Epiphytic and endophytic bacteria on Camellia oleifera phyllosphere: exploring region and cultivar effect.

The epiphytic and endophytic bacteria play an important role in the healthy growth of plants. Both plant species and growth environmental influence the bacterial population diversity, yet it is inconclusive whether it is the former or the latter that has a greater impact. To explore the communities of the epiphytic and endophytic microbes in Camellia oleifera, this study assessed three representative C. oleifera cultivars from three areas in Hunan, China by Illumina high-throughput sequencing. The results showed that the diversity and species richness of endophytic microbial community in leaves were significantly higher than those of microbial community in the epiphytic. The diversity and species richness of epiphytic and endophytic microbes are complex when the same cultivar was grown in different areas. The C. oleifera cultivars grown in Youxian had the highest diversity of epiphytic microbial community, but the lowest abundance, while the cultivars grown in Changsha had the highest diversity and species richness of endophytic microbes in leaves. It was concluded that the dominant phylum mainly included Proteobacteria, Actinobacteriota and Firmicutes through the analysis of the epiphytic and endophytic microbial communities of C. oleifera. The species and relative abundances of epiphytic and endophytic microbial community were extremely different at the genus level. The analysis of NMDS map and PERMANOVA shows that the species richness and diversity of microbial communities in epiphytes are greatly influenced by region. However, the community structure of endophytic microorganisms in leaves is influenced by region and cultivated varieties, but the influence of cultivars is more significant. Molecular ecological network analysis showed that the symbiotic interaction of epiphytic microbial community was more complex.

Endophytic species of Nigrospora from grasses and shrubs of Shadegan International Wetland, with new species and records from Iran.

The "Shadegan International Wetland" (SIW) is one of the wetlands internationally recognized in the Ramsar convention. The vegetation of this wetland ecosystem consists of mostly grasses and shrubs that host a large number of fungi including endophytes. In this study, Nigrospora isolates were obtained from healthy plants of this wetland and its surrounding salt marshes and identified based on morphological features and multilocus phylogenetic analyses based on three DNA loci, namely the internal transcribed spacer regions 1 and 2 including the intervening 5.8S nuclear ribosomal DNA (ITS), ß-tubulin (tub2), and elongation factor 1-α (tef1-α). Accordingly, the following Nigrospora species were identified: N. lacticolonia, N. oryzae, N. osmanthi, N. pernambucoensis and a novel taxon N. shadeganensis sp. nov., which is described and illustrated. To the best of our knowledge, 10 new hosts for Nigrospora species are here reported, namely Aeluropus lagopoides, Allenrolfea occidentalis, Anthoxanthum monticola, Arthrocnemum macrostachyum, Cressa cretica, Halocnemum strobilaceum, Seidlitzia rosmarinus, Suaeda vermiculata, Tamarix passerinoides, and Typha latifolia. Moreover, the species N. lacticolonia and N. pernambucoensis are new records for the mycobiota of Iran.

Luteibacter mycovicinus sp. nov., a yellow-pigmented gammaproteobacterium found as an endohyphal symbiont of endophytic Ascomycota.

We isolated and described a yellow-pigmented strain of bacteria (strain 9143T), originally characterized as an endohyphal inhabitant of an endophytic fungus in the Ascomycota. Although the full-length sequence of its 16S rRNA gene displays 99 % similarity to Luteibacter pinisoli, genomic hybridization demonstrated <30 % genomic similarity between 9143T and its closest named relatives, further supported by average nucleotide identity results. This and related endohyphal strains form a well-supported clade separate from L. pinisoli and other validly named species including the most closely related Luteibacter rhizovicinus. The name Luteibacter mycovicinus sp. nov. is proposed, with type strain 9143T (isolate DBL433), for which a genome has been sequenced and is publicly available from the American Type Culture Collection (ATCC TSD-257T) and from the Leibniz Institute DSMZ (DSM 112764T). The type strain reliably forms yellow colonies across diverse media and growth conditions (lysogeny broth agar, King's Medium B, potato dextrose agar, trypticase soy agar and Reasoner's 2A (R2A) agar). It forms colonies readily at 27 °C on agar with a pH of 6-8, and on salt (NaCl) concentrations up to 2 %. It lacks the ability to utilize sulphate as a sulphur source and thus only forms colonies on minimal media if supplemented with alternative sulphur sources. It is catalase-positive and oxidase-negative. Although it exhibits a single polar flagellum, motility was only clearly visible on R2A agar. Its host range and close relatives, which share the endohyphal lifestyle, are discussed.

