Fungal Communities in the Native New Zealand Medicinal Plant Pseudowintera colorata (Horopito) Are Determined by Plant Organ Type and Host Maturity with Key Members Promoting Plant Growth

The plant Pseudowintera colorata is well known for its antimicrobial and medicinal properties and is endemic to New Zealand. Using PCR-Denaturing gradient gel electrophoresis (DGGE), we investigated the factors influencing the composition of endophytic fungal communities in P. colorata from ten distinct sites across New Zealand. Our results showed that plant organs of P. colorata influenced the diversity and richness of endophytic fungi (PERMANOVA, p < 0.05). In addition, plant maturity and its interactions revealed that endophytic fungal communities formed discrete clusters in leaves, stems, and roots of mature and immature P. colorata plants (PERMANOVA; p = 0.002, p = 0.001 and p = 0.039, respectively). For identifying isolates with biocontrol potential, dual culture tests were set up against four different phytopathogenic fungi. Isolates with high activity (zone of inhibition >10 mm) were sequenced and identified as Trichoderma harzianum, Pezicula neosporulosa, Fusarium tricinctum, Metarhizium sp., and Chaetomium sp. Applying selected endophytic fungi (n = 7) as soil drenchers significantly increased the growth of P. colorata seedlings and produced more internodes. Seedling shoots treated with Trichoderma sp. PRY2BA21 were 2.2 × longer (8.36 cm) than the untreated controls (3.72 cm). Our results elucidate the main plant factors influencing fungal community composition and demonstrate a role for endophytic fungi in P. colorata growth and further demonstrate that medicinal plants are a rich source of endophytes with potential as biocontrol agents.

The Root Mycobiota of Betula aetnensis Raf., an Endemic Tree Species Colonizing the Lavas of Mt. Etna (Italy)

Betula aetnensis is an endemic tree of high conservation value, which thrives on the nutrient-poor volcanic soils of Mount Etna. Since plant–microbe interactions could play a crucial role in plant growth, resource uptake, and resistance to abiotic stresses, we aimed to characterize the root and rhizosphere microbial communities. Individuals from natural habitat (NAT) and forest nursery (NURS) were surveyed through microscopy observations and molecular tools: bacterial and fungal automated ribosomal intergenic spacer analysis (ARISA), fungal denaturing gradient gel electrophoresis (DGGE). B. aetnensis was found to be simultaneously colonized by arbuscular (AM), ectomycorrhizal (ECM), ericoid (ERM) fungi, and dark septate endophytes (DSE). A high diversity of the bacterial community was observed whilst the root fungal assemblage of NAT plants was richer than that of NURS. Root and rhizosphere fungal communities from NAT plants were characterized by Illumina MiSeq sequencing. Most of the identified sequences were affiliated to Helotiales, Pezizales, and Malasseziales. Ascomycota and Basidiomycota dominated roots and rhizosphere but differed in community structure and composition. ECM in the roots mainly belonged to Tylospora and Leccinum, while Rhizopogon was abundant in the rhizosphere. The Helotiales, including ERM (mostly Oidiodendron) and DSE (mostly Phialocephala), appeared the dominant component of the fungal community. B. aetnensis harbors an extraordinarily wide array of root-associated soil microorganisms, which are likely to be involved in the adaptation and resistance mechanisms to the extreme environmental conditions in volcano Etna. We argue that nursery-produced seedlings could lack the necessary microbiota for growth and development in natural conditions.

An Early Diagnosis of Thalassemia: A Boon to a Healthy Society

The β-thalassemia is a hereditary blood disorders, characterized by reduced or absent synthesis of the hemoglobin beta chain that cause microcytic hypochromic anemia. An early diagnosis, economical test, awareness programs and prenatal screening will be a milestone for the eradication of this genetic disorder and to reduce burden of the health sector of a country subsequently the economics. Initially, the diagnosis of β-thalassemia depends on the hematological tests with red cell indices that disclosed the microcytic hypochromic anemia.Hemoglobin analysis shows the abnormal peripheral blood smear with nucleated red blood cells, and reduced amounts of hemoglobin A (HbA). In severe anemia, the hemoglobin analysis by HPLC reveals decreased quantities of HbA and increased the level of hemoglobin F (HbF).The decrease level of MCV and MCH are also associated with β-thalassemia. There are various different molecular techniques such as ARMS PCR, allele-specific PCR, Gap PCR, denaturing gradient gel electrophoresis, reverse dot blotting, DGGE, SSCP, HRM, MLPA, sequencing technology and microarray available to identify the globin chain gene mutations. These molecular techniques can be clustered for detection by mutation types and alteration in gene sequences.

