Rhizophagus irregularis, the model fungus in arbuscular mycorrhiza research, forms dimorphic spores.

Rhizophagus irregularis is the model species for arbuscular mycorrhizal fungi (AMF) research and the most widely propagated species for commercial plant biostimulants. Using asymbiotic and symbiotic cultivation systems initiated from single spores, advanced microscopy, Sanger sequencing of the glomalin gene, and PacBio sequencing of the partial 45S rRNA gene, we show that four strains of R. irregularis produce spores of two distinct morphotypes, one corresponding to the morphotype described in the R. irregularis protologue and the other having the phenotype of R. fasciculatus. The two spore morphs are easily distinguished by spore colour, thickness of the subtending hypha, thickness of the second wall layer, lamination of the innermost layer, and the dextrinoid reaction of the two outer spore wall layers to Melzer's reagent. The glomalin gene of the two spore morphs is identical and that of the PacBio sequences of the partial SSU-ITS-LSU region (2780 bp) obtained from single spores of the R. cf fasciculatus morphotype has a median pairwise similarity of 99.8% (SD = 0.005%) to the rDNA ribotypes of R. irregularis DAOM 197198. Based on these results, we conclude that the model AMF species R. irregularis is dimorphic, which has caused taxonomic confusion in culture collections and possibly in AMF research.

Phylogenomic Insights on the Xanthomonas translucens Complex, and Development of a TaqMan Real-Time Assay for Specific Detection of pv. translucens on Barley.

The reemergence and spread of Xanthomonas translucens, the causal agent of bacterial leaf streak in cereal crops and wilt in turfgrass and forage species, is a concern to growers in the United States and Canada. The pathogen is seedborne and listed as an A2 quarantine organism by EPPO, making it a major constraint to international trade and exchange of germplasm. The pathovar concept of the X. translucens group is confusing due to overlapping of plant host ranges and specificity. Here, comparative genomics, phylogenomics, and 81 up-to-date bacterial core gene set (ubcg2) were used to assign the pathovars of X. translucens into three genetically and taxonomically distinct clusters. The study also showed that whole genome-based digital DNA-DNA hybridization unambiguously can differentiate the pvs. translucens and undulosa. Orthologous gene and proteome matrix analyses suggest that the cluster consisting of graminis, poae, arrhenatheri, phlei, and phleipratensis is very divergent. Whole-genome data were exploited to develop the first pathovar-specific TaqMan real-time PCR tool for detection of pv. translucens on barley. Specificity of the TaqMan assay was validated using 62 Xanthomonas and non-Xanthomonas strains as well as growth chamber-inoculated and naturally infected barley leaves. Sensitivity levels of 0.1 pg (purified DNA) and 23 CFUs per reaction (direct culture) compared favorably with other previously reported real-time PCR assays. The phylogenomics data reported here suggest that the clusters could constitute novel taxonomic units or new species. Finally, the pathovar-specific diagnostic tool will have significant benefits to growers and facilitate international exchange of barley germplasm and trade.

Corrigendum: Molecular Analysis of Bacterial Isolates From Necrotic Wheat Leaf Lesions Caused by Xanthomonas translucens, and Description of Three Putative Novel Species, Sphingomonas albertensis sp. nov., Pseudomonas triticumensis sp. nov. and Pseudomonas foliumensis sp. nov.

[This corrects the article DOI: 10.3389/fmicb.2021.666689.].

Root Suberin Plays Important Roles in Reducing Water Loss and Sodium Uptake in Arabidopsis thaliana.

Suberin is a cell-wall-associated hetero-polymer deposited in specific plant tissues. The precise role of its composition and lamellae structure in protecting plants against abiotic stresses is unclear. In Arabidopsis thaliana, we tested the biochemical and physiological responses to water deficiency and NaCl treatment in mutants that are differentially affected in suberin composition and lamellae structure. Chronic drought stress increased suberin and suberin-associated waxes in wild-type plants. Suberin-deficient mutants were not more susceptible than the wild-type to the chronic drought stress imposed in this study. Nonetheless, the cyp86a1-1 cyp86b1-1 mutant, which had a severely altered suberin composition and lamellae structure, exhibited increased water loss through the root periderm. Cyp86a1-1 cyp86b1-1 also recorded lower relative water content in leaves. The abcg2-1 abcg6-1 abcg20-1 mutant, which has altered suberin composition and lamellae, was very sensitive to NaCl treatment. Furthermore, cyp86a1-1 cyp86b1-1 recorded a significant drop in the leaf K/Na ratio, indicating salt sensitivity. The far1-2 far4-1 far5-1 mutant, which did not show structural defects in the suberin lamellae, had similar responses to drought and NaCl treatments as the wild-type. Our results provide evidence that the suberin amount and lamellae structure are key features in the barrier function of suberin in reducing water loss and reducing sodium uptake through roots for better performance under drought and salt stresses.