High-throughput sequencing-based analysis of the composition and diversity of the endophyte community in roots of Stellera chamaejasme.

Stellera chamaejasme (S. chamaejasme) is an important medicinal plant with heat-clearing, detoxifying, swelling and anti-inflammatory effects. At the same time, it is also one of the iconic plants of natural grassland degradation in northwest China, playing a key role in the invasion process. Plant endophytes live in healthy plant tissues and can synthesize substances needed for plant growth, induce disease resistance in host plants, and enhance plant resistance to environmental stress. Therefore, studying the root endophytes of S. chamaejasme is of great significance for mining beneficial microbial resources and biological prevention and control of S. chamaejasme. This study used Illumina MiSeq high-throughput sequencing technology to analyze the composition and diversity of endophytes in the roots of S. chamaejasme in different alpine grasslands (BGC, NMC and XGYZ) in Tibet. Research results show that the main phylum of endophytic fungi in the roots of S. chamaejasme in different regions is Ascomycota, and the main phyla of endophytic bacteria are Actinobacteria, Proteobacteria and Firmicutes (Bacteroidota). Overall, the endophyte diversity of the NMC samples was significantly higher than that of the other two sample sites. Principal coordinate analysis (PCoA) and permutational multivariate analysis of variance (PERMANOVA) results showed significant differences in the composition of endophytic bacterial and fungal communities among BGC, NMC and XGYZ samples. Co-occurrence network analysis of endophytes showed that there were positive correlations between fungi and some negative correlations between bacteria, and the co-occurrence network of bacteria was more complex than that of fungi. In short, this study provides a vital reference for further exploring and utilizing the endophyte resources of S. chamaejasme and an in-depth understanding of the ecological functions of S. chamaejasme endophytes.

Characterization of the olive endophytic community in genotypes displaying a contrasting response to Xylella fastidiosa.

BACKGROUND: Endophytes mediate the interactions between plants and other microorganisms, and the functional aspects of interactions between endophytes and their host that support plant-growth promotion and tolerance to stresses signify the ecological relevance of the endosphere microbiome. In this work, we studied the bacterial and fungal endophytic communities of olive tree (Olea europaea L.) asymptomatic or low symptomatic genotypes sampled in groves heavily compromised by Xylella fastidiosa subsp. pauca, aiming to characterize microbiota in genotypes displaying differential response to the pathogen. RESULTS: The relationships between bacterial and fungal genera were analyzed both separately and together, in order to investigate the intricate correlations between the identified Operational Taxonomic Units (OTUs). Results suggested a dominant role of the fungal endophytic community compared to the bacterial one, and highlighted specific microbial taxa only associated with asymptomatic or low symptomatic genotypes. In addition, they indicated the occurrence of well-adapted genetic resources surviving after years of pathogen pressure in association with microorganisms such as Burkholderia, Quambalaria, Phaffia and Rhodotorula. CONCLUSIONS: This is the first study to overview endophytic communities associated with several putatively resistant olive genotypes in areas under high X. fastidiosa inoculum pressure. Identifying these negatively correlated genera can offer valuable insights into the potential antagonistic microbial resources and their possible development as biocontrol agents.

Efficient gene editing in the slow-growing, non-sporulating, melanized, endophytic fungus Berkleasmium sp. Dzf12 using a CRISPR/Cas9 system.

