PCR-denaturing Gradient Gel Electrophoresis Research Articles

Impact of whole grain highland hull-less barley on the denaturing gradient gel electrophoresis profiles of gut microbial communities in rats fed high-fat diets.

While next-generation sequencing has overshadowed polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), the latter still holds promise for advancing gut microbiota analysis due to its unique advantages. In this study, we used optimized nested PCR-DGGE to investigate the gut microbiota profile of high-fat diet rats after administering whole grain highland hull-less barley. High-throughput sequencing was employed to validate the DGGE results. Our results proved the reliability of PCR-DGGE for analyzing the dominant taxonomic composition while also providing visual evidence of a notable relationship between the composition of cecal and fecal microbial communities, highlighting substantial differences in both richness and abundance.

Mangroves: A hotspot for novel bacterial and archaeal diversity

Mangrove ecosystems are highly productive ecosystems that harbor diverse microbial lineage playing multiple roles in nutrient cycling. Though several novel mangrove rhizospheres associated with bacterial genera and species have been reported from these unexplored wetland ecosystems, but yet to be studied for their richness and diversity including actinobacterial and archaeal communities. Therefore, this study was undertaken to decode the bacterial and archaeal communities associated with the rhizosphere regions of true mangrove species-Avicennia marina and Rhizophora mucronata and their intersecting regions; and associated halophytic species -Suaeda maritima, and Salicornia brachiata of Pichavaram mangroves, Tamil Nadu, India. Amplified ribosomal DNA restriction analysis (ARDRA) and PCR denaturing gradient gel electrophoresis (PCR-DGGE) analysis techniques were adopted. About 567 culturable bacterial isolates with distinct morphology were purified, and nearly 88 distinct Operational Taxonomic Units (OTUs) were identified by ARDRA analysis. Phylogenetic analysis of representative groups indicated that the majority of the isolates belonged to Firmicutes (10 genera), followed by Proteobacteria- Alphaproteobacteria (10 genera) and Gammaproteobacteria (8 genera), Actinobacteria (8 genera), Bacteroidetes (1genera), and Halomonas (9 genera) representing 38 genera with 105 species. In addition, a few phylogenetically distinct novel groups belonging to the genera Mangrovibacter, Vibrio, Demequina, and Catenococcus were also isolated. PCR-DGGE with the RDP-Classifier program identified the prominent DGGE bands up to genus level namely Bacillus, Pseudomonas, Vibrio, Halomonas, etc. In addition, this study for the first time describes the genera Bhargavaea, Exiguobacterium, Hoeflea, Catenococcus, Albirhodobacter, Dietzia, Brachybacterium, Aerococcus, and Myroides association with mangrove rhizospheres. Likewise, DGGE analysis of archaea represented 2 major groups such as Crenarchaeota and Euryarchaeota, which were further classified into 7 different genera.

A Novel: Low-cost Method for the Isolation of Anaerobic Hydrogen Producing Bacteria

The hydrogen content of the two sludge samples namely TDS and PLS were increased after the heat shock pretreatment from 239.23 ± 10.23 mL/L to 663.87 ± 15.99 mL/L at 80°C and from 173.61 ± 17.21 mL/L to 496.74 ± 30.21 mL/L at 60°C, respectively. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) indicated that Enterococcus and Clostridium were the dominant bacteria in the TDS and PLS consortium. Two strains were successfully isolated from TDS and PLS using a simple method. Based on Bergey’s key identification and 16S rDNA sequencing, they were identified as Clostridium butyricum strain TD and Clostridium acetobutylicum strain PL. The hydrogen content, glucose consumed yield, and hydrogen yield of C. butyricum TD and C. acetobutylicum PL were 545.24 ± 7.31 mL/L, 90 ± 0.41 %, 0.89 ± 0.01 mol H2/mol glucose, and 729.34 ± 22.81 mL/L, 92.77 ± 1.39 %, 1.26 ± 0.05 mol H2/mol glucose, respectively.

Bacterial and fungal community composition and community-level physiological profiles in forest soils.

