Taxonomic classification of the bacterial endosymbiont Wolbachia based on next-generation sequencing: is there molecular evidence for its presence in tardigrades?

We used high-throughput sequencing of 16S rRNA to test whether tardigrade species are infected with Wolbachia parasites. We applied SILVA and Greengenes databases that allowed taxonomic classification of bacterial sequences to OTUs. The results obtained from both databases differed considerably in the number of OTUs, and only the Greengenes database allowed identification of Wolbachia (infection was also supported by comparison of sequences to NCBI database). The putative bacterial endosymbiont Wolbachia was discovered only in adult eutardigrades, while bacteria identified down to the order Rickettsiales were detected in both eutardigrade eggs and adult specimens. Nevertheless, the frequency of Wolbachia in the bacterial communities of the studied eutardigrades was low. Similarly, in our positive control, i.e., a fairy shrimp Streptocephalus cafer, which was found to be infected with Wolbachia in our previous study using Sanger sequencing, only the Rickettsiales were detected. We also carried out phylogenetic reconstruction using Wolbachia sequences from the SILVA and Greengenes databases, Alphaproteobacteria putative endosymbionts and Rickettsiales OTUs obtained in previous studies on the microbial community of tardigrades, and Rickettsiales and Wolbachia OTUs obtained in the current study. Our discovery of Wolbachia in tardigrades can fuel new research to uncover the specifics of this interaction.

Microenvironmental Interplay Predominated by Beneficial Aspergillus Abates Fungal Pathogen Incidence in Paddy Environment.

Rice fungal pathogens, responsible for severe rice yield loss and biotoxin contamination, cause increasing concerns on environmental safety and public health. In the paddy environment, we observed that the asymptomatic rice phyllosphere microenvironment was dominated by an indigenous fungus, Aspergillus cvjetkovicii, which positively correlated with alleviated incidence of Magnaporthe oryzae, one of the most aggressive plant pathogens. Through the comparative metabolic profiling for the rice phyllosphere microenvironment, two metabolites were assigned as exclusively enriched metabolic markers in the asymptomatic phyllosphere and increased remarkably in a population-dependent manner with A. cvjetkovicii. These two metabolites evidenced to be produced by A. cvjetkovicii in either a phyllosphere microenvironment or artificial media were purified and identified as 2(3H)-benzofuranone and azulene, respectively, by gas chromatography coupled to triple quadrupole mass spectrometry and nuclear magnetic resonance analyses. Combining with bioassay analysis in vivo and in vitro, we found that 2(3H)-benzofuranone and azulene exerted dissimilar actions at the stage of infection-related development of M. oryzae. A. cvjetkovicii produced 2(3H)-benzofuranone at the early stage to suppress MoPer1 gene expression, leading to inhibited mycelial growth, while azulene produced lately was involved in blocking of appressorium formation by downregulation of MgRac1. More profoundly, the microenvironmental interplay dominated by A. cvjetkovicii significantly blocked M. oryzae epidemics in the paddy environment from 54.7 to 68.5% (p < 0.05). Our study first demonstrated implication of the microenvironmental interplay dominated by indigenous and beneficial fungus to ecological balance and safety of the paddy environment.

Effect of Exogenous Application of Methyl Jasmonate on the Lipid and Carbohydrate Content and Composition of Winter Triticale ( Triticosecale Wittm.) Grain and the Severity of Fungal Infections in Triticale Plants and Grain.

Kernels of winter triticale ( Triticosecale Wittm. cv. Dinaro) were analyzed. In the autumn of 2015, the effect of methyl jasmonate (MJ) on the germination of triticale kernels and the development of triticale seedlings was analyzed in a laboratory before kernels were sown in experimental plots. Kernels harvested from plots in August 2016 were analyzed to determine their lipid and carbohydrate content and composition and the severity of fungal infections. Triticale grain was harvested at full maturity. The plots were sprayed with MJ at concentrations of 10-6 to 10-3 M in the stem elongation stage (200 L/ha) and in the early milk stage (300 L/ha). Other preventive treatments, fungicides, pesticides, or foliar fertilizers were not applied. Lipids of triticale kernels contained 20 fatty acids (FAs) with the highest proportion of linoleic acid. Methyl jasmonate did not exert a significant effect on the FA composition of kernel lipids treated with the plant hormone during the growing season. Statistical analysis did not reveal significant ( p < 0.05) differences in the total content of soluble carbohydrates in control kernels and in the kernels collected from triticale plants treated with MJ. Methyl jasmonate applied at a concentration of 10-3 M in BBCH stages 54 and 73 reduced the prevalence of stem base, leaf, and spike diseases. However, the severity of grain infections caused by mycotoxin-producing fungi increased in treatments where MJ was applied at a concentration of 10-5 M relative to the control treatment. The study describes the results noted in naturally infected plants and provides valuable inputs for agricultural practice, but further research is required to validate the presented findings.

Effect of jasmonic acid-methyl ester on the composition of carbohydrates and germination of yellow lupine (Lupinus luteus L.) seeds.

Mature seeds of yellow lupine contained sucrose, raffinose family oligosaccharides (RFOs), and galactosyl cyclitols as major soluble carbohydrates. The study showed that RFOs dominated in lupine seeds (16% DW). The disappearance of both types of alpha-d-galactosides in germinating lupine seeds was strongly inhibited by the presence of jasmonic acid-methyl ester (JA-Me) at a concentration of 10(-3)M in the incubation medium. JA-Me inhibited the activity of alpha-D-galactosidase (fraction I) during seed germination. Anatomical studies of lupine roots have shown certain cell structure differences between control and JA-Me-treated seedlings. The cross-sections of plant roots treated with JA-Me showed a characteristic folding of the cell walls in all root tissues, starting from the rhyzodermis, cortex and vascular cylinder. In water-treated (control) plants, the cell walls were rounded with no folding.