The endophytic fungus Berkleasmium sp. Dzf12 that was isolated from Dioscorea zingiberensis, is a proficient producer of palmarumycins, which are intriguing polyketides of the spirobisnaphthalene class. These compounds displayed a wide range of bioactivities, including antibacterial, antifungal, and cytotoxic activities. However, conventional genetic manipulation of Berkleasmium sp. Dzf12 is difficult and inefficient, partially due to the slow-growing, non-sporulating, and highly pigmented behavior of this fungus. Herein, we developed a CRISPR/Cas9 system suitable for gene editing in Berkleasmium sp. Dzf12. The protoplast preparation was optimized, and the expression of Cas9 in Berkleasmium sp. Dzf12 was validated. To assess the gene disruption efficiency, a putative 1, 3, 6, 8-tetrahydroxynaphthalene synthase encoding gene, bdpks, involved in 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, was selected as the target for gene disruption. Various endogenous sgRNA promoters were tested, and different strategies to express sgRNA were compared, resulting in the construction of an optimal system using the U6 snRNA-1 promoter as the sgRNA promoter. Successful disruption of bdpks led to a complete abolishment of the production of spirobisnaphthalenes and melanin. This work establishes a useful gene targeting disruption system for exploration of gene functions in Berkleasmium sp. Dzf12, and also provides an example for developing an efficient CRISPR/Cas9 system to the fungi that are difficult to manipulate using conventional genetic tools.

Enhancing Pisum sativum growth and symbiosis under heat stress: the synergistic impact of co-inoculated bacterial consortia and ACC deaminase-lacking Rhizobium.

The 1-aminocyclopropane-1-carboxylate (ACC) deaminase is a crucial bacterial trait, yet it is not widely distributed among rhizobia. Hence, employing a co-inoculation approach that combines selected plant growth-promoting bacteria with compatible rhizobial strains, especially those lacking ACC deaminase, presents a practical solution to alleviate the negative effects of diverse abiotic stresses on legume nodulation. Our objective was to explore the efficacy of three non-rhizobial endophytes, Phyllobacterium salinisoli (PH), Starkeya sp. (ST) and Pseudomonas turukhanskensis (PS), isolated from native legumes grown in Tunisian arid regions, in improving the growth of cool-season legume and fostering symbiosis with an ACC deaminase-lacking rhizobial strain under heat stress. Various combinations of these endophytes (ST + PS, ST + PH, PS + PH, and ST + PS + PH) were co-inoculated with Rhizobium leguminosarum 128C53 or its ΔacdS mutant derivative on Pisum sativum plants exposed to a two-week heat stress period.Our findings revealed that the absence of ACC deaminase activity negatively impacted both pea growth and symbiosis under heat stress. Nevertheless, these detrimental effects were successfully mitigated in plants co-inoculated with ΔacdS mutant strain and specific non-rhizobial endophytes consortia. Our results indicated that heat stress significantly altered the phenolic content of pea root exudates. Despite this, there was no impact on IAA production. Interestingly, these changes positively influenced biofilm formation in consortia containing the mutant strain, indicating synergistic bacteria-bacteria interactions. Additionally, no positive effects were observed when these endophytic consortia were combined with the wild-type strain. This study highlights the potential of non-rhizobial endophytes to improve symbiotic performance of rhizobial strains lacking genetic mechanisms to mitigate stress effects on their legume host, holding promising potential to enhance the growth and yield of targeted legumes by boosting symbiosis.

Selection of the Dominant Endophytes Based on Illumina Sequencing Analysis for Controlling Bacterial Wilt of Patchouli Caused by Ralstonia solanacearum.

Bacterial wilt caused by Ralstonia solanacearum (RS) is one of the most devastating diseases in patchouli (Pogostemon cablin [Blanco] Benth.), which results in low yield and quality of patchouli. However, no stable and effective control methods have been developed yet. To evaluate the potential of dominant bacterial endophytes in biocontrol, the endophytic bacterial diversity of patchouli was investigated based on Illumina sequencing analysis, and the ability of isolates belonging to the dominant bacterial genera to control RS wilt of patchouli was explored in pot experiments. A total of 245 bacterial genera were detected in patchouli plants, with the highest relative abundance of operational taxonomic units belonging to the genus Pseudomonas detected in roots, leaves, and stems. The Pseudomonas isolates S02, S09, and S26 showed antagonistic activity against RS in vitro and displayed many plant growth-promoting characteristics, including production of indole-3-acetic acid, siderophores, and 1-aminocyclopropane-1-carboxylic acid deaminase and phosphate- and potassium-solubilizing capability. Inoculation of patchouli plants with the isolates S02, S09, and S26 significantly improved shoot growth and decreased the incidence of bacterial wilt caused by RS. The results suggest that screening of dominant bacterial endophytes for effective biocontrol agents based on Illumina sequencing analysis is more efficient than random isolation and screening procedures.

Bacterial endophytic community composition varies by hemp cultivar in commercially sourced seed.

