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.

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.

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.

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.

The biotransformation of brewer's spent grain into biogas by anaerobic microbial communities.

The present study reports on the biotransformation of the brewer's spent grain (BSG) in co-digestion with Jerusalem artichoke (JA, Helianthus tuberosus L.) phytomass by thermophilic (+55 °C) and mesophilic (+30 °C) anaerobic methanogenic communities. BSG is a by-product of the beer-brewing process generated in large amounts, in which utilization provokes a negative effect on the environment. In this study, we will show an effective conversion of BSG into biogas by selected microbial communities, obtained from different sources (animal manure and previously isolated microbial consortia). The stimulation of methanogenesis was reached by the co-digestion of JA's phytomass (stem and leaves). The optimized conditions for microbial stable cultivation included the use of nutrient medium, containing yeast extract and trace element solution. The optimal BSG concentration in biogas production was 50 and 100 g L(-1). Under thermophilic conditions, the maximum total methane production reached 64%, and it comprised around 6-8 and 9-11 of L CH4 per 100 g of fermented BSG without and with co-digested JA, respectively, when the fresh inoculum was added. Although, after a year of re-cultivation, the values reduced to around 6-7, and 6-10 L CH4/100 g BSG, correspondingly, the selected microbial communities showed effective biotransformation of BSG. The supplementation of soil with the residual fermented BSG (10%, w/w) resulted in the promotion of lettuce (Lepidium sativum L.) growth. The results obtained demonstrate a potential for complete BSG utilization via biogas production and application as a soil additive.

Antibiotic resistance differentiates Echinacea purpurea endophytic bacterial communities with respect to plant organs.

Recent findings have shown that antibiotic resistance is widespread in multiple environments and multicellular organisms, as plants, harboring rich and complex bacterial communities, could be hot spot for emergence of antibiotic resistances as a response to bioactive molecules production by members of the same community. Here, we investigated a panel of 137 bacterial isolates present in different organs of the medicinal plant Echinacea purpurea, aiming to evaluate if different plant organs harbor strains with different antibiotic resistance profiles, implying then the presence of different biological interactions in the communities inhabiting different plant organs. Data obtained showed a large antibiotic resistance variability among strains, which was strongly related to the different plant organs (26% of total variance, P < 0.0001). Interestingly this uneven antibiotic resistance pattern was present also when a single genus (Pseudomonas), ubiquitous in all organs, was analyzed and no correlation of antibiotic resistance pattern with genomic relatedness among strains was found. In conclusion, we speculate that antibiotic resistance patterns are tightly linked to the type of plant organ under investigation, suggesting the presence of differential forms of biological interaction in stem/leaves, roots and rhizosphere.

Assessing quantitative resistance against Leptosphaeria maculans (phoma stem canker) in Brassica napus (oilseed rape) in young plants.

Quantitative resistance against Leptosphaeria maculans in Brassica napus is difficult to assess in young plants due to the long period of symptomless growth of the pathogen from the appearance of leaf lesions to the appearance of canker symptoms on the stem. By using doubled haploid (DH) lines A30 (susceptible) and C119 (with quantitative resistance), quantitative resistance against L. maculans was assessed in young plants in controlled environments at two stages: stage 1, growth of the pathogen along leaf veins/petioles towards the stem by leaf lamina inoculation; stage 2, growth in stem tissues to produce stem canker symptoms by leaf petiole inoculation. Two types of inoculum (ascospores; conidia) and three assessment methods (extent of visible necrosis; symptomless pathogen growth visualised using the GFP reporter gene; amount of pathogen DNA quantified by PCR) were used. In stage 1 assessments, significant differences were observed between lines A30 and C119 in area of leaf lesions, distance grown along veins/petioles assessed by visible necrosis or by viewing GFP and amount of L. maculans DNA in leaf petioles. In stage 2 assessments, significant differences were observed between lines A30 and C119 in severity of stem canker and amount of L. maculans DNA in stem tissues. GFP-labelled L. maculans spread more quickly from the stem cortex to the stem pith in A30 than in C119. Stem canker symptoms were produced more rapidly by using ascospore inoculum than by using conidial inoculum. These results suggest that quantitative resistance against L. maculans in B. napus can be assessed in young plants in controlled conditions. Development of methods to phenotype quantitative resistance against plant pathogens in young plants in controlled environments will help identification of stable quantitative resistance for control of crop diseases.

Searching for artemisinin production improvement in plants and microorganisms.

The endoperoxide sesquiterpene lactone artemisinin which is isolated from the plant Artemisia annua, and its semi-synthetic derivatives, are potent, novel, antimalarial drugs. They are effective against multidrug-resistant Plasmodium strains and have become essential components of the so-called Artemisinin-based Combination Therapy, that is recommended by the World Health Organization as the treatment of choice for malaria tropica. Moreover, artemisinin and its derivatives show additional anti-parasite, antitumor, and anti-viral properties. The plants, however, are very poor resources for the drug, as the content of artemisinin is low (from 0,1 to 1,5 % of dried leaves) and dependent on seasonal and somatic variations as well as the infestation of bacteria, fungi and insects. A chemical synthesis of the compound is complex and uneconomic. Therefore, artemisinin is in short supply and remains unaffordable for most people in malaria-endemic countries. Thus, many researchers have focused on enhancing the production of artemisinin, first, through traditional breeding and in in vitro plant tissue cultures and, then, by heterologous expression systems (a semi-synthetic approach) with the use of genetically-modified or transgenic microbes. In this review, we summarize the progress made in the production of artemisinin by the biotechnological approach.

