Volatile Organic Compounds from Pythium oligandrum Play a Role in Its Parasitism on Plant-Pathogenic Pythium myriotylum.

The oomycete Pythium oligandrum is a soil-inhabiting parasite and predator of both fungi and oomycetes, and uses hydrolytic enzymes extensively to penetrate and hydrolyze its host or prey. Other mechanisms have been studied less, and we investigated the contribution of P. oligandrum-produced volatile organic compounds (VOCs) to parasitism. The growth-inhibiting activity of P. oligandrum VOCs was tested on Pythium myriotylum-a host or prey of P. oligandrum-coupled with electron microscopy, and biochemical and transcriptomic analyses. The P. oligandrum-produced VOCs reduced P. myriotylum growth by 80% and zoospore levels by 60%. Gas chromatography-mass spectrometry (GC-MS) identified 23 VOCs, and methyl heptenone, d-limonene, 2-undecanone, and 1-octanal were potent inhibitors of P. myriotylum growth and led to increased production of reactive oxygen species at a concentration that did not inhibit P. oligandrum growth. Exposure to the P. oligandrum VOCs led to shrinkage of P. myriotylum hyphae and lysis of the cellular membranes and organelles. Transcriptomics of P. myriotylum exposed to the P. oligandrum VOCs at increasing levels of growth inhibition initially showed a strong upregulation of putative detoxification-related genes that was not maintained later. The inhibition of P. myriotylum growth continued immediately after the exposure to the VOCs was discontinued and led to the reduced infection of its plant hosts. The VOCs produced by P. oligandrum could be another factor alongside hydrolytic enzymes contributing to its ecological role as a microbial parasite in particular ecological niches such as in soil, and may also contribute to the biocontrol of diseases using P. oligandrum commercial preparations. IMPORTANCE Microbe-microbe interactions in nature are multifaceted, with multiple mechanisms of action, and are crucial to how plants interact with microbes. Volatile organic compounds (VOCs) have diverse functions, including contributing to parasitism in ecological interactions and potential applications in biocontrol. The microbial parasite P. oligandrum is well known for using hydrolytic enzymes as part of its parasitism. We found that P. oligandrum VOCs reduced the growth of, and caused major damage to, the hyphae of P. myriotylum (a host or prey of P. oligandrum). Transcriptomic analyses of P. myriotylum exposed to the VOCs revealed the upregulation of genes potentially involved in an attempt to detoxify the VOCs. The inhibitory effects of the VOCs had a knock-on effect by reducing the virulence of P. myriotylum toward its plant hosts. The P. oligandrum VOCs could contribute to its ecological role as a microbial parasite. The VOCs analyzed here may also contribute to the biocontrol of diseases using P. oligandrum commercial preparations.

Novel Genetic Dysregulations and Oxidative Damage in Fusarium graminearum Induced by Plant Defense Eliciting Psychrophilic Bacillus atrophaeus TS1.

This study elaborates inter-kingdom signaling mechanisms, presenting a sustainable and eco-friendly approach to combat biotic as well as abiotic stress in wheat. Fusarium graminearum is a devastating pathogen causing head and seedling blight in wheat, leading to huge yield and economic losses. Psychrophilic Bacillus atrophaeus strain TS1 was found as a potential biocontrol agent for suppression of F. graminearum under low temperature by carrying out extensive biochemical and molecular studies in comparison with a temperate biocontrol model strain Bacillus amyloliquefaciens FZB42 at 15 and 25 °C. TS1 was able to produce hydrolytic extracellular enzymes as well as antimicrobial lipopeptides, i.e., surfactin, bacillomycin, and fengycin, efficiently at low temperatures. The Bacillus strain-induced oxidative cellular damage, ultrastructural deformities, and novel genetic dysregulations in the fungal pathogen as the bacterial treatment at low temperature were able to downregulate the expression of newly predicted novel fungal genes potentially belonging to necrosis inducing protein families (fgHCE and fgNPP1). The wheat pot experiments conducted at 15 and 25 °C revealed the potential of TS1 to elicit sudden induction of plant defense, namely, H2O2 and callose enhanced activity of plant defense-related enzymes and induced over-expression of defense-related genes which accumulatively lead to the suppression of F. graminearum and decreased diseased leaf area.