The seed-endophytic bacterial community is a potentially beneficial and heritable fraction of the plant microbiome. Its utilization as a sustainable crop improvement strategy could be especially valuable for species such as hemp, where production is being scaled up and new challenges will be faced in managing crop productivity and health. However, little is known about the makeup and variation of the hemp seed microbiome. This study profiled the endophytic bacterial communities harboured by 16 hemp cultivars sourced from commercial suppliers in Europe. A 16S rDNA amplicon sequencing approach identified 917 amplicon sequence variants across samples. Taxonomic classification of sequences revealed 4 phyla and 87 genera to be represented in the dataset. Several genera were widespread while some were specific to one or a few cultivars. Flavobacterium, Pseudomonas, and Pantoea were notable in their high overall abundance and prevalence, but community composition was variable and no one taxon was universally abundant, suggesting a high degree of flexibility in community assembly. Taxonomic composition and alpha diversity differed among cultivars, though further work is required to understand the relative influence of hemp genetic factors on community structure. The taxonomic profiles presented here can be used to inform further work investigating the functional characteristics and potential plant-growth-promoting traits of seed-borne bacteria in hemp.

In silico biotechnological potential of Bacillus sp. strain MHSD_37 bacterial endophyte.

BACKGROUND: Endophytic bacteria possess a range of unique characteristics that enable them to successfully interact with their host and survive in adverse environments. This study employed in silico analysis to identify genes, from Bacillus sp. strain MHSD_37, with potential biotechnological applications. RESULTS: The strain presented several endophytic lifestyle genes which encode for motility, quorum sensing, stress response, desiccation tolerance and root colonisation. The presence of plant growth promoting genes such as those involved in nitrogen fixation, nitrate assimilation, siderophores synthesis, seed germination and promotion of root nodule symbionts, was detected. Strain MHSD_37 also possessed genes involved in insect virulence and evasion of defence system. The genome analysis also identified the presence of genes involved in heavy metal tolerance, xenobiotic resistance, and the synthesis of siderophores involved in heavy metal tolerance. Furthermore, LC-MS analysis of the excretome identified secondary metabolites with biological activities such as anti-cancer, antimicrobial and applications as surfactants. CONCLUSIONS: Strain MHSD_37 thereby demonstrated potential biotechnological application in bioremediation, biofertilisation and biocontrol. Moreover, the strain presented genes encoding products with potential novel application in bio-nanotechnology and pharmaceuticals.

Isolation of endophytes from Dioscorea nipponica Makino for stimulating diosgenin production and plant growth.

KEY MESSAGE: Both bacterial and fungal endophytes exhibited one or more plant growth-promoting (PGP) traits. Among these strains, the Paenibacillus peoriae SYbr421 strain demonstrated the greatest activity in the direct biotransformation of tuber powder from D. nipponica into diosgenin. Endophytes play crucial roles in shaping active metabolites within plants, significantly influencing both the quality and yield of host plants. Dioscorea nipponica Makino accumulates abundant steroidal saponins, which can be hydrolyzed to produce diosgenin. However, our understanding of the associated endophytes and their contributions to plant growth and diosgenin production is limited. The present study aimed to assess the PGP ability and potential of diosgenin biotransformation by endophytes isolates associated with D. nipponica for the efficient improvement of plant growth and development of a clean and effective approach for producing the valuable drug diosgenin. Eighteen bacterial endophytes were classified into six genera through sequencing and phylogenetic analysis of the 16S rDNA gene. Similarly, 12 fungal endophytes were categorized into 5 genera based on sequencing and phylogenetic analysis of the ITS rDNA gene. Pure culture experiments revealed that 30 isolated endophytic strains exhibited one or more PGP traits, such as nitrogen fixation, phosphate solubilization, siderophore synthesis, and IAA production. One strain of endophytic bacteria, P. peoriae SYbr421, effectively directly biotransformed the saponin components in D. nipponica. Moreover, a high yield of diosgenin (3.50%) was obtained at an inoculum size of 4% after 6 days of fermentation. Thus, SYbr421 could be used for a cleaner and more eco-friendly diosgenin production process. In addition, based on the assessment of growth-promoting isolates and seed germination results, the strains SYbr421, SYfr1321, and SYfl221 were selected for greenhouse experiments. The results revealed that the inoculation of these promising isolates significantly increased the plant height and fresh weight of the leaves and roots compared to the control plants. These findings underscore the importance of preparing PGP bioinoculants from selected isolates as an additional option for sustainable diosgenin production.