Bacterioplankton Community as a Biological Element for Reservoirs Water Quality Assessment

The Water Framework Directive (WFD) is used to evaluate the water quality of aquatic ecosystems. Phytoplankton is the only biological element considered in the reservoirs water quality assessment. In this study, we aimed to assess the use of the bacterioplankton community as an indicator of water quality, using a culture-independent assay (denaturing gradient gel electrophoresis, DGGE). Four Portuguese reservoirs (Miranda, Pocinho, Aguieira and Alqueva) were analysed in four periods (autumn 2018, spring and autumn 2019, and spring 2020). Bacterial total abundance had similar values for Miranda, Pocinho and Aguieira, and generally lower values for Alqueva. Diversity and richness values did not show a clear trend. Negative correlations were observed between some nutrients and the bacterial community. Overall, members of Actinobacteria, Bacteroidetes, Cyanobacteria, Nitrospirae and Proteobacteria were identified in all sampling sites. In Alqueva, no spatial, temporal or water body quality relationships with bacterial community were observed, which may be due to its higher size, low water velocity rate and higher residence times. However, in Miranda, Pocinho and Aguieira, a strong spatial and temporal bacterial community dynamic was observed. Furthermore, the presence of some species (e.g., Acinetobacter sp.) may reflect the poor water quality that was not detected by the WFD approach.

Impact of Hinoki Cypress Wood on Diversity of Microflora: A Case Study from Owase City Hall

Hinoki cypress (Chamaecyparis obtusa) shows durability against termites and wood decay-causing fungi and is used as a construction material in Japan. However, the effects of the material are still not fully understood. The aim of this study was to evaluate whether Hinoki cypress has antimicrobial effects against airborne microorganisms. We examined the influence of Hinoki cypress on the growth of airborne bacteria and fungi using culture-based methods. The growth of bacterial colonies was observed after day 3 in the control group without Hinoki material. In contrast, the growth of bacterial colonies was observed after day 13 in the experimental group containing Hinoki material. In the experimental group, the number of fungal colonies was smaller than that in the control group, suggesting the antifungal effect of Hinoki cypress to some extent. In addition, we characterized the community structure of airborne bacteria in two rooms with and without cypress wood by the culture-independent method of PCR-denaturing gradient gel electrophoresis. This also suggested differences in the community structure of airborne bacteria depending on the presence or absence of Hinoki cypress wood. These results indicate that Hinoki cypress might be a useful functional material in building environments.

Analysis of microflora in dentinal tubule by denaturing gradient gel electrophoresis

This study was performed to apply denaturing gradient gel electrophoresis to microbiological examination of endodontic infections. The method was used to assess the bacterial communities in dentinal tubules. Samples were collected using #15, #35, and #60 K-type files from five infected root canals during treatment. Deoxyribonucleic acid(DNA) was extracted from the samples, and 16S ribosomal DNA was amplified by PCR using universal primers. The polymerase chain reaction(PCR) products were separated in the denaturing gel and band patterns were compared between the deep (#60 file) and superficial layers (#15 file) of the dentin. The major bands were then excised and DNA fragments in the gel were cloned and sequenced. The sequence data were subjected to BLAST search in the GenBank database for determination of bacterial species or closest relatives. In three root canals, similar band patterns were observed in both superficial and deep layers of the dentin, while several specific bands remained in the deep layer in two roots. The bacteria isolated from the deep layers were Porphyromonas gingivalis, Olsenella profuse, Atopobium rimae, and Prevotella, Flexistipes, Firmicutes, Peptostreptococcus, Dialister, and Eubacterium spp. Unlike previous studies, gram-negative anaerobic rods were isolated from the deep layers. Clone library analysis was simultaneously performed and similar results were obtained. The method utilized here will be useful for microbiological examination of endodontic infections. In addition, although it is still unknown whether they were viable, this study demonstrated the presence of gram-negative rods in dentinal tubules.

Responsiveness of Elite Cultivars vs. Ancestral Genotypes of Barley to Beneficial Rhizosphere Microbiome, Supporting Plant Defense Against Root-Lesion Nematodes

Harnessing plant-microbe interactions to advance crop resistance to pathogens could be a keystone in sustainable agriculture. The breeding of crops to maximize yield in intensive agriculture might have led to the loss of traits that are necessary for beneficial plant-soil feedback. In this study, we tested whether the soil microbiome can induce a stronger plant defense against root-lesion nematodes in ancestral genotypes of barley than in elite cultivars. Plants were grown in a sterile substrate with or without the inoculation of rhizosphere microbiomes, and Pratylenchus neglectus was inoculated to the roots. Unexpectedly, elite cultivars profited significantly more from the microbiome than ancestral genotypes, by the reduction of nematodes in roots and the increased shoot weight relative to control plants. The elite cultivars had higher microbial densities in the rhizosphere, which were correlated with root weight. The structure of the bacterial and fungal community of elite and ancestral genotypes differed, as compared by 16S rDNA or internal transcribed spacer amplicon profiles in denaturing gradient gel electrophoresis. The elite cultivars differed in responsiveness to the microbiome. For the most responsive cultivars Beysehir and Jolgeh, the strong microbe-induced suppression of nematodes coincided with the strongest microbe-dependent increase in transcripts of salicylic acid-regulated defense genes after nematode invasion, while the jasmonate-regulated genes LOX2 and AOS were downregulated in roots with the inoculated microbiome. The microbe-triggered modulation of defense gene expression differed significantly between elite and ancestral genotypes of barley. Soil microbiomes conditioned by maize roots suppressed the nematodes in elite cultivars, while the corresponding bulk soil microbiome did not. In conclusion, cultivars Beysehir and Jolgeh harbor the genetic background for a positive plant-microbiome feedback. Exploiting these traits in breeding for responsiveness to beneficial soil microbiomes, accompanied by soil biome management for compatible plant-microbe interactions, will support low-input agriculture and sustainability.

