers studied in a natural, highly diverse microbiome.

BACKGROUND: Antimicrobial resistance (AMR) is a major threat to public health. Microorganisms equipped with AMR genes are suggested to have partially emerged from natural habitats; however, this hypothesis remains inconclusive so far. To understand the consequences of the introduction of exogenic antimicrobials into natural environments, we exposed lichen thalli of Peltigera polydactylon, which represent defined, highly diverse miniature ecosystems, to clinical (colistin, tetracycline), and non-clinical (glyphosate, alkylpyrazine) antimicrobials. We studied microbiome responses by analysing DNA- and RNA-based amplicon libraries and metagenomic datasets. RESULTS: The analyzed samples consisted of the thallus-forming fungus that is associated with cyanobacteria as well as other diverse and abundant bacterial communities (up to 108 16S rRNA gene copies ng-1 DNA) dominated by Alphaproteobacteria and Bacteroidetes. Moreover, the natural resistome of this meta-community encompassed 728 AMR genes spanning 30 antimicrobial classes. Following 10 days of exposure to the selected antimicrobials at four different concentrations (full therapeutic dosage and a gradient of sub-therapeutic dosages), we observed statistically significant, antimicrobial-specific shifts in the structure and function but not in bacterial abundances within the microbiota. We observed a relatively lower response after the exposure to the non-clinical compared to the clinical antimicrobial compounds. Furthermore, we observed specific bacterial responders, e.g., Pseudomonas and Burkholderia to clinical antimicrobials. Interestingly, the main positive responders naturally occur in low proportions in the lichen holobiont. Moreover, metagenomic recovery of the responders' genomes suggested that they are all naturally equipped with specific genetic repertoires that allow them to thrive and bloom when exposed to antimicrobials. Of the responders, Sphingomonas, Pseudomonas, and Methylobacterium showed the highest potential. CONCLUSIONS: Antimicrobial exposure resulted in a microbial dysbiosis due to a bloom of naturally low abundant taxa (positive responders) with specific AMR features. Overall, this study provides mechanistic insights into community-level responses of a native microbiota to antimicrobials and suggests novel strategies for AMR prediction and management. Video Abstract.

Bacterial seed endophyte shapes disease resistance in rice.

Cereal crop production is severely affected by seed-borne bacterial diseases across the world. Locally occurring disease resistance in various crops remains elusive. Here, we have observed that rice plants of the same cultivar can be differentiated into disease-resistant and susceptible phenotypes under the same pathogen pressure. Following the identification of a seed-endophytic bacterium as the resistance-conferring agent, integration of high-throughput data, gene mutagenesis and molecular interaction assays facilitated the discovery of the underlying mode of action. Sphingomonas melonis that is accumulated and transmitted across generations in disease-resistant rice seeds confers resistance to disease-susceptible phenotypes by producing anthranilic acid. Without affecting cell growth, anthranilic acid interferes with the sigma factor RpoS of the seed-borne pathogen Burkholderia plantarii, probably leading to impairment of upstream cascades that are required for virulence factor biosynthesis. The overall findings highlight the hidden role of seed endophytes in the phytopathology paradigm of 'disease triangles', which encompass the plant, pathogens and environmental conditions. These insights are potentially exploitable for modern crop cultivation threatened by globally widespread bacterial diseases.

Increased Yield and High Resilience of Microbiota Representatives With Organic Soil Amendments in Smallholder Farms of Uganda.

Organic matter inputs positively affect soil fertility and quality but management effects on the soil and plant microbiome are less understood. Therefore, we studied the response of microbial colonization of the East African highland banana cultivar "Mpologoma" (AAA genome) under different mulch and manure treatments on three representative smallholder farms in Uganda. In general, the gammaproteobacterial community appeared stable with no significant response to organic matter inputs after 24 months of treatment. Significant differences (p < 0.05) in the plant-associated carpo-, phyllo-, and rhizosphere microbial community composition and diversity were found among individual sampled farms, independent of added soil inputs. Across farms, banana fruit harbored a richer and more balanced gammaproteobacterial community than the rhizo- and endospheres. Gammaproteobacterial beta diversity was shaped by the microenvironment (44%) as well as the sampling site (4%). Global effects of treatments in the rhizosphere analyzed using linear discriminant analysis effect size showed significantly enriched genera, such as Enterobacter, under manure and mulch treatments. As shown in previous works, bunch size and total yield were highly increased with manure and mulch, however, our results highlight general short-term microbial stability of Ugandan banana cropping systems with increases in the gammaproteobacterial community.

Microbiome Management by Biological and Chemical Treatments in Maize Is Linked to Plant Health.