Molecular Analysis of Bacterial Isolates From Necrotic Wheat Leaf Lesions Caused by Xanthomonas translucens, and Description of Three Putative Novel Species, Sphingomonas albertensis sp. nov., Pseudomonas triticumensis sp. nov. and Pseudomonas foliumensis sp. nov.

Xanthomonas translucens is the etiological agent of the wheat bacterial leaf streak (BLS) disease. The isolation of this pathogen is usually based on the Wilbrink's-boric acid-cephalexin semi-selective medium which eliminates 90% of other bacteria, some of which might be novel species. In our study, a general purpose nutrient agar was used to isolate 49 bacterial strains including X. translucens from necrotic wheat leaf tissues. Maximum likelihood cluster analysis of 16S rRNA sequences grouped the strains into 10 distinct genera. Pseudomonas (32.7%) and Pantoea (28.6%) were the dominant genera while Xanthomonas, Clavibacter and Curtobacterium had 8.2%, each. Erwinia and Sphingomonas had two strains, each. BLAST and phylogenetic analyses of multilocus sequence analysis (MLSA) of specific housekeeping genes taxonomically assigned all the strains to validly described bacterial species, except three strains (10L4B, 12L4D and 32L3A) of Pseudomonas and two (23L3C and 15L3B) of Sphingomonas. Strains 10L4B and12L4D had Pseudomonas caspiana as their closest known type strain while strain 32L3A was closest to Pseudomonas asturiensis. Sphingomonas sp. strains 23L3C and 15L3B were closest to S. faeni based on MLSA analysis. Our data on MLSA, whole genome-based cluster analysis, DNA-DNA hybridization and average nucleotide identity, matrix-assisted laser desorption/ionization-time-of-flight, chemotaxonomy and phenotype affirmed that these 5 strains constitute three novel lineages and are taxonomically described in this study. We propose the names, Sphingomonas albertensis sp. nov. (type strain 23L3CT = DOAB 1063T = CECT 30248T = LMG 32139T), Pseudomonas triticumensis sp. nov. (type strain 32L3AT = DOAB 1067T = CECT 30249T = LMG 32140T) and Pseudomonas foliumensis sp. nov. (type strain 10L4BT = DOAB 1069T = CECT 30250T = LMG 32142T). Comparative genomics of these novel species, relative to their closest type strains, revealed unique repertoires of core secretion systems and secondary metabolites/antibiotics. Also, the detection of CRISPR-Cas systems in the genomes of these novel species suggests an acquired mechanism for resistance against foreign mobile genetic elements. The results presented here revealed a cohabitation, within the BLS lesions, of diverse bacterial species, including novel lineages.

Taxonomic revision of Blumeria based on multi-gene DNA sequences, host preferences and morphology.

A taxonomic revision of the hitherto monotypic genus Blumeria was conducted incorporating multi-gene sequence analyses, host preference data and morphological criteria. The sequenced loci included rDNA ITS, partial chitin synthase gene (CHS1), as well as fragments of two unnamed orthologous genes (Bgt-1929, Bgt-4572). The combined evidence led to a reassessment and a new neotypification of B. graminiss. str. (emend.), and the description of seven additional species, viz. B. americana sp. nov. (mainly on hosts of the Triticeae), B. avenae sp. nov. (on Avena spp.), B. bromi-cathartici sp. nov. (on Bromus catharticus), B. bulbigera comb. nov. (on Bromus spp.), B. dactylidis sp. nov. (on Dactylis glomerata as the main host, but also on various other hosts), B. graminicola sp. nov. (on Poa spp. as principal hosts, but also on various other hosts), and B. hordei sp. nov. (on Hordeum spp.). Synonyms were assessed, some were lectotypified, and questionable names previously associated with powdery mildew on monocots were discussed although their identities remained unresolved. Keys to the described species were developed.

