The Toxic Mechanism of Gliotoxins and Biosynthetic Strategies for Toxicity Prevention

Gliotoxin is a kind of epipolythiodioxopiperazine derived from different fungi that is characterized by a disulfide bridge. Gliotoxins can be biosynthesized by a gli gene cluster and regulated by a positive GliZ regulator. Gliotoxins show cytotoxic effects via the suppression the function of macrophage immune function, inflammation, antiangiogenesis, DNA damage by ROS production, peroxide damage by the inhibition of various enzymes, and apoptosis through different signal pathways. In the other hand, gliotoxins can also be beneficial with different doses. Low doses of gliotoxin can be used as an antioxidant, in the diagnosis and treatment of HIV, and as an anti-tumor agent in the future. Gliotoxins have also been used in the control of plant pathogens, including Pythium ultimum and Sclerotinia sclerotiorum. Thus, it is important to elucidate the toxic mechanism of gliotoxins. The toxic mechanism of gliotoxins and biosynthetic strategies to reduce the toxicity of gliotoxins and their producing strains are summarized in this review.

Phage co-transport with hyphal-riding bacteria fuels bacterial invasion in a water-unsaturated microbial model system

Nonmotile microorganisms often enter new habitats by co-transport with motile microorganisms. Here, we report that also lytic phages can co-transport with hyphal-riding bacteria and facilitate bacterial colonization of a new habitat. This is comparable to the concept of biological invasions in macroecology. In analogy to invasion frameworks in plant and animal ecology, we tailored spatially organized, water-unsaturated model microcosms using hyphae of Pythium ultimum as invasion paths and flagellated soil-bacterium Pseudomonas putida KT2440 as carrier for co-transport of Escherichia virus T4. P. putida KT2440 efficiently dispersed along P. ultimum to new habitats and dispatched T4 phages across air gaps transporting ≈0.6 phages bacteria−1. No T4 displacement along hyphae was observed in the absence of carrier bacteria. If E. coli occupied the new habitat, T4 co-transport fueled the fitness of invading P. putida KT2440, while the absence of phage co-transport led to poor colonization followed by extinction. Our data emphasize the importance of hyphal transport of bacteria and associated phages in regulating fitness and composition of microbial populations in water-unsaturated systems. As such co-transport seems analogous to macroecological invasion processes, hyphosphere systems with motile bacteria and co-transported phages could be useful models for testing hypotheses in invasion ecology.

Evaluation of damping-off tolerance in spinach cultivars in field soils and in a standardized lab assay with Pythium ultimum

Spinach growers face increasing problems of damping off in the production of fresh market (baby-leaf) spinach due to increasing restrictions on chemical treatments. Damping-off tolerant cultivars are increasingly important, requiring proper evaluation methods. From three locations in the Netherlands with a history of spinach cultivation and from one location in France, potential damping-off pathogens were isolated from the soil, identified to species or genus, and tested for their pathogenicity. Pythium ultimum was the most prevalent pathogen in those fields, causing spinach pre- and post-emergence damping off. Eight spinach cultivars with two or three seed lots each, were evaluated at the same field locations and in a greenhouse with soil sampled from one of the Dutch field sites. Pre-emergence damping off was more discriminating for differences among the cultivars than post-emergence damping off. Variation in levels of infection among trials, replicates, and seed lots of same cultivars, emphasized the need for a more standardized phenotyping assay. For such an assay, a cornmeal/sand-based inoculum with a pathogenic P. ultimum isolate was added to a substrate mixture of sand, perlite, and vermiculite, moistened until 50% water holding capacity, in which spinach seeds were incubated for ten days in a dark climate cabinet at 15°C, including a control treatment without P. ultimum inoculum. The assay showed reproducible results for discriminating differences in pre-emergence damping-off tolerance levels among seed lots. However, cultivar differences in pre-emergence damping-off tolerance levels could not be confirmed due to a large variation among seed lots that needs further investigation.