The targeted application of plant growth-promoting rhizobacteria (PGPR) provides the key for a future sustainable agriculture with reduced pesticide application. PGPR interaction with the indigenous microbiota is poorly understood, but essential to develop reliable applications. Therefore, Stenotrophomonas rhizophila SPA-P69 was applied as a seed coating and in combination with a fungicide based on the active ingredients fludioxonil, metalaxyl-M, captan and ziram. The plant performances and rhizosphere compositions of treated and non-treated maize plants of two field trials were analyzed. Plant health was significantly increased by treatment; however, overall corn yield was not changed. By applying high-throughput amplicon sequencing of the 16S rRNA and the ITS genes, the bacterial and fungal changes in the rhizosphere due to different treatments were determined. Despite the fact that treatments had a significant impact on the rhizosphere microbiota (9-12%), the field site was identified as the main driver (27-37%). The soil microbiota composition from each site was significantly different, which explains the site-specific effects. In this study we were able to show the first indications how PGPR treatments increase plant health via microbiome shifts in a site-specific manner. This way, first steps towards a detailed understanding of PGPRs and developments of consistently efficient applications in diverse environments are made.

Microbiome approaches provide the key to biologically control postharvest pathogens and storability of fruits and vegetables.

Microbes play an important role in plants and interact closely with their host starting from sprouting seeds, continuing during growth and after harvest. The discovery of their importance for plant and postharvest health initiated a biotechnological development of various antagonistic bacteria and fungi for disease control. Nevertheless, their application often showed inconsistent effects. Recently, high-throughput sequencing-based techniques including advanced microscopy reveal fruits and vegetables as holobionts. At harvest, all fruits and vegetables harbor a highly abundant and specific microbiota including beneficial, pathogenic and spoilage microorganisms. Especially, a high microbial diversity and resilient microbial networks were shown to be linked to fruit and vegetable health, while diseased products showed severe dysbiosis. Field and postharvest handling of fruits and vegetables was shown to affect the indigenous microbiome and therefore has a substantial impact on the storability of fruits and vegetables. Microbiome tracking can be implemented as a new tool to evaluate and assess all postharvest processes and contribute to fruit and vegetable health. Here, we summarize current research advancements in the emerging field of postharvest microbiomes and elaborate its importance. The generated knowledge provides profound insights into postharvest microbiome dynamics and sets a new basis for targeted, microbiome-driven and sustainable control strategies.

Trichomes form genotype-specific microbial hotspots in the phyllosphere of tomato.

BACKGROUND: The plant phyllosphere is a well-studied habitat characterized by low nutrient availability and high community dynamics. In contrast, plant trichomes, known for their production of a large number of metabolites, are a yet unexplored habitat for microbes. We analyzed the phyllosphere as well as trichomes of two tomato genotypes (Solanum lycopersicum LA4024, S. habrochaites LA1777) by targeting bacterial 16S rRNA gene fragments. RESULTS: Leaves, leaves without trichomes, and trichomes alone harbored similar abundances of bacteria (108-109 16S rRNA gene copy numbers per gram of sample). In contrast, bacterial diversity was found significantly increased in trichome samples (Shannon index: 4.4 vs. 2.5). Moreover, the community composition was significantly different when assessed with beta diversity analysis and corresponding statistical tests. At the bacterial class level, Alphaproteobacteria (23.6%) were significantly increased, whereas Bacilli (8.6%) were decreased in trichomes. The bacterial family Sphingomonadacea (8.4%) was identified as the most prominent, trichome-specific feature; Burkholderiaceae and Actinobacteriaceae showed similar patterns. Moreover, Sphingomonas was identified as a central element in the core microbiome of trichome samples, while distinct low-abundant bacterial families including Hymenobacteraceae and Alicyclobacillaceae were exclusively found in trichome samples. Niche preferences were statistically significant for both genotypes and genotype-specific enrichments were further observed. CONCLUSION: Our results provide first evidence of a highly specific trichome microbiome in tomato and show the importance of micro-niches for the structure of bacterial communities on leaves. These findings provide further clues for breeding, plant pathology and protection as well as so far unexplored natural pathogen defense strategies.

Microbiome Response to Hot Water Treatment and Potential Synergy With Biological Control on Stored Apples.

Postharvest food decay is one major issue for today's food loss along the supply chain. Hot water treatment (HWT), a sustainable method to reduce pathogen-induced postharvest fruit decay, has been proven to be effective on a variety of crops. However, the microbiome response to HWT is still unknown, and the role of postharvest microbiota for fruit quality is largely unexplored. To study both, we applied a combined approach of metabarcoding analysis and real time qPCR for microbiome tracking. Overall, HWT was highly effective in reducing rot symptoms on apples under commercial conditions, and induced only slight changes to the fungal microbiota, and insignificantly affected the bacterial community. Pathogen infection, however, significantly decreased the bacterial and fungal diversity, and especially rare taxa were almost eradicated in diseased apples. Here, about 90% of the total fungal community was composed by co-occurring storage pathogens Neofabraea alba and Penicillium expansum. Additionally, the prokaryote to eukaryote ratio, almost balanced in apples before storage, was shifted to 0.6% bacteria and 99.4% fungi in diseased apples, albeit the total bacterial abundance was stable across all samples. Healthy stored apples shared 18 bacterial and 4 fungal taxa that were not found in diseased apples; therefore, defining a health-related postharvest microbiome. In addition, applying a combined approach of HWT and a biological control consortium consisting of Pantoea vagans 14E4, Bacillus amyloliquefaciens 14C9 and Pseudomonas paralactis 6F3, were proven to be efficient in reducing both postharvest pathogens. Our results provide first insights into the microbiome response to HWT, and suggest a combined treatment with biological control agents.