A triticale tapetal non-specific lipid transfer protein (nsLTP) is translocated to the pollen cell wall.

KEY MESSAGE: A Triticeae type III non-specific lipid transfer protein (nsLTP) was shown for the first time to be translocated from the anther tapetum to the pollen cell wall. Two anther-expressed non-specific lipid transfer proteins (nsLTPs) were identified in triticale (× Triticosecale Wittmack). LTPc3a and LTPc3b contain a putative signal peptide sequence and eight cysteine residues in a C-Xn-C-Xn-CC-Xn-CXC-Xn-C-Xn-C pattern. These proteins belong to the type III class of nsLTPs which are expressed exclusively in the inflorescence of angiosperms. The level of LTPc3 transcript in the anther was highest at the tetrad and uninucleate microspore stages, and absent in mature pollen. In situ hybridization showed that LTPc3 was expressed in the tapetal layer of the developing triticale anther. The expression of the LTPc3 protein peaked at the uninucleate microspore stage, but was also found to be associated with the mature pollen. Accordingly, an LTPc3a::GFP translational fusion expressed in transgenic Brachypodium distachyon first showed activity in the tapetum, then in the anther locule, and later on the mature pollen grain. Altogether, these results represent the first detailed characterization of a Triticeae anther-expressed type III nsLTP with possible roles in pollen cell wall formation.

The peptidoglycan and biofilm matrix of Staphylococcus epidermidis undergo structural changes when exposed to human platelets.

Staphylococcus epidermidis is a bacterium frequently isolated from contaminated platelet concentrates (PCs), a blood product used to treat bleeding disorders in transfusion patients. PCs offer an accidental niche for colonization of S. epidermidis by forming biofilms and thus avoiding clearance by immune factors present in this milieu. Using biochemical and microscopy techniques, we investigated the structural changes of the peptidoglycan (PG) and the biofilm matrix of S. epidermidis biofilms formed in whole-blood derived PCs compared to biofilms grown in glucose-supplemented trypticase soy broth (TSBg). Both, the PG and the biofilm matrix are primary mechanisms of defense against environmental stress. Here we show that in PCs, the S. epidermidis biofilm matrix is mainly of a proteinaceous nature with extracellular DNA, in contrast to the predominant polysaccharide nature of the biofilm matrix formed in TSBg cultures. PG profile studies demonstrated that the PG of biofilm cells remodels during PC storage displaying fewer muropeptides variants than those observed in TSBg. The PG muropeptides contain two chemical modifications (amidation and O-acetylation) previously associated with resistance to antimicrobial agents by other staphylococci. Our study highlights two key structural features of S. epidermidis that are remodeled when exposed to human platelets and could be used as targets to reduce septic transfusions events.

Redox signalling from NADPH oxidase targets metabolic enzymes and developmental proteins in Fusarium graminearum.

NADPH oxidase (NOX) is one of the sources of reactive oxygen species (ROS) that modulates the activity of proteins through modifications of their cysteine residues. In a previous study, we demonstrated the importance of NOX in both the development and pathogenicity of the phytopathogen Fusarium graminearum. In this article, comparative proteomics between the wild-type and a Nox mutant of F. graminearum was used to identify active cysteine residues on candidate redox-sensing proteins. A two-dimensional gel approach based on labelling with monobromobimane (mBBR) identified 19 candidate proteins, and was complemented with a gel-free shotgun approach based on a biotin switch method, which yielded 99 candidates. The results indicated that, in addition to temporal regulation, a large number of primary metabolic enzymes are potentially targeted by NoxAB-generated ROS. Targeted disruption of these metabolic genes showed that, although some are dispensable, others are essential. In addition to metabolic enzymes, developmental proteins, such as the Woronin body major protein (FGSG_08737) and a glycosylphosphatidylinositol (GPI)-anchored protein (FGSG_10089), were also identified. Deletion of either of these genes reduced the virulence of F. graminearum. Furthermore, changing the redox-modified cysteine (Cys325 ) residue in FGSG_10089 to either serine or phenylalanine resulted in a similar phenotype to the FGSG_10089 knockout strain, which displayed reduced virulence and altered cell wall morphology; this underscores the importance of Cys325 to the function of the protein. Our results indicate that NOX-generated ROS act as intracellular signals in F. graminearum and modulate the activity of proteins affecting development and virulence in planta.

