Expression Profiles of Alkaloid-Related Genes across the Organs of Narrow-Leafed Lupin (Lupinus angustifolius L.) and in Response to Anthracnose Infection.

The main restraint obstructing the wider adoption of lupins as protein crops is the presence of bitter and toxic quinolizidine alkaloids (QAs), whose contents might increase under exposure to stressful environmental conditions. A poor understanding of how QAs accumulate hinders the breeding of sweet varieties. Here, we characterize the expression profiles of QA-related genes, along with the alkaloid content, in various organs of sweet and bitter narrow-leafed lupin (NLL, Lupinus angustifolius L.). Special attention is paid to the RAP2-7 transcription factor, a candidate regulator of the QA pathway. We demonstrate the upregulation of RAP2-7 and other QA-related genes, across the aerial organs of a bitter cultivar and the significant correlations between their expression levels, thus supporting the role of RAP2-7 as an important regulatory gene in NLL. Moreover, we showed that the initial steps of QA synthesis might occur independently in all aerial plant organs sharing common regulatory mechanisms. Nonetheless, other regulatory steps might be involved in RAP2-7-triggered QA accumulation, given its expression pattern in leaves. Finally, the examination of QA-related gene expression in plants infected with Colletotrichum lupini evidenced no connection between QA synthesis and anthracnose resistance, in contrast to the important role of polyamines during plant-pathogen interactions.

Major changes in grapevine wood microbiota are associated with the onset of esca, a devastating trunk disease.

Esca, a major grapevine trunk disease in old grapevines, is associated with the colonization of woody tissues by a broad range of plant pathogenic fungi. To identify which fungal and bacterial species are involved in the onset of this disease, we analysed the microbiota from woody tissues of young (10-year-old) grapevines at an early stage of esca. Using meta-barcoding, 515 fungal and 403 bacterial operational taxonomic units (OTUs) were identified in woody tissues. In situ hybridization showed that these fungi and bacteria co-inhabited in grapevine woody tissues. In non-necrotic woody tissues, fungal and bacterial microbiota varied according to organs and seasons but not diseased plant status. Phaeomoniella chlamydospora, involved in the Grapevine trunk disease, was the most abundant species in non-necrotic tissues from healthy plants, suggesting a possible non-pathogenic endophytic behaviour. Most diseased plants (70%) displayed cordons, with their central white-rot necrosis colonized essentially by two plant pathogenic fungi (Fomitiporia mediterranea: 60%-90% and P. chlamydospora: 5%-15%) and by a few bacterial taxa (Sphingomonas spp. and Mycobacterium spp.). The occurrence of a specific association of fungal and bacterial species in cordons from young grapevines expressing esca-foliar symptoms strongly suggests that that microbiota is involved in the onset of this complex disease.

Culture-independent analysis of an endophytic core microbiome in two species of wheat: Triticum aestivum L. (cv. 'Hondia') and the first report of microbiota in Triticum spelta L. (cv. 'Rokosz').

The main goal of the study was to determine the structure of endophytic bacteria inhabiting different parts (endosperm, germ, roots, coleoptiles, and leaves) of two wheat species, Triticum aestivum L. (cv. 'Hondia') and Triticum spelta L. (cv. 'Rokosz'), in order to provide new knowledge about the stability and/or changeability of the core microbiome in different plant organs. The endophytic core microbiome is associated with plants throughout their whole life cycle; however, plant organs can determine the actual endophytic community. Therefore, next generation sequencing with MiSeq Illumina technology was applied to identify the endophytic microbiome of T. aestivum and T. spelta. Bioinformatic analyses were performed with the use of the DADA2(1.8) package and R software (3.5.1). It was demonstrated that wheat, which is an important crop plant, was associated with beneficial endophytic bacteria inside the endosperms, germs, roots, leaves, and coleoptiles. Importantly, for the first time, biodiversity was recognized in the coleoptiles of the investigated wheat species. Flavobacterium, Pseudomonas and Janthinobacterium were shown to be common genera for both tested wheat cultivars. Among them, Pseudomonas was found to be the only endophytic genus accompanying both wheat species from the endosperm stage to the development of the leaf. Paenibacillus was recognized as a core genus for the 'Hondia' cv., whereas Pedobacter and Duganella constituted the core microbiome in the 'Rokosz' cv. In addition, the first insight into the unique and yet unrecognized endophytic microbiome of T. spelta is presented.