We characterized the potential functioning and composition of the bacterial and fungal communities in the O and A horizons of forest soils using community-level physiological profile (CLPP) based on BIOLOG analysis, and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of 16S and 18S rDNA fragments, respectively. In addition, relationships between the potential functioning and the community composition in each horizon, and between the O and A horizons, were assessed using Procrustes analysis. For the bacterial and fungal communities, the CLPP and DGGE profile were clearly separated between the O and A horizons in a principal coordinate analysis except for the fungal CLPP. No significant links for CLPP and DGGE profile between the O and A horizons were observed for either bacterial or fungal communities, suggesting that different factors had considerable influence on the microbial communities between the O and A horizons. Significant couplings between bacterial and fungal DGGE profiles (p <0.05 for O horizon; p <0.01 for A horizon), and between bacterial and fungal CLPPs (p = 0.001 for O horizon; p <0.01 for A horizon), were observed in the O and A horizons, implying that common factors strongly influenced the bacterial and fungal communities in each horizon. Although a significant correlation was observed between bacterial community composition and the potential functioning in the A horizon (p <0.01), such a correlation was not observed for the fungal community in the A horizon, and for the bacterial and fungal communities in the O horizon. This finding suggested that potential functioning, which would reflect only rapidly growing microorganisms, was not strongly associated with the composition of the entire microbial community. Further studies are needed to unravel the factors shaping the composition and functioning of microbial communities in forest soils.

Effects of pavements on diversity and activity of mycorrhizal symbionts associated with urban trees

This study investigated the impact of different types of soil sealing on the communities of a group of beneficial plant symbionts, arbuscular mycorrhizal fungi (AMF), colonizing the roots of two shade trees, Celtis australis and Fraxinus ornus, frequently grown in urbanized sites. Such plants were grown in an experimental site, in northern Italy, established in November 2011 and subjected to four different pavement treatments: impermeable monolithic asphalt, permeable pavers, permeable concrete and unpaved soil. The diversity and composition of root AMF communities were assessed by PCR denaturating gradient gel electrophoresis of partial 18S rRNA gene, AMF taxa were identified by amplicon sequencing and mycorrhizal colonization was evaluated after root clearing and staining. For the first time, our molecular work revealed that impermeable pavements induced shifts in the composition of AMF communities associated to the roots of C. australis and F. ornus and impacted on the percentage of mycorrhizal root length. When the root-zone was covered with permeable pavements, a similar AMF community as that observed in the unpaved soil was detected, providing novel information to be utilised for reducing the disturbance caused by specific types of soil sealing on AMF symbionts, which play a key role in plant nutrition and health. A total of 45 AMF sequence types were detected, with Sclerocystis and Septoglomus as the most abundant phylotypes, accounting for 84% of the sequences. The predominance of Sclerocystis species in the roots of both tree species under impermeable pavements indicated their high and unforeseen tolerance towards harsh environmental conditions. Such species could be utilized as AMF inocula specifically selected for their proven resilience in paved sites, in order to exploit their ability to boost biogeochemical processes fundamental for energy fluxes and plant nutrition and health.

Removal of Hydrogen Sulfide from Swine-Waste Biogas on a Pilot Scale Using Immobilized Paracoccus versutus CM1.

Hydrogen sulfide (H2S) is a toxic and corrosive component that commonly occurs in biogas. In this study, H2S removal from swine-waste biogas using sulfur-oxidizing Paracoccus versutus CM1 immobilized in porous glass (PG) and polyurethane foam (PUF) biofilters was investigated. Bacterial compositions in the biofilters were also determined using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The biofilters were first tested on a laboratory scale under three space velocities (SV): 20, 30, and 40 h-1. Within 24 h, at an SV of 20 h-1, PG and PUF biofilters immobilized with P. versutus CM1 removed 99.5% and 99.7% of H2S, respectively, corresponding to the elimination capacities (EC) of 83.5 and 86.2 gm-3 h-1. On a pilot scale, with the horizontal PG-P. versutus CM1 biofilter operated at an SV of 30 h-1, a removal efficiency of 99.7% and a maximum EC of 113.7 gm-3 h-1 were achieved. No reduction in methane content in the outlet biogas was observed under these conditions. The PCR-DGGE analysis revealed that Paracoccus,&amp;nbsp;Acidithiobacillus, and Thiomonas were the predominant bacterial genera in the biofilters, which might play important roles in H2S removal. This PG-P. versutus CM1 biofiltration system is highly efficient for H2S removal from swine-waste biogas.