Microbiome-driven identification of microbial indicators for postharvest diseases of sugar beets.

BACKGROUND: Sugar loss due to storage rot has a substantial economic impact on the sugar industry. The gradual spread of saprophytic fungi such as Fusarium and Penicillium spp. during storage in beet clamps is an ongoing challenge for postharvest processing. Early detection of shifts in microbial communities in beet clamps is a promising approach for the initiation of targeted countermeasures during developing storage rot. In a combined approach, high-throughput sequencing of bacterial and fungal genetic markers was complemented with cultivation-dependent methods and provided detailed insights into microbial communities colonizing stored roots. These data were used to develop a multi-target qPCR technique for early detection of postharvest diseases. RESULTS: The comparison of beet microbiomes from six clamps in Austria and Germany highlighted regional differences; nevertheless, universal indicators of the health status were identified. Apart from a significant decrease in microbial diversity in decaying sugar beets (p ≤ 0.01), a distinctive shift in the taxonomic composition of the overall microbiome was found. Fungal taxa such as Candida and Penicillium together with the gram-positive Lactobacillus were the main disease indicators in the microbiome of decaying sugar beets. In contrast, the genera Plectosphaerella and Vishniacozyma as well as a higher microbial diversity in general were found to reflect the microbiome of healthy beets. Based on these findings, a qPCR-based early detection technique was developed and confirmed a twofold decrease of health indicators and an up to 10,000-fold increase of disease indicators in beet clamps. This was further verified with analyses of the sugar content in storage samples. CONCLUSION: By conducting a detailed assessment of temporal microbiome changes during the storage of sugar beets, distinct indicator species were identified that reflect progressing rot and losses in sugar content. The insights generated in this study provide a novel basis to improve current or develop next-generation postharvest management techniques by tracking disease indicators during storage.

Microbiome-Guided Exploration of the Microbial Assemblage of the Exotic Beverage "Insect Tea" Native to Southwestern China.

Insect tea is a unique beverage that is native to Southwestern China and traditionally produced by local farmers in an elaborate process. It consists of insect larvae excrements that are commonly obtained from meal moths (Pyralis farinalis Linnaeus 1758) reared on a specific plant-based diet. We have reconstructed the whole production process under laboratory conditions in order to obtain microbiome-level insights into this uncommon beverage and to trace back the origin of the prevalent bacteria in the final product. The bacterial community composition was specific for each production stage, with a high proportion of Streptomycetacea, Pseudonocaridaceae, Enterococcaceae, and Enterobacteriaceae in the insect tea. A large proportion of the constituents was traced back to the producing insect (13.2%) and its excrements (43.8%), while the initial plant-based substrate for tea production was found to contribute only 0.6% of the traceable bacteria in the final product. Moreover, an enrichment of Enterobactericeae was observed during the analyzed process steps and verified with complementary analyses. The cultivation experiments indicated a high occurrence of viable bacteria in the tea at 2.7 × 105 ± 1.2 × 105 cfu g-1. The isolated bacteria included Bordetella petrii and Enterococcus spp. that were recovered from a commercial product. By implementing an integrative approach, the insect tea was shown to harbor a species-rich bacterial community that can be traced back to certain plant and insect microbiome constituents from distinct production steps. Moreover, the microbial profile of the insect tea was found to be unique for a food product so far and contained several bacterial groups that are considered from the current perspective as food contaminants or yet unreported in other beverages. Due to the high number of viable bacteria, the tea harbors a so far undescribed dynamic component that might have implications for human health.

Replacing conventional decontamination of hatching eggs with a natural defense strategy based on antimicrobial, volatile pyrazines.

The treatment of hatching eggs relies on classic yet environmentally harmful decontamination methods such as formaldehyde fumigation. We evaluated bacteria-derived volatiles as a replacement within a fundamentally novel approach based on volatile organic compounds (VOCs), which are naturally involved in microbial communication and antagonism due to their high antimicrobial efficiency. Pyrazine (5-isobutyl-2,3-dimethylpyrazine) was applied passively and actively in prototypes of a pre-industry-scale utilization. Altogether, pyrazine decontamination rates of up to 99.6% were observed, which is comparable to formaldehyde fumigation. While active evaporation was highly efficient in all experiments, passive treatment showed reducing effects in two of four tested groups only. These results were confirmed by visualization using LIVE/DEAD staining microscopy. The natural egg shell microbiome was characterized by an unexpected bacterial diversity of Pseudomonadales, Enterobacteriales, Sphingomonadales, Streptophyta, Burkholderiales, Actinomycetales, Xanthomonadales, Rhizobiales, Bacillales, Clostridiales, Lactobacillales, and Flavobacteriales members. Interestingly, we found that especially low pyrazine concentrations lead to a microbiome shift, which can be explained by varying antimicrobial effects on different microorganisms. Micrococcus spp., which are linked to embryonic death and reduced hatchability, was found to be highly sensitive to pyrazines. Taken together, pyrazine application was shown to be a promising, environmentally friendly alternative for fumigation treatments of hatchery eggs.