Diversity of bacteria associated with corn roots inoculated with Canadian woodland soils, and description of Pseudomonas aylmerense sp. nov.

Bacteria associated with corn roots inoculated with soils collected from the Canadian woodlands were isolated and characterized. Genus-level identification based on 16S rRNA sequence analysis classified the 161 isolates in 19 genera. The majority (64%) of the isolates were affiliated with the genus Pseudomonas. Further analysis of the Pseudomonas isolates based on BLASTn and rpoD-rpoB-gyrB concatenated gene phylogeny revealed three unique clusters that could not be assigned to known species. This study reports the taxonomic description of one of the distinct lineages represented by two strains (S1E40T and S1E44) with P. lurida LMG 21995T, P. costantinii LMG 22119T, P. palleroniana LMG 23076T, P. simiae CCUG 50988T and P. extremorientalis LMG 19695T as the closest taxa. Both strains showed low ANIm (<90%) and genome-based DNA-DNA hybridization (<50%) values, which unequivocally delineated the new strains from the closest relatives. These findings were supported by multilocus sequence analysis (MLSA) and DNA fingerprinting. In addition, growth characteristics and biochemical tests revealed patterns that differed from the related species. Strains S1E40T and S1E44 are Gram-negative, aerobic, rod-shaped and motile by at least one flagellum; and grew optimally at 30 °C. The predominant polar lipid is phosphatidylethanolamine while the major respiratory quinone is ubiquinone-9. Based on phenotypic and genotypic data presented here, strains S1E40T and S1E44 represent a novel species for which the name Pseudomonas aylmerense sp. nov. is proposed. The type strain is S1E40T (= LMG 30784T = DOAB 703T = HAMI 3696T) with a G + C content of 61.6%.

Tapetal oleosins play an essential role in tapetosome formation and protein relocation to the pollen coat.

The Arabidopsis pollen grain is covered by a lipidic pollen coat representing select constituents released upon the programmed cell death of the anther secretory tapetum. These constituents originate primarily from two specialized tapetal organelles, elaioplasts and tapetosomes. Tapetosomes are distinctive Brassicaceae organelles derived from the endoplasmic reticulum that store triacylglycerols, flavonoids, alkanes, and proteins. The tapetosome triacylglycerols are found within lipid droplets surrounded by the highly variable tapetal oleosins that eventually generate the most abundant proteins of the pollen coat. Many questions remain regarding the sub-cellular targeting of tapetal oleosins as well as their role in tapetosome formation. Translational fusions of different tapetal oleosins or their derived domains to marker proteins were introduced into Arabidopsis thaliana to investigate their localization, processing and function. Arabidopsis tapetal oleosins were shown to be proteolytically cleaved following tapetum degeneration and different protein domains were targeted to the pollen coat despite vast differences in composition and size. Importantly, specific fusions were discovered to affect distinct aspects of tapetosome formation. This report not only highlighted the critical role of individual tapetal oleosin domains in Arabidopsis tapetosome formation, but revealed translational fusions to be a valuable tool in deciphering this evidently complex developmental process.

Diverted secondary metabolism and improved resistance to European corn borer (Ostrinia nubilalis) in maize (Zea mays L.) transformed with wheat oxalate oxidase.

An alteration in the secondary metabolism of maize (Zea mays L.) genetically modified with the wheat oxalate oxidase (OxO) gene was observed using HPLC and fluorescence microscopy. Phenolic concentrations in the OxO lines were significantly increased, but DIMBOA synthesis was reduced due to a diversion in the shikimate pathway leading to phenolic and hydroxamic acids. Ferulic acid exhibited the largest increase and accounted for 80.4% of the total soluble phenolics. Transcription of a 13-lipoxygenase gene, coding for a key enzyme involved in the regulation of secondary metabolism, was substantially higher in the OxO line than in the null line. To test whether the high levels of soluble phenolic acids, in particular ferulic acid, contributed to the insect resistance in the OxO maize, ferulic acid was administered in meridic diets to European corn borer (ECB). A significant negative correlation between ferulic acid concentration and ECB larval growth rate was found. Field testing during 2001 showed that OxO maize was more resistant to ECB, with leaf consumption and stalk-tunneling damage significantly reduced by 28-34 and 37-39%, respectively, on all of the OxO lines tested and confirming published 2000 findings.