The Latest Research Advances of Danggui Buxue Tang as an Effective Prescription for Various Diseases: A Comprehensive Review.

Danggui Buxue Tang (DBT) is composed of Astragali Radix and Angelicae Sinensis Radix in a weight ratio of 5:1. The recipe of the decoction is simple, and DBT has been widely used in the treatment of blood deficiency syndrome for more than 800 years in China. Studies on its chemical constituents show that saponins, flavonoids, volatile oils, organic acids, and polysaccharides are the main components of DBT. Many techniques such as third-generation sequencing, PCR-denaturing gradient gel electrophoresis, and HPLC-MS have been used for the quality control of DBT. DBT has a wide range of biological activities, including blood enhancement, antagonizing diabetic nephropathy, cardiovascular protection, immunity stimulation, estrogen-like effect, and antifibrosis, among others. In this paper, we summarize the recent research advances of DBT in terms of its components, pharmacological activities, and possible mechanisms of action as well as provide suggestions for further research.

Lactobacillus acidophilus CGMCC 878 impacts colorectal cancer in Sprague-Dawley rats through changing the gut microbiota

A main cause of cancer-related mortality, colorectal cancer (CRC) is one of the most prevalent cancers in the world. A number of variables, including a poor dietary and lifestyle, genetics, metabolic problems, and inheritance, are linked to the risk of CRC. However, it has been studied extensively about the involvement of various environmental factors which facilitates the development of this particular type of cancer in both genders. These elements includes excessive consumption of meat, refined grain and their products, starch, sugars in distinct forms, and alcohol are at top of the list. Hence, to execute the significance of Lactobacillus acidophilus CGMCC 878 (L.A 878) which may possibly hinder the development as well as progression of chemically induce colorectal tumor in in-vivo model via transformation of gut microbiota. We had established CRC in male Sprague-Dawley rats through subcutaneously inject the carcinogen named 1,2 dimethylhydrazine hydrochloride (DMH). We had detected the microbial diversity among different groups which included in the study by evaluation of stool samples by the virtue of molecular genetic computation. Although, we had targeted the bacterial V3 region by using classic molecular technique of PCR-DGGE (Polymerase chain reaction- Denaturing Gradient Gel Electrophoresis) followed by excision of amplified bands and were cloned for sequencing. The DGGE profiles of distinct groups demonstrated significant alterations between DMH group, short-term L. acidophilus group (p ​< ​0.05), and the long-term L. acidophilus group (P 0.001). The fecal-glucuronidase was significantly overexpressed in DMH group associated with long-term L. acidophilus group (p ​< ​0.05). Hence, prolong administration of L.A 878 could reduce the development of colorectal tumors in rats by altering the intestinal pathogenic bacteria (Ruminococcus obeum, Clostridium thermocellum, Bacteroides vulgates, Mycoplasma leachii, Porphyromonas asaccharolytica) and beneficial bacteria (Lactobacillus reuteri, Lactobacillus). These observations suggested that Lactobacillus acidophilus CGMCC 878 may have therapeutic potential in attenuating carcinogenesis of gastrointestinal (GI) tract.