Characteristics and Diversity of Endophytic Bacteria in Endangered Chinese Herb Glehnia littoralis Based on Illumina Sequencing.

Glehnia littoralis is an endangered medicinal plant growing in the coastal ecological environment and plays an important role in coastal ecosystems. The endophytes in the plant have a significant role in promoting plant growth and enhancing plant stress resistance. However, the endophytic bacterial structure associated with halophyte G. littoralis is still not revealed. In this project, the construction and diversity of endophytic bacterial consortium associated with different tissues of G. littoralis were illustrated with high throughput sequencing of the V3-V4 region of the bacterial 16S rRNA. The results resolved that the diversity and richness of endophytic bacteria were significantly higher in root than in leaf and stem. The operational taxonomic units (OTU) analysis demonstrated that the Actinobacteria and Proteobacteria were dominant in all the samples at the phylum level, and Pseudomonas, Bacillus, Rhizobium were the dominant genera. Our results unraveled that the bacterial communities differed among different tissues of G. littoralis. Endophytic bacterial communities in leaf and stem shared more similarity than that in the root. Furthermore, the difference of bacteria community and structure among different tissues were also detected by principal coordinate analysis. Taken altogether, we can conclude that the bacterial communities of different tissues are unique, which could facilitate understanding the diversity of endophytic bacteria in G. littoralis.

Imidazole and Methoxybenzylamine Growth Inhibitors Reduce Salmonella Persistence in Tomato Plant Tissues.

HIGHLIGHTS: Small molecules (SMs) 1, 3, 4, and 5 are novel growth inhibitors of Salmonella enterica. These SMs are not toxic to tomato plant tissues including fruits. Combining biocontrol agents and SMs enhanced the control of Salmonella in infected plants. These SMs may be safe bactericides against Salmonella and phytopathogens in produce.

Sugarcane apoplast fluid modulates the global transcriptional profile of the diazotrophic bacteria Paraburkholderia tropica strain Ppe8.

The stalk apoplast fluid of sugarcane contains different sugars, organic acids and amino acids that may supply the demand for carbohydrates by endophytic bacteria including diazotrophs P. tropica (syn. B. tropica) strain Ppe8, isolated from sugarcane, is part of the bacterial consortium recommended as inoculant to sugarcane. However, little information has been accumulated regarding this plant-bacterium interaction considering that it colonizes internal sugarcane tissues. Here, we made use of the RNA-Seq transcriptomic analysis to study the influence of sugarcane stalk apoplast fluid on Ppe8 gene expression. The bacterium was grown in JMV liquid medium (100 ml), divided equally and then supplemented with 50 ml of fresh JMV medium or 50 ml of apoplast fluid extracted from sugarcane variety RB867515. Total RNA was extracted 2 hours later, the rRNAs were depleted and mRNAs used to construct libraries to sequence the fragments using Ion Torrent technology. The mapping and statistical analysis were carried out with CLC Genomics Workbench software. The RNA-seq data was validated by RT-qPCR using the reference genes fliP1, paaF, and groL. The data analysis showed that 544 genes were repressed and 153 genes were induced in the presence of apoplast fluid. Genes that induce plant defense responses, genes related to chemotaxis and movements were repressed in the presence of apoplast fluid, indicating that strain Ppe8 recognizes the apoplast fluid as a plant component. The expression of genes involved in bacterial metabolism was regulated (up and down), suggesting that the metabolism of strain Ppe8 is modulated by the apoplast fluid. These results suggest that Ppe8 alters its gene expression pattern in the presence of apoplast fluid mainly in order to use compounds present in the fluid as well as to avoid the induction of plant defense mechanisms. This is a pioneer study showing the role played by the sugarcane apoplast fluid on the global modulation of genes in P. tropica strain Ppe8.

Investigating the antifungal activity and mechanism of a microbial pesticide Shenqinmycin against Phoma sp.