Integrative metagenomic dissection of last-resort antibiotic resistance genes and mobile genetic elements in hospital wastewaters.

Hospital wastewater is a critical source of antimicrobial resistance (AMR), which facilitates the proliferation and spread of clinically significant antimicrobial resistance genes (ARGs) and pathogenic bacteria. This study utilized metagenomic approaches, including advanced binning techniques, such as MetaBAT2, MaxBin2, and CONCOCT, which offer significant improvements in accuracy and completeness over traditional binning methods. These methods were used to comprehensively assess the dynamics and composition of resistomes and mobilomes in untreated wastewater samples taken from two general hospitals and one cancer hospital. This study revealed a diverse bacterial landscape, largely consisting of Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, with notable variations in microbial composition among hospitals. Analysis of the top 15 genera showed unique microbial pattern distribution in each hospital: Aeromonas was predominant in 1stHWTS (49.39 %), Acidovorax in the CAHWTS at 16.85 %, and Escherichia and Bacteroides in the 2ndHWTS at 11.44 % and 11.33 %, respectively. A total of 114 pathogenic bacteria were identified, with drug-resistant Aeromonas caviae and Escherichia coli being the most prevalent. The study identified 34 types and 1660 subtypes of ARGs, including important last-resort antibiotic resistance genes (LARGs), such as blaNDM, mcr, and tet(X). Using metagenomic binning, this study uncovered distinct patterns of host-resistance associations, particularly with Proteobacteria and Firmicutes. Network analysis highlighted the complex interactions among ARGs, mobile genetic elements (MGEs), and bacterial species, all contributing to the dissemination of AMR. These findings emphasize the intricate nature of AMR in hospital wastewater and the influence of hospital-specific factors on microbial resistance patterns. This study provides support for implementing integrated management strategies, including robust surveillance, advanced wastewater treatment, and strict antibiotic stewardship, to control the dissemination of AMR. Understanding the interplay among bacterial communities, ARGs, and MGEs is important for developing effective public health measures against AMR.

Composition and assembly mechanisms of prokaryotic communities in wetlands, and their relationships with different vegetation and reclamation methods.

Coastal wetlands are undergoing substantial transformations globally as a result of increased human activities. However, compared to other ecosystems, diversity and functional characteristics of microbial communities in reclaimed coastal wetlands are not well studied compared to other ecosystems. This is important because it is known that microorganisms can play a crucial role in biogeochemical cycling within coastal wetland ecosystems. Hence, this study utilized the high-throughput sequencing technique to investigate the structure and assembly processes of microbial communities in reclaimed coastal wetlands. The results revealed a substantial change in soil properties following coastal wetland reclamation. Remarkably, the reclaimed soil exhibited significantly lower pH, soil organic carbon (SOC), and total salinity (TS) values (p < 0.05). The dominant phyla included Proteobacteria, Chloroflexi, Bacteroidetes, Acidobacteria, and Planctomycetes among study sites. However, the relative abundance of Proteobacteria increased from un-reclaimed coastal wetlands to reclaimed ones. The Proteobacteria, Chloroflexi, and Acidobacteria showed higher relative abundance in vegetated soil compared to bare soil, while Bacteroidetes and Planctomycetes exhibited the opposite trend. Notably, vegetation types exerted the strongest influence on microbial diversity, surpassing the effects of soil types and depth (F = 34.49, p < 0.001; F = 25.49, p < 0.001; F = 3.173, p < 0.078, respectively). Stochastic assembly processes dominated in un-reclaimed soil, whereas deterministic processes governed the assembly in artificial sea embankment wetlands (SEW). The presence of Spartina alterniflora in all soil types (except SEW soils) indicated stochastic assembly, while Phragmites australis in reclaimed soils pointed toward deterministic microbial assembly. Furthermore, environmental factors such as pH, soil water content (SWC), SOC, total carbon (TC), total nitrogen (TN), total phosphorus (TP), NH4+-N, vegetation types, soil depth, and geographic distance exhibited significant effects on microbial beta diversity indices. Co-occurrence network analysis revealed a stronger association between taxa in SEW compared to land reclaimed from wetlands (LRW) and natural coastal wetlands (NCW). The bottom soil layer exhibited more complex network interactions than the topsoil layer. Besides soil parameters, reclamation and varieties of vegetation were also substantial factors influencing the composition, diversity, and assembly processes of microbial communities in coastal wetlands.