Molecular techniques for the assessment of Cr (VI) reduction by Bacillus thuringiensis

Effluent pollution with Cr (VI) is a worldwide environmental problem. In the Pasto River (southeastern, Colombia), previous studies reported contamination with this metal at points near tanneries. To establish the role of Bacillus thuringiensis in Cr (VI) reduction in water from Pasto River, experiments were carried out with untreated Pasto River water (treatment 1), sterile Pasto River water inoculated with B. thuringiensis (treatment 2), and unsterilized Pasto River water inoculated with B. thuringiensis (treatment 3). All experiments were conducted in bioreactors with a controlled temperature of 20 °C and constant agitation for 156 h. Samples of 20 mL were taken every 12 h from each treatment to track Cr (VI) reduction levels and to confirm microorganism identity via molecular methods involving denaturing gradient gel electrophoresis (DGGE), restriction enzyme digestion profiles (RFLP), and bioinformatic analysis. Cr (VI) reduction was higher in treatment 3 (99:42 %) as opposed to treatment 2 (76:12 %) and treatment 1 (74:46 %). The molecular identity of B. thuringiensis was determined via sequencing of the 16SrRNA gene, and RFLP assessments in all three treatments revealed B. thuringiensis profiles. Since B. thuringiensis was present in all three treatments trough time, Cr (VI) reduction can be attributed to this bacterium.

The Influence of Applying Microbial Amendments to Soil and Plants on the Microbial Diversity in the Rhizosphere Soil of Garlic

With the environmental problems brought about by the excessive use of fertilizers and pesticides, numerous biological amendments have been developed and used in recent years. This study, through in-depth analysis of the effects of two different microbial amendments on the microbial diversity in the garlic rhizosphere, provides a theoretical basis and data support for farmers to select microbial amendments. In the experiment, two different microbial amendments were applied to garlic, and its rhizosphere soil was collected after 10, 20, 30, and 40 days. The polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze bacterial diversity in the garlic rhizosphere soil. The total abundance and diversity of microbial flora in the rhizosphere soil of garlic increased after application of microbial amendments to soil or plants. Plant growth was significantly better in the soil treatment than the plant treatment and the water control. The two dominant bacteria of uncultured gamma proteobacterium and Uncultured Gemmatimonadete existed only in the soil treatment. After 40 day of treatment, the abundance in the rhizosphere soil of these four bacterial strains (Uncultured Bacillus sp. clone D.an-22, Mesorhizobium sp., uncultured gamma proteobacterium, and Pseudomonas boreopolis) was greater in the soil than the plant treatment and the control. The irrigation of microbial amendments not only influenced the structure and abundance of rhizosphere microorganisms, and also promoted the colonization by microorganisms. The five bacterial strains of uncultured gamma proteobacterium, Uncultured Gemmatimonadetes, Uncultured Bacillus sp. clone D.an-22, Mesorhizobium sp., Pseudomonas boreopolis could facilitate the growth and enhance resistance of garlic, establishing a foundation for the use of microbial amendments and providing new ideas and methods for environmental management and protection.

Microbial Communities of Hydrothermal Guaymas Basin Surficial Sediment Profiled at 2 Millimeter-Scale Resolution

The surficial hydrothermal sediments of Guaymas Basin harbor complex microbial communities where oxidative and reductive nitrogen, sulfur, and carbon-cycling populations and processes overlap and coexist. Here, we resolve microbial community profiles in hydrothermal sediment cores of Guaymas Basin on a scale of 2 millimeters, using Denaturing Gradient Gel Electrophoresis (DGGE) to visualize the rapid downcore changes among dominant bacteria and archaea. DGGE analysis of bacterial 16S rRNA gene amplicons identified free-living and syntrophic deltaproteobacterial sulfate-reducing bacteria, fermentative Cytophagales, members of the Chloroflexi (Thermoflexia), Aminicenantes, and uncultured sediment clades. The DGGE pattern indicates a gradually changing downcore community structure where small changes on a 2-millimeter scale accumulate to significantly changing populations within the top 4 cm sediment layer. Functional gene DGGE analyses identified anaerobic methane-oxidizing archaea (ANME) based on methyl-coenzyme M reductase genes, and members of the Betaproteobacteria and Thaumarchaeota based on bacterial and archaeal ammonia monooxygenase genes, respectively. The co-existence and overlapping habitat range of aerobic, nitrifying, sulfate-reducing and fermentative bacteria and archaea, including thermophiles, in the surficial sediments is consistent with dynamic redox and thermal gradients that sustain highly complex microbial communities in the hydrothermal sediments of Guaymas Basin.