Roughage biodegradation by natural co-cultures of rumen fungi and methanogens from Qinghai yaks

Anaerobic fungus–methanogen co-cultures from rumen liquids and faeces can degrade lignocellulose efficiently. In this study, 31 fungus–methanogen co-cultures were first obtained from the rumen of yaks grazing in Qinghai Province, China, using the Hungate roll-tube technique. The fungi were identified according to morphological characteristics and internal transcribed spacer (ITS) sequences. The methanogens associated with each fungus were identified by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and 16S rRNA gene sequencing. They were five co-culture types: Neocallimastix frontalis + Methanobrevibacter ruminantium, Neocallimastix frontalis + Methanobrevibacter gottschalkii, Orpinomyces joyonii + Methanobrevibacter ruminantium, Caecomyces communis + Methanobrevibacter ruminantium, and Caecomyces communis + Methanobrevibacter millerae. Among the 31 co-cultures, during the 5-day incubation, the N. frontalis + M. gottschalkii co-culture YakQH5 degraded 59.0%–68.1% of the dry matter (DM) and 49.5%–59.7% of the neutral detergent fiber (NDF) of wheat straw, corn stalk, rice straw, oat straw and sorghum straw to produce CH4 (3.0–4.6 mmol/g DM) and acetate (7.3–8.6 mmol/g DM) as end-products. Ferulic acid (FA) released at 4.8 mg/g DM on corn stalk and p-coumaric acid (PCA) released at 11.7 mg/g DM on sorghum straw showed the highest values, with the following peak values of enzyme activities: xylanase at 12,910 mU/mL on wheat straw, ferulic acid esterase (FAE) at 10.5 mU/mL on corn stalk, and p-coumaric acid esterase (CAE) at 20.5 mU/mL on sorghum straw. The N. frontalis + M. gottschalkii co-culture YakQH5 from Qinghai yaks represents a new efficient combination for lignocellulose biodegradation, performing better than previously reported fungus–methanogen co-cultures from the digestive tract of ruminants.

Mapping Archaeal Diversity in Soda Lakes by Coupling 16S rRNA PCR-DGGE Analysis with Remote Sensing and GIS Technology

The haloarchaeal diversity of four hypersaline alkaline lakes from the Wadi El-Natrun depression (Northern Egypt) was investigated using culture-independent polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S rRNA gene phylotypes, which was combined with remote sensing and geographic information system (GIS) data to highlight the distribution pattern of the microbial diversity in water and sediment samples. The majority of archaeal sequences identified in all four lakes belonged to the phyla Euryarchaeota and Crenarchaeota. Sediment samples from Beida Lake and water samples from El-Hamra Lake showed the highest levels of archaeal diversity. Sequence similarities ≥ 95% were found between six of the acquired clones and uncultured Halorhabdus, Euryarchaeota, and archaeon clones. In addition, two clones shared a high level of sequence similarity (97%) with unclassified archaea, while other nine clones exhibited 96% to 99% sequence similarity with uncultured archaeon clones, and only one clone showed 97% identity with an uncultured Crenarchaeota. Likewise, 7 DGGE bands presented a sequence similarity of 90 to 98% to Halogranum sp., Halalkalicoccus tibetensis, Halalkalicoccus jeotgali, uncultured Halorubrum, Halobacteriaceae sp., or uncultured haloarchaeon. In conclusion, while the variety of alkaliphilic haloarchaea in the examined soda lakes was restricted, the possibility of uncovering novel species for biotechnological applications from these extreme habitats remains promising.

Further culture-independent characterization of the lactic microbiota of Serro artisanal cheese.

This study aimed to provide a further characterization of the lactic microbiota present in Minas artisanal cheese (MAC) from the Serro region by using culture-independent methods, as a complementary analysis of a previous study. The total DNA extracted from MAC samples (n = 55) was subjected to repetitive extragenic palindromic-PCR (rep-PCR) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Rep-PCR analysis showed that core microbiota of Serro MAC was closely related, independent of the production town, farm size, or time of production. The sequencing of PCR-DGGE bands identified the prevalence of Lactococcus lactis in all samples, and Streptococcus salivarius was also identified. Thus, we conclude that when more accurate methods are unavailable, rep-PCR can be used as a culture-independent method to demonstrate if the microbiota is closely related or not among the samples. PCR-DGGE results also matched to the main findings of high-throughput sequencing, previously presented, confirming its confidence to detect the main microbial groups present in the raw milk cheeses.