Plant-Microbes Interaction: Exploring the Impact of Cold-Tolerant Bacillus Strains RJGP41 and GBAC46 Volatiles on Tomato Growth Promotion through Different Mechanisms.

The interaction between plant and bacterial VOCs has been extensively studied, but the role of VOCs in growth promotion still needs to be explored. In the current study, we aim to explore the growth promotion mechanisms of cold-tolerant Bacillus strains GBAC46 and RJGP41 and the well-known PGPR strain FZB42 and their VOCs on tomato plants. The result showed that the activity of phytohormone (IAA) production was greatly improved in GBAC46 and RJGP41 as compared to FZB42 strains. The in vitro and in-pot experiment results showed that the Bacillus VOCs improved plant growth traits in terms of physiological parameters as compared to the CK. The VOCs identified through gas chromatography-mass spectrometry (GC-MS) analysis, namely 2 pentanone, 3-ethyl (2P3E) from GBAC46, 1,3-cyclobutanediol,2,2,4,4-tetramethyl (CBDO) from RJGP41, and benzaldehyde (BDH) from FZB42, were used for plant growth promotion. The results of the partition plate (I-plate) and in-pot experiments showed that all the selected VOCs (2P3E, CBDO, and BDH) promoted plant growth parameters as compared to CK. Furthermore, the root morphological factors also revealed that the selected VOCs improved the root physiological traits in tomato plants. The plant defense enzymes (POD, APX, SOD, and CAT) and total protein contents were studied, and the results showed that the antioxidant enzymes and protein contents significantly increased as compared to CK. Similarly, plant growth promotion expression genes (IAA4, ARF10A, GA2OX2, CKX2, and EXP1) were significantly upregulated and the ERF gene was downregulated as compared to CK. The overall findings suggest that both Bacillus isolates and their pure VOCs positively improved plant growth promotion activities by triggering the antioxidant enzyme activity, protein contents, and relative gene expressions in tomato plants.

Volatile Organic Compounds Emitted by the Biocontrol Agent Pythium oligandrum Contribute to Ginger Plant Growth and Disease Resistance.