Tea white scab (TWS) is a major disease affecting tea trees in mid-elevation regions and often occurs during rainy seasons with low temperatures. This disease is caused by the fungal pathogen Phoma sp. TWS can infect young stems, tender leaves, and tender shoots and lead to the production of low-quality tea. Owing to the absence of an effective control, TWS can result in substantial loss in tea production. In this study, we isolated and identified the pathogen from tea leaves infected by TWS and then evaluated in vitro the antifungal activity of Shenqinmycin, polyoxin, azoxystrobin, oligosaccharins, and tebuconazole against Phoma sp. Our results indicated that Shenqinmycin can inhibit the growth of Phoma sp. mycelia, with the EC50 value of 0.74µg/mL. After Phoma sp. being incubated in PDB liquid medium with Shenqinmycin, its mycelia were distorted and distended at 1.56µg/mL of minimum inhibitory concentration for 6h. Crucial genes associated with cell redox homeostasis, proteins synthesis, energy metabolism, and cytoskeleton were studied at mRNA and protein levels through RT-qPCR and Nano-LC-MS/MS. The results showed that the genes of 3-phosphate-glyceraldehyde dehydrogenase, citrate synthase, NADH-ubiquinone oxidoreductase subunit (NADH-subunit), ribosomal protein, eukaryotic initiation factor 4A-I, ß-tubulin, and α-tubulin were up-regulated. Meanwhile, the genes of formate dehydrogenase (FDH), malate dehydrogenase, mitochondrial heat shock protein, and protein disulfide-isomerase (PDI) were up-regulated at mRNA level but down-regulated at protein level. These results indicated that Shenqinmycin contribute to cell redox homeostasis by up- or down-regulating NADH-subunit, FDH, and PDI.

Mycobiome analysis of asymptomatic and symptomatic Norway spruce trees naturally infected by the conifer pathogens Heterobasidion spp.

Plant microbiome plays an important role in maintaining the host fitness. Despite a significant progress in our understanding of the plant microbiome achieved in the recent years, very little is known about the effect of plant pathogens on composition of microbial communities associated with trees. In this study, we analysed the mycobiome of different anatomic parts of asymptomatic and symptomatic Norway spruce trees naturally infected by Heterobasidion spp. We also investigated the primary impact of the disease on the fungal communities, which are associated with Norway spruce trees. Our results demonstrate that the structure of fungal communities residing in the wood differed significantly among symptomatic and asymptomatic Heterobasidion-infected trees. However, no significant differences were found in the other anatomic regions of the trees. The results also show that not only each of individual tree tissues (wood, bark, needles and roots) harbours a unique fungal community, but also that symptomatic trees were more susceptible to co-infection by other wood-degrading fungi compared to the asymptomatic ones.

Bioactive and biocontrol potential of endophytic fungi associated with Brugmansia aurea Lagerh.

This study describes 32 fungal endophytes isolated from different tissues of Brugmansia aurea Lagerh. Each fungal strain was authenticated based on internal transcribed spacer rDNA sequence. Phylogenetic analysis showed that these fungi are distributed in three classes, seven orders and 12 genera. The dichloromethane extracts of endophytic strains were screened for anticancer and antimicrobial activity. Anticancer activity of extracts against human cancer cell lines revealed that 50% strains are active with IC 50  < 10 µg/mL. While analysing antimicrobial potential against both Gram-positive and Gram-negative bacteria, 56.25% endophytic strains displayed activity at least against one of the tested human pathogenic bacteria with minimum inhibitory concentration of 12.5-100 µg/mL. In vitro antagonistic activity of endophytes was analysed against Sclerotinia sp ., Aspergillus fumigatus, Fusarium solani, A. flavus and F. oxysporum pathogen . The broad-spectrum anti-phytopathogenic activity was shown by R2BA. The presence of ketoacyl synthase domain of polyketide synthase gene and high degree of bioactivity shown by endophytic fungi suggested that they have potential to produce therapeutic compounds and to serve as biocontrol agent.

The Metagenome of Utricularia gibba's Traps: Into the Microbial Input to a Carnivorous Plant.

The genome and transcriptome sequences of the aquatic, rootless, and carnivorous plant Utricularia gibba L. (Lentibulariaceae), were recently determined. Traps are necessary for U. gibba because they help the plant to survive in nutrient-deprived environments. The U. gibba's traps (Ugt) are specialized structures that have been proposed to selectively filter microbial inhabitants. To determine whether the traps indeed have a microbiome that differs, in composition or abundance, from the microbiome in the surrounding environment, we used whole-genome shotgun (WGS) metagenomics to describe both the taxonomic and functional diversity of the Ugt microbiome. We collected U. gibba plants from their natural habitat and directly sequenced the metagenome of the Ugt microbiome and its surrounding water. The total predicted number of species in the Ugt was more than 1,100. Using pan-genome fragment recruitment analysis, we were able to identify to the species level of some key Ugt players, such as Pseudomonas monteilii. Functional analysis of the Ugt metagenome suggests that the trap microbiome plays an important role in nutrient scavenging and assimilation while complementing the hydrolytic functions of the plant.