Paenibacillus sp. strain UY79, isolated from a root nodule of Arachis villosa , displays a broad spectrum of antifungal activity

A nodule-inhabiting Paenibacillus sp. strain (UY79) isolated from wild peanut ( Arachis villosa ) was screened for its antagonistic activity against diverse fungi and oomycetes ( Botrytis cinerea , Fusarium verticillioides , Fusarium oxysporum , Fusarium graminearum , Fusarium semitectum , Macrophomina phaseolina , Phomopsis longicolla , Pythium ultimum, Phytophthora sojae, Rhizoctonia solani , Sclerotium rolfsii and Trichoderma atroviride ). Results obtained show that Paenibacillus sp. UY79 was able to antagonize these fungi/oomycetes and that agar-diffusible compounds and volatile compounds (different from HCN), participate in the antagonism exerted. Acetoin, 2,3-butanediol and 2-methyl-1-butanol were identified among the volatile compounds produced by UY79 strain with possible antagonistic activity against fungi/oomycetes. Paenibacillus sp. strain UY79 did not affect symbiotic association or growth promotion of alfalfa plants when co-inoculated with rhizobia. By whole genome sequence analysis, we determined that strain UY79 is a new species of Paenibacillus within the Paenibacillus polymyxa complex. Diverse genes putatively involved in biocontrol activity were identified in the UY79 genome. Furthermore, according to genome mining and antibiosis assays, strain UY79 would have the capability to modulate the growth of bacteria commonly found in soil/plant communities. IMPORTANCE Phytopathogenic fungi and oomycetes are responsible for causing devastating losses in agricultural crops. Therefore, there is an enormous interest in the development of effective and complementary strategies that allow the control of the phytopathogens, reducing the input of agrochemicals in croplands. Discovery of new strains with expanded antifungal activities and with a broad spectrum of action is challenging and of great future impact. Diverse strains belonging to the P. polymyxa complex have been reported to be effective biocontrol agents. Results presented here show that the novel discovered strain of Paenibacillus sp. presents diverse traits involved in antagonistic activity against a broad spectrum of pathogens and would be a potential and valuable strain to be further assessed for the development of biofungicides.

Specific interaction of an RNA-binding protein with the 3′-UTR of its target mRNA is critical to oomycete sexual reproduction

Sexual reproduction is an essential stage of the oomycete life cycle. However, the functions of critical regulators in this biological process remain unclear due to a lack of genome editing technologies and functional genomic studies in oomycetes. The notorious oomycete pathogen Pythium ultimum is responsible for a variety of diseases in a broad range of plant species. In this study, we revealed the mechanism through which PuM90, a stage-specific Puf family RNA-binding protein, regulates oospore formation in P. ultimum. We developed the first CRISPR/Cas9 system-mediated gene knockout and in situ complementation methods for Pythium. PuM90-knockout mutants were significantly defective in oospore formation, with empty oogonia or oospores larger in size with thinner oospore walls compared with the wild type. A tripartite recognition motif (TRM) in the Puf domain of PuM90 could specifically bind to a UGUACAUA motif in the mRNA 3′ untranslated region (UTR) of PuFLP, which encodes a flavodoxin-like protein, and thereby repress PuFLP mRNA level to facilitate oospore formation. Phenotypes similar to PuM90-knockout mutants were observed with overexpression of PuFLP, mutation of key amino acids in the TRM of PuM90, or mutation of the 3′-UTR binding site in PuFLP. The results demonstrated that a specific interaction of the RNA-binding protein PuM90 with the 3′-UTR of PuFLP mRNA at the post-transcriptional regulation level is critical for the sexual reproduction of P. ultimum.

Novel Fusari- and Toti-like Viruses, with Probable Different Origins, in the Plant Pathogenic Oomycete Globisporangiumultimum

To further classify the oomycete viruses that have been discovered in recent years, we investigated virus infection in the plant-parasitic oomycete Globisporangium ultimum in Japan. Double-stranded RNA detection, high-throughput sequencing, and RT-PCR revealed that the G. ultimum isolate UOP226 contained two viruses related to fusarivirus and totivirus, named Pythium ultimum RNA virus 1 (PuRV1) and Pythium ultimum RNA virus 2 (PuRV2), respectively. Phylogenetic analysis of the deduced amino acid sequence of the RNA-dependent RNA polymerase (RdRp) showed that fusari-like PuRV1 belonged to a different phylogenetic group than Plasmopara viticola lesion-associated fusari virus (PvlaFV) 1–3 from oomycete Plasmopara viticola. Codon usage bias of the PuRV1 RdRp gene was more similar to those of fungi than Globisporangium and Phytophthora, suggesting that the PuRV1 ancestor horizontally transmitted to G. ultimum ancestor from fungi. Phylogenetic analysis of the deduced amino acid sequence of the RdRp of toti-like PuRV2 showed a monophyletic group with the other toti-like oomycete viruses from Globisporangium, Phytophthora, and Pl. viticola. However, the nucleotide sequences of toti-like oomycete viruses were not so homologous, suggesting the possibility of convergent evolution of toti-like oomycete viruses.