The oomycete Pythium oligandrum is a potential biocontrol agent to control a wide range of fungal and oomycete-caused diseases, such as Pythium myriotylum-caused rhizome rot in ginger, leading to reduced yields and compromised quality. Previously, P. oligandrum has been studied for its plant growth-promoting potential by auxin production and induction of disease resistance by elicitors such as oligandrin. Volatile organic compounds (VOCs) play beneficial roles in sustainable agriculture by enhancing plant growth and resistance. We investigated the contribution of P. oligandrum-produced VOCs on plant growth and disease suppression by initially using Nicotiana benthamiana plants for screening. P. oligandrum VOCs significantly enhanced tobacco seedling and plant biomass contents. Screening of the individual VOCs showed that 3-octanone and hexadecane promoted the growth of tobacco seedlings. The total VOCs from P. oligandrum also enhanced the shoot and root growth of ginger plants. Transcriptomic analysis showed a higher expression of genes related to plant growth hormones and stress responses in the leaves of ginger plants exposed to P. oligandrum VOCs. The concentrations of plant growth hormones such as auxin, zeatin, and gibberellic acid were higher in the leaves of ginger plants exposed to P. oligandrum VOCs. In a ginger disease biocontrol assay, the VOC-exposed ginger plants infected with P. myriotylum had lower levels of disease severity. We conclude that this study contributes to understanding the growth-promoting mechanisms of P. oligandrum on ginger and tobacco, priming of ginger plants against various stresses, and the mechanisms of action of P. oligandrum as a biocontrol agent. IMPORTANCE Plant growth promotion plays a vital role in enhancing production of agricultural crops, and Pythium oligandrum is known for its plant growth-promoting potential through production of auxins and induction of resistance by elicitors. This study highlights the significance of P. oligandrum-produced VOCs in plant growth promotion and disease resistance. Transcriptomic analyses of leaves of ginger plants exposed to P. oligandrum VOCs revealed the upregulation of genes involved in plant growth hormone signaling and stress responses. Moreover, the concentration of growth hormones significantly increased in P. oligandrum VOC-exposed ginger plants. Additionally, the disease severity was reduced in P. myriotylum-infected ginger plants exposed to P. oligandrum VOCs. In ginger, P. myriotylum-caused rhizome rot disease results in severe losses, and biocontrol has a role as part of an integrated pest management strategy for rhizome rot disease. Overall, growth enhancement and disease reduction in plants exposed to P. oligandrum-produced VOCs contribute to its role as a biocontrol agent.

Corrigendum: Zinc essentiality, toxicity, and its bacterial bioremediation: A comprehensive insight.

[This corrects the article DOI: 10.3389/fmicb.2022.900740.].

Zinc Essentiality, Toxicity, and Its Bacterial Bioremediation: A Comprehensive Insight.

Zinc (Zn) is one of the most abundantly found heavy metals in the Earth's crust and is reported to be an essential trace metal required for the growth of living beings, with it being a cofactor of major proteins, and mediating the regulation of several immunomodulatory functions. However, its essentiality also runs parallel to its toxicity, which is induced through various anthropogenic sources, constant exposure to polluted sites, and other natural phenomena. The bioavailability of Zn is attributable to various vegetables, beef, and dairy products, which are a good source of Zn for safe consumption by humans. However, conditions of Zn toxicity can also occur through the overdosage of Zn supplements, which is increasing at an alarming rate attributing to lack of awareness. Though Zn toxicity in humans is a treatable and non-life-threatening condition, several symptoms cause distress to human activities and lifestyle, including fever, breathing difficulty, nausea, chest pain, and cough. In the environment, Zn is generally found in soil and water bodies, where it is introduced through the action of weathering, and release of industrial effluents, respectively. Excessive levels of Zn in these sources can alter soil and aquatic microbial diversity, and can thus affect the bioavailability and absorption of other metals as well. Several Gram-positive and -negative species, such as Bacillus sp., Staphylococcus sp., Streptococcus sp., and Escherichia coli, Pseudomonas sp., Klebsiella sp., and Enterobacter sp., respectively, have been reported to be promising agents of Zn bioremediation. This review intends to present an overview of Zn and its properties, uses, bioavailability, toxicity, as well as the major mechanisms involved in its bioremediation from polluted soil and wastewaters.

Genome of Pythium myriotylum Uncovers an Extensive Arsenal of Virulence-Related Genes among the Broad-Host-Range Necrotrophic Pythium Plant Pathogens.