Current Status and Future Perspectives on Distribution of Fungal Endophytes and Their Utilization for Plant Growth Promotion and Management of Grapevine Diseases.

Grapevine is one of the economically most important fruit crops cultivated worldwide. Grape production is significantly affected by biotic constraints leading to heavy crop losses. Changing climatic conditions leading to widespread occurrence of different foliar diseases in grapevine. Chemical products are used for managing these diseases through preventive and curative application in the vineyard. High disease pressure and indiscriminate use of chemicals leading to residue in the final harvest and resistance development in phytopathogens. To mitigate these challenges, the adoption of potential biocontrol control agents is necessary. Moreover, multifaceted benefits of endophytes made them eco-friendly, and environmentally safe approach. The genetic composition, physiological conditions, and ecology of their host plant have an impact on their dispersion patterns and population diversity. Worldwide, a total of more than 164 fungal endophytes (FEs) have been characterized originating from different tissues, varieties, crop growth stages, and geographical regions of grapevine. These diverse FEs have been used extensively for management of different phytopathogens globally. The FEs produce secondary metabolites, lytic enzymes, and organic compounds which are known to possess antimicrobial and antifungal properties. The aim of this review was to understand diversity, distribution, host-pathogen-endophyte interaction, role of endophytes in disease management and for enhanced, and quality production.

Isolation and characterization of Paenibacillus peoriae JC-3jx from Dendrobium nobile.

Dendrobium is a rich source of high-value natural components. Endophytic fungi are well studied, yet bacteria research is limited. In this study, endophytic bacteria from Dendrobium nobile were isolated using an improved method, showing inhibition of pathogens and growth promotion. JC-3jx, identified as Paenibacillus peoriae, exhibited significant inhibitory activity against tested fungi and bacteria, including Escherichia coli. JC-3jx also promoted corn seed rooting and Dendrobium growth, highlighting its excellent biocontrol and growth-promoting potential.

Unraveling endophytic diversity in dioecious Siraitia grosvenorii: implications for mogroside production.

Host and tissue-specificity of endophytes are important attributes that limit the endophyte application on multiple crops. Therefore, understanding the endophytic composition of the targeted crop is essential, especially for the dioecious plants where the male and female plants are different. Here, efforts were made to understand the endophytic bacterial composition of the dioecious Siraitia grosvenorii plant using 16S rRNA amplicon sequencing. The present study revealed the association of distinct endophytic bacterial communities with different parts of male and female plants. Roots of male and female plants had a higher bacterial diversity than other parts of plants, and the roots of male plants had more bacterial diversity than the roots of female plants. Endophytes belonging to the phylum Proteobacteria were abundant in all parts of male and female plants except male stems and fruit pulp, where the Firmicutes were most abundant. Class Gammaproteobacteria predominated in both male and female plants, with the genus Acinetobacter as the most dominant and part of the core microbiome of the plant (present in all parts of both, male and female plants). The presence of distinct taxa specific to male and female plants was also identified. Macrococcus, Facklamia, and Propionibacterium were the distinct genera found only in fruit pulp, the edible part of S. grosvenorii. Predictive functional analysis revealed the abundance of enzymes of secondary metabolite (especially mogroside) biosynthesis in the associated endophytic community with predominance in roots. The present study revealed bacterial endophytic communities of male and female S. grosvenorii plants that can be further explored for monk fruit cultivation, mogroside production, and early-stage identification of male and female plants. KEY POINTS: • Male and female Siraitia grosvenorii plants had distinct endophytic communities • The diversity of endophytic communities was specific to different parts of plants • S. grosvenorii-associated endophytes may be valuable for mogroside biosynthesis and monk fruit cultivation.

©Relationship between endophytic fungal diversity and colonization and soil factors of cultured blueberry roots in Guizhou Province, Southwest China.