Biosynthesis of Zinc Nanocomplex Employing for Plant Growth Promotion and Bio-Control of Pythium ultimum

Green Biosynthesis method was used for the preparation of Zn(II) nano complex from the reaction of the schiff base ligand 2,2′-((1E,1′E)-(1,2-phenylenebis (azanylylidene)) bis(methanylylidene))bis(4-bromophenol) and Zn(II)sulphate. The nano complex was characterized by different physicochemical methods. Zinc nanoparticles (ZnNP-T) will be studied as an antifungal agent. In this study, we will investigate the ability of the myogenic Zinc nanoparticles for plant Growth Promotion and Bio-control of Pythium ultimum.

Laccase Directed Lignification Is One of the Major Processes Associated With the Defense Response Against Pythium ultimum Infection in Apple Roots

Apple replant disease (ARD), incited by a pathogen complex including Pythium ultimum, causes stunted growth or death of newly planted trees at replant sites. Development and deployment of resistant or tolerant rootstocks offers a cost-effective, ecologically friendly, and durable approach for ARD management. Maximized exploitation of natural resistance requires integrated efforts to identify key regulatory mechanisms underlying resistance traits in apple. In this study, miRNA profiling and degradome sequencing identified major miRNA pathways and candidate genes using six apple rootstock genotypes with contrasting phenotypes to P. ultimum infection. The comprehensive RNA-seq dataset offered an expansive view of post-transcriptional regulation of apple root defense activation in response to infection from P. ultimum. Several pairs of miRNA families and their corresponding targets were identified for their roles in defense response in apple roots, including miR397-laccase, miR398-superoxide dismutase, miR10986-polyphenol oxidase, miR482-resistance genes, and miR160-auxin response factor. Of these families, the genotype-specific expression patterns of miR397 indicated its fundamental role in developing defense response patterns to P. ultimum infection. Combined with other identified copper proteins, the importance of cellular fortification, such as lignification of root tissues by the action of laccase, may critically contribute to genotype-specific resistance traits. Our findings suggest that quick and enhanced lignification of apple roots may significantly impede pathogen penetration and minimize the disruption of effective defense activation in roots of resistant genotypes. The identified target miRNA species and target genes consist of a valuable resource for subsequent functional analysis of their roles during interaction between apple roots and P. ultimum.

Stress Detection Using Proximal Sensing of Chlorophyll Fluorescence on the Canopy Level

Chlorophyll fluorescence is interesting for phenotyping applications as it is rich in biological information and can be measured remotely and non-destructively. There are several techniques for measuring and analysing this signal. However, the standard methods use rather extreme conditions, e.g., saturating light and dark adaption, which are difficult to accommodate in the field or in a greenhouse and, hence, limit their use for high-throughput phenotyping. In this article, we use a different approach, extracting plant health information from the dynamics of the chlorophyll fluorescence induced by a weak light excitation and no dark adaption, to classify plants as healthy or unhealthy. To evaluate the method, we scanned over a number of species (lettuce, lemon balm, tomato, basil, and strawberries) exposed to either abiotic stress (drought and salt) or biotic stress factors (root infection using Pythium ultimum and leaf infection using Powdery mildew Podosphaera aphanis). Our conclusions are that, for abiotic stress, the proposed method was very successful, while, for powdery mildew, a method with spatial resolution would be desirable due to the nature of the infection, i.e., point-wise spread. Pythium infection on the roots is not visually detectable in the same way as powdery mildew; however, it affects the whole plant, making the method an interesting option for Pythium detection. However, further research is necessary to determine the limit of infection needed to detect the stress with the proposed method.