The Pythium (Peronosporales, Oomycota) genus includes devastating plant pathogens that cause widespread diseases and severe crop losses. Here, we have uncovered a far greater arsenal of virulence factor-related genes in the necrotrophic Pythium myriotylum than in other Pythium plant pathogens. The genome of a plant-virulent P. myriotylum strain (~70 Mb and 19,878 genes) isolated from a diseased rhizome of ginger (Zingiber officinale) encodes the largest repertoire of putative effectors, proteases, and plant cell wall-degrading enzymes (PCWDEs) among the studied species. P. myriotylum has twice as many predicted secreted proteins than any other Pythium plant pathogen. Arrays of tandem duplications appear to be a key factor of the enrichment of the virulence factor-related genes in P. myriotylum. The transcriptomic analysis performed on two P. myriotylum isolates infecting ginger leaves showed that proteases were a major part of the upregulated genes along with PCWDEs, Nep1-like proteins (NLPs), and elicitin-like proteins. A subset of P. myriotylum NLPs were analyzed and found to have necrosis-inducing ability from agroinfiltration of tobacco (Nicotiana benthamiana) leaves. One of the heterologously produced infection-upregulated putative cutinases found in a tandem array showed esterase activity with preferences for longer-chain-length substrates and neutral to alkaline pH levels. Our results allow the development of science-based targets for the management of P. myriotylum-caused disease, as insights from the genome and transcriptome show that gene expansion of virulence factor-related genes play a bigger role in the plant parasitism of Pythium spp. than previously thought. IMPORTANCE Pythium species are oomycetes, an evolutionarily distinct group of filamentous fungus-like stramenopiles. The Pythium genus includes several pathogens of important crop species, e.g., the spice ginger. Analysis of our genome from the plant pathogen Pythium myriotylum uncovered a far larger arsenal of virulence factor-related genes than found in other Pythium plant pathogens, and these genes contribute to the infection of the plant host. The increase in the number of virulence factor-related genes appears to have occurred through the mechanism of tandem gene duplication events. Genes from particular virulence factor-related categories that were increased in number and switched on during infection of ginger leaves had their activities tested. These genes have toxic activities toward plant cells or activities to hydrolyze polymeric components of the plant. The research suggests targets to better manage diseases caused by P. myriotylum and prompts renewed attention to the genomics of Pythium plant pathogens.

Dual-Transcriptomic, Microscopic, and Biocontrol Analyses of the Interaction Between the Bioeffector Pythium oligandrum and the Pythium Soft-Rot of Ginger Pathogen Pythium myriotylum.

Biological control is a promising approach to suppress diseases caused by Pythium spp. such as Pythium soft rot of ginger caused by P. myriotylum. Unusually for a single genus, it also includes species that can antagonize Pythium plant pathogens, such as Pythium oligandrum. We investigated if a new isolate of P. oligandrum could antagonize P. myriotylum, what changes occurred in gene expression when P. oligandrum (antagonist) and P. myriotylum (host) interacted, and whether P. oligandrum could control soft-rot of ginger caused by P. myriotylum. An isolate of P. oligandrum, GAQ1, recovered from soil could antagonize P. myriotylum in a plate-based confrontation assay whereby P. myriotylum became non-viable. The loss of viability of P. myriotylum coupled with how P. oligandrum hyphae could coil around and penetrate the hyphae of P. myriotylum, indicated a predatory interaction. We investigated the transcriptional responses of P. myriotylum and P. oligandrum using dual-RNAseq at a stage in the confrontation where similar levels of total transcripts were measured from each species. As part of the transcriptional response of P. myriotylum to the presence of P. oligandrum, genes including a subset of putative Kazal-type protease inhibitors were strongly upregulated along with cellulases, elicitin-like proteins and genes involved in the repair of DNA double-strand breaks. In P. oligandrum, proteases, cellulases, and peroxidases featured prominently in the upregulated genes. The upregulation along with constitutive expression of P. oligandrum proteases appeared to be responded to by the upregulation of putative protease inhibitors from P. myriotylum, suggesting a P. myriotylum defensive strategy. Notwithstanding this P. myriotylum defensive strategy, P. oligandrum had a strong disease control effect on soft-rot of ginger caused by P. myriotylum. The newly isolated strain of P. oligandrum is a promising biocontrol agent for suppressing the soft-rot of ginger. The dual-RNAseq approach highlights responses of P. myriotylum that suggests features of a defensive strategy, and are perhaps another factor that may contribute to the variable success and durability of biological attempts to control diseases caused by Pythium spp.

Genetic Screening and Expression Analysis of Psychrophilic Bacillus spp. Reveal Their Potential to Alleviate Cold Stress and Modulate Phytohormones in Wheat.