Dark septate endophytes (DSEs) inhabit plant roots and soil in ecosystems and host plants worldwide. DSE colonization is influenced by cultivars, soil factors, and specific habitat conditions. The regular diversity of DSEs in blueberries in Guizhou, China, is still unclear. In this study, four cultivars (Gardenblue, Powderblue, O'Neal, and Legacy) in three areas (Gaopo, Majiang, and Fenggang) in Guizhou were used to identify DSEs by morphological and molecular biological methods and to clarify the relationship between DSE diversity and DSE colonization and soil factors of cultivated blueberries in Guizhou. The DSEs isolated from cultivated blueberry roots in 3 areas in Guizhou Province were different, belonging to 17 genera, and the dominant genera were Penicillium, Phialocephala, and Thozetella. DSEs isolated from Majiang belonged to 12 genera and 16 species, those from Gaopo belonged to 7 genera and 15 species, and those from Fenggang belonged to 5 genera and 7 species. Among the different blueberry varieties, 11 genera were isolated from O'Neal, 12 genera were isolated from Powderblue, 11 genera were isolated from Legacy and 13 genera were isolated from Gardenblue. Coniochaeta is endemic to O'Neal, Chaetomium and Curvularia are endemic to Powderblue, and Thielavia is endemic to Legacy. Correlation analysis showed that DSE diversity was significantly correlated with DSE colonization and soil factors.

Effects of Microplastics on Endophytes in Different Niches of Chinese Flowering Cabbage (Brassica campestris).

Microplastics (MPs) are present in soil as emerging contaminants and pose a threat to soil as well as plants. Here, the effects of MPs on Chinese flowering cabbage from a microbiology perspective were explored. MP size and concentration significantly affected endophytic communities of plant root and petiole (p < 0.05). Under MP treatments, the root, petiole, and leaf exhibited a substantial abundance of pathogenic biomarkers, such as Pseudomonas, Burkholderia, Ralstonia, and Escherichia, resulting in the slow growth and morbidity of the plant. Difference analysis of metabolic pathways revealed that MPs significantly upregulated the pathogenic metabolic pathways (p < 0.05), and the presence of Vibrio infectious and pathogenic metabolic pathways was detected in all three niches of the plant. Moreover, MPs significantly inhibited the contents of carotenoids, iron, vitamin C, and calcium in edible niches of the plant (p < 0.05), and most of the high-abundant biomarkers were negatively correlated with their nutritional qualities.

Dynamic nitrogen fixation in an aerobic endophyte of Populus.

Biological nitrogen fixation by microbial diazotrophs can contribute significantly to nitrogen availability in non-nodulating plant species. In this study of molecular mechanisms and gene expression relating to biological nitrogen fixation, the aerobic nitrogen-fixing endophyte Burkholderia vietnamiensis, strain WPB, isolated from Populus trichocarpa served as a model for endophyte-poplar interactions. Nitrogen-fixing activity was observed to be dynamic on nitrogen-free medium with a subset of colonies growing to form robust, raised globular like structures. Secondary ion mass spectrometry (NanoSIMS) confirmed that N-fixation was uneven within the population. A fluorescent transcriptional reporter (GFP) revealed that the nitrogenase subunit nifH is not uniformly expressed across genetically identical colonies of WPB and that only ~11% of the population was actively expressing the nifH gene. Higher nifH gene expression was observed in clustered cells through monitoring individual bacterial cells using single-molecule fluorescence in situ hybridization. Through 15N2 enrichment, we identified key nitrogenous metabolites and proteins synthesized by WPB and employed targeted metabolomics in active and inactive populations. We cocultivated WPB Pnif-GFP with poplar within a RhizoChip, a synthetic soil habitat, which enabled direct imaging of microbial nifH expression within root epidermal cells. We observed that nifH expression is localized to the root elongation zone where the strain forms a unique physical interaction with the root cells. This work employed comprehensive experimentation to identify novel mechanisms regulating both biological nitrogen fixation and beneficial plant-endophyte interactions.

Comamonas endophytica sp. nov., a novel indole acetic acid producing endophyte isolated from bamboo in China.