Growth response of Pinus densiflora seedlings inoculated with three indigenous ectomycorrhizal fungi in combination

Pinus densiflora seedlings were inoculated with three indigenous ectomycorrhizal fungi (Cenococcum geophilum, Rhizopogon roseolus and Russula densifolia) in single-, two-, and three-species treatments. After 8 months, the colonization rates of each ectomycorrhizal species, seedling growth and the nutrition were assessed in each treatment. P. densiflora seedlings inoculated with different ECM species composition showed an increase in height and basal diameter and improved seedling root and shoot nutrition concentrations compared to control treatment. Generally, combined inoculation had a more positive influence on the seedlings than the single inoculation. The three-species inoculation presented the highest growth and basal diameter and concentration of most nutrients except potassium. In conclusion, the results provided strong evidence for benefits of combined inoculation with the indigenous ectomycorrhizal fungi on P. densiflora seedlings under controlled conditions.

No tillage affects the phosphorus status, isotopic composition and crop yield of Phaseolus vulgaris in a rain-fed farming system.

BACKGROUND: Conservation tillage promotes the accretion of soil organic matter and often leads to improved soil fertility and moisture availability. However, few studies have looked at the physiological response of crop plants to different tillage practices. It was therefore hypothesised that measuring the nutrient concentrations and stable isotope composition (δ(13)C, δ(18)O, δ(15)N) of shoots could help evaluate the physiological response of common bean (Phaseolus vulgaris L.) to different tillage treatments (no tillage (NT) and mouldboard ploughing (MP)) in a rain-fed farming system in northern Mexico. RESULTS: NT significantly enhanced shoot phosphorus concentration in bean plants. Tillage exerted a negative effect on the extent of root colonisation (%) by arbuscular mycorrhizal fungi (AMF). Lower shoot δ(18)O but unchanged δ(13)C values in plants from the NT system suggest enhanced stomatal conductance but also enhanced photosynthetic rate, which overall resulted in unchanged water use efficiency. Bean plants in the NT system showed lower shoot δ(15)N values, which suggests that a larger proportion of total plant nitrogen was obtained through atmospheric nitrogen fixation in this treatment. CONCLUSION: Greater diversity of AMF soil communities and heavier colonisation of roots by AMF in the NT compared with the MP system appeared to contribute to improved crop nutrition, water relations and yield in this rain-fed agroecosystem.

Proteomic analysis of the maize rachis: potential roles of constitutive and induced proteins in resistance to Aspergillus flavus infection and aflatoxin accumulation.

Infection of the maize (Zea mays L.) with aflatoxigenic fungus Aspergillus flavus and consequent contamination with carcinogenic aflatoxin is a persistent and serious agricultural problem causing disease and significant crop losses worldwide. The rachis (cob) is an important structure of maize ear that delivers essential nutrients to the developing kernels and A. flavus spreads through the rachis to infect kernels within the ear. Therefore, rachis plays an important role in fungal proliferation and subsequent kernel contamination. We used proteomic approaches and investigated the rachis tissue from aflatoxin accumulation resistant (Mp313E and Mp420) and susceptible (B73 and SC212m) maize inbred lines. First, we compared rachis proteins from resistant and susceptible inbred lines, which revealed that the young resistant rachis contains higher levels of abiotic stress-related proteins and proteins from phenylpropanoid metabolism, whereas susceptible young rachis contains pathogenesis-related proteins, which are generally inducible upon biotic stress. Second, we identified A. flavus-responsive proteins in rachis of both resistant and susceptible genotypes after 10- and 35-day infection. Differential expression of many stress/defense proteins during rachis juvenility, maturation and after A. flavus challenge demonstrates that resistant rachis relies on constitutive defenses, while susceptible rachis is more dependent on inducible defenses.

Atomic force microscope observation on ultrastructures in plant cells.

AFM is being applied in increasingly wide research fields and extracting more biochemical/biophysical information that is beyond the capability of traditional SEM and TEM. Due to its inherent features, AFM is rarely used to observe the subcellular details within cells. Although subcellular features were recently observed on thin sections of plant tissues using AFM, this method might introduce unexpected artifacts during sample processing. Here we try to observe plant cells still embedded in resin block. This modified method minimizes the possibility of artifacts. The comparison among outcomes of AFM, SEM, TEM and LM on the same single cell suggest that this modified method is a good, applicable, efficient and faithful way applying AFM on biological materials.

Diversity and antimicrobial activity of endophytic fungi associated with the alpine plant Saussurea involucrata.

Endophytic fungi are rich in species diversity and may play an important role in the fitness of their host plants. This study investigated the diversity and antimicrobial potential of endophytic fungi obtained from Saussurea involucrata KAR. et KIR. A total of 49 endophytic fungi were isolated from S. involucrata and identified using morphological and molecular techniques. Extracts of fermentation broth from the 49 fungi were tested for antimicrobial activity against pathogenic microorganisms using the agar diffusion method. Forty-eight out of the 49 endophytic fungi were identified and grouped into 14 taxa. Cylindrocarpon sp. was the dominant species isolated from S. involucrata, followed by Phoma sp. and Fusarium sp. Among the 49 endophytic fungi, 9 root isolates having darkly pigmented, septate hyphae were identified as dark septate endophytic (DSE) fungus, and 12 fungi inhibited at least one test microorganism. Moreover, 5 strains showed a broader spectrum of antimicrobial activity and 4 strains displayed strong inhibition (+++) against pathogenic fungi. The results indicate that endophytic fungi isolated from S. involucrata are diverse in species and a potential source of antimicrobial agents.