Abiotic stress in plants pose a major threat to cereal crop production worldwide and cold stress is also notorious for causing a decrease in plant growth and yield in wheat. The present study was designed to alleviate cold stress on plants by inoculating psychrophilic PGPR bacteria belonging to Bacillus genera isolated from extreme rhizospheric environments of Qinghai-Tibetan plateau. The genetic screening of psychrophilic Bacillus spp. CJCL2, RJGP41 and temperate B. velezensis FZB42 revealed presence of genetic features corresponding to cold stress response, membrane transport, signal transduction and osmotic regulation. Subsequently, the time frame study for the expression of genes involved in these pathways was also significantly higher in psychrophilic strains as analyzed through qPCR analysis at 4 ℃. The inoculated cold tolerant Bacillus strains also aided in inducing stress response in wheat by regulating abscisic acid, lipid peroxidation and proline accumulation pathways in a beneficial manner. Moreover, during comparative analysis of growth promotion in wheat all three Bacillus strains showed significant results at 25 ℃. Whereas, psychrophilic Bacillus strains CJCL2 and RJGP41 were able to positively regulate the expression of phytohormones leading to significant improvement in plant growth under cold stress.

The Type III Accessory Protein HrpE of Xanthomonas oryzae pv. oryzae Surpasses the Secretion Role, and Enhances Plant Resistance and Photosynthesis.

Many species of plant-pathogenic gram-negative bacteria deploy the type III (T3) secretion system to secrete virulence components, which are mostly characteristic of protein effectors targeting the cytosol of the plant cell following secretion. Xanthomonas oryzae pv. oryzae (Xoo), a rice pathogen causing bacterial blight disease, uses the T3 accessory protein HrpE to assemble the pilus pathway, which in turn secretes transcription activator-like (TAL) effectors. The hrpE gene can execute extensive physiological and pathological functions beyond effector secretion. As evidenced in this study, when the hrpE gene was deleted from the Xoo genome, the bacteria incur seriouimpairments in multiplication, motility, and virulence. The virulence nullification is attributed to reduced secretion and translocation of PthXo1, which is a TAL effector that determines the bacterial virulence in the susceptible rice varieties. When the HrpE protein produced by prokaryotic expression is applied to plants, the recombinant protein is highly effective at inducing the defense response. Moreover, leaf photosynthesis efficiency is enhanced in HrpE-treated plants. These results provide experimental avenues to modulate the plant defense and growth tradeoff by manipulating a bacterial T3 accessory protein.

Suppression of Sclerotinia sclerotiorum by the Induction of Systemic Resistance and Regulation of Antioxidant Pathways in Tomato Using Fengycin Produced by Bacillus amyloliquefaciens FZB42.

Lipopeptides from Bacillus species exhibit promising biological control activity against plant pathogens. This study aimed to explore the potential of purified fengycin to induce systemic resistance in tomato against Sclerotinia sclerotiorum. Bacillus amyloliquefaciens FZB42, its mutant AK1S, and their corresponding metabolites showed in vitro inhibition of S. sclerotiorum mycelium. Fengycin derived from an AK1S mutant was purified and identified through HPLC and MALDI-TOF-MS, respectively. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed structural deformities in the fungal mycelium. Moreover, fengycin induced the accumulation of reactive oxygen species (ROS) in S. sclerotiorum mycelium and downregulated the expression of ROS-scavenging genes viz., superoxide dismutase (SsSOD1), peroxidase (SsPO), and catalase (SsCAT1) compared to the untreated control. Furthermore, the lesion size was dramatically reduced in fengycin-treated tomato plants compared to plants infected with S. sclerotiorum only in a greenhouse experiment. Additionally, the transcriptional regulation of defense-related genes GST, SOD, PAL, HMGR, and MPK3 showed the highest upsurge in expression at 48 h post-inoculation (hpi). However, their expression was subsequently decreased at 96 hpi in fengycin + S. sclerotiorum treatment compared to the plants treated with fengycin only. Conversely, the expression of PPO increased in a linear manner up to 96 hpi.