Two novel indole acetic acid-producing strains, 5MLIRT and D4N7, were isolated from Indosasa shibataeoides in Yongzhou, Hunan province, and Phyllostachys edulis in Hangzhou, Zhejiang province, respectively. Based on their 16S rRNA sequences, strains 5MLIRT and D4N7 were closely related to Comamonas antarcticus 16-35-5T (98.4 % sequence similarity), and the results of 92-core gene phylogenetic trees showed that strains 5MLIRT and D4N7 formed a phylogenetic lineage within the clade comprising Comamonas species. The complete genome size of strain 5MLIRT was 4.49 Mb including two plasmids, and the DNA G+C content was 66.5 mol%. The draft genome of strain D4N7 was 4.26 Mb with 66.7 mol% G+C content. The average nucleotide identity and digital DNA-DNA hybridization values among strain 5MLIRT and species in the genus Comamonas were all below the species delineation threshold. The colonies of strain 5MLIRT and D4N7 were circular with regular margins, convex, pale yellow and 1.0-2.0 mm in diameter when incubated at 30 °C for 3 days. Strains 5MLIRT and D4N7 grew optimally at 30 °C, pH 7.0 and 1.0 % NaCl. The respiratory isoprenoid quinone was ubiquinone-8. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Polyphasic analyses indicated that strains 5MLIRT and D4N7 could be distinguished from related validly named Comamonas species and represent a novel species of the genus Comamonas, for which the name Comamonas endophytica sp. nov. is proposed. The type strain is 5MLIRT (=ACCC 62069T=GDMCC 1.2958T=JCM 35331T).

Multiomics Reveals Mechanisms of Alternaria oxytropis Inhibiting Pathogenic Fungi in Oxytropis ochrocephala.

Endophytic fungi can benefit the host plant and increase the plant resistance. Now, there is no in-depth study of how Alternaria oxytropis (A. oxytropis) is enhancing the ability of inhibiting pathogenic fungi in Oxytropis ochrocephala (O. ochrocephala). In this study, the fungal community and metabolites associated with endophyte-infected (EI) and endophyte-free (EF) O. ochrocephala were compared by multiomics. The fungal community indicated that there was more A. oxytropis, less phylum Ascomycota, and less genera Leptosphaeria, Colletotrichum, and Comoclathris in the EI group. As metabolic biomarkers, the levels of swainsonine and apigenin-7-O-glucoside-4-O-rutinoside were significantly increased in the EI group. Through in vitro validation experiments, swainsonine and apigenin-7-O-glucoside-4-O-rutinoside can dramatically suppress the growth of pathogenic fungi Leptosphaeria sclerotioides and Colletotrichum americae-borealis by increasing the level of oxidative stress. This work suggested that O. ochrocephala containing A. oxytropis could increase the resistance to fungal diseases by markedly enhancing the content of metabolites inhibiting pathogenic fungi.

Colonization dynamic and distribution of the endophytic fungus Microdochium bolleyi in plants measured by qPCR.

Microdochium bolleyi is a fungal endophyte of cereals and grasses proposed as an ideal model organism for studying plant-endophyte interactions. A qPCR-based diagnostic assay was developed to detect M. bolleyi in wheat and Brachypodium distachyon tissues using the species-specific primers MbqITS derived from the ITS of the ribosomal gene. Specificity was tested against 20 fungal organisms associated with barley and wheat. Colonization dynamics, endophyte distribution in the plant, and potential of the seed transmission were analyzed in the wheat and model plant B. distachyon. The colonization of plants by endophyte starts from the germinating seed, where the seed coats are first strongly colonized, then the endophyte spreads to the adjacent parts, crown, roots near the crown, and basal parts of the stem. While in the lower distal parts of roots, the concentration of M. bolleyi DNA did not change significantly in successive samplings (30, 60, 90, 120, and 150 days after inoculation), there was a significant increase over time in the roots 1 cm under crown, crowns and stem bases. The endophyte reaches the higher parts of the base (2-4 cm above the crown) 90 days after sowing in wheat and 150 days in B. distachyon. The endophyte does not reach both host species' leaves, peduncles, and ears. Regarding the potential for seed transmission, endophyte was not detected in harvested grains of plants with heavily colonized roots. Plants grown from seeds derived from parental plants heavily colonized by endophyte did not exhibit any presence of the endophyte, so transmission by seeds was not confirmed. The course of colonization dynamics and distribution in the plant was similar for both hosts tested, with two differences: the base of the wheat stem was colonized earlier, but B. distachyon was occupied more intensively and abundantly than wheat. Thus, the designed species-specific primers could detect and quantify the endophyte in planta.