Simultaneous removal of ammonia nitrogen and sulfide by coupled anammox and sulfur autotrophic denitrification process from industrial wastewater

This work presents an experimental study on the coupling between anammox and sulfur autotrophic denitrification for simultaneous removal of ammonia nitrogen and sulfide in industrial wastewater, leading to less environmental pollution. In a low-load continuous operation mode hybrid anaerobic baffled reactor (HABR) was started with 7 <pH<8.2 and temperature 30–35 °C. After 60 d of continuous operation successful enrichment of methanogenic granular sludge (MGS) size 7 and 5 mm was achieved. In the mature granular sludge phase (HRT 10 h), with influent chemical oxygen demand (COD) 1100 mg/L, the effluent COD 90 mg/L and removal efficiency was 90%. The MLSS, MLVSS, and f values were 9520, 6980, and 0.7. After 42 d of continuous operation with influent COD 600–700 mg/L, NO3−-N 250-300 mg/L, and COD/NO3−-N 2 to 2.8 (temperature 30–35 °C and pH 6.5–7.5) successful enrichment of denitrifying granular sludge (DGS) was achieved. In the research, the anammox start-up periods in MGS and DGS reactors are 86 and 118 d. An experimental comparative analysis to explore the influence of COD/TN 1:1,1:1.8,1:4.5, and 1:8.5 on the two reactors was performed. For COD/TN 1:1.8, the NH4+-N, NO2−-N, and TN removal efficiency were 54%, 73%, and 52%. The removal efficiency of the #2 reactors NH4+-N, NO2−-N, and TN were 50%, 60%, and 55%. S2−/NO3−-N of 2:5, 3.5:5, 5:5, 6.5:5 were investigated. Influent pH 7.5–8.0, temperature 30–35 °C and HRT 6.5 h, with average influent NH4+-N, NO3−-N, S2−, COD concentration 60, 25, 75, and 25 mg/L, the maximum removal efficiency of #1 reactor was 70%, 97%, 82%, and 100%. For 2# reactor 82%, 99%, 84%, and 100%. When MGS as carrier, the NH4+-N, NO3−-N, and S2− removal efficiency reached 67.67%, 98.59%, and 99.1%. With DGS as carrier, the NH4+-N, NO3−-N, and S2− removal efficiency reached 81.82%, 98.69%, and 84.18%. However, the coupling phase S0 production and productivity results showed that the DGS reactor was significantly better than the MGS reactor. Under the same hydraulic conditions the two reactors successfully coupled after 54 d of operation. Additionally, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) analysis, revealed that the microbial community change is related to the concentration of organic matter, nitrogen and sulfur in the water. Building on these findings, future study will focus on the optimization of the mix design to provide practical engineering guidance.

Dynamics of Water and Biofilm Bacterial Community Composition in a Mediterranean Recirculation Aquaculture System

Recirculation technology has been emerging in the marine aquaculture industry. The microbiome developed in recirculation aquaculture systems (RASs) is an important factor for the optimal operation of these systems and fish welfare. In this study, the microbial community dynamics in the water column and the biofilms of a marine RAS with Mediterranean species of gilthead sea bream and sea bass were investigated, while physicochemical conditions were also monitored. Microbiological, culture, and non-culture analyses based on PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) fingerprints were performed on the water column and biofilm developed on stainless-steel surfaces. According to the obtained results, feed administration seemed to cause changes in pH and TAN, as well as drive changes in the bacterial abundance in the water column. Tested surfaces were colonized within 24 h and sessile cells were stabilized in terms of density within 6 days. DGGE fingerprints indicated the stability of the microbial community in water and a dynamic succession in the community of the biofilms. The fish pathogen Tenacibaculum discolor was found to colonize the biofilm and the water column. The main findings confirmed that RAS technology can be used as a control strategy for the stability of the water microbial community, that there is a dynamic succession of the dominant species in the biofilm communities, and that pathogenic bacteria can be dominant in the latter.

Impact of sheep wool residues as soil amendments on olive beneficial symbionts and bacterial diversity

In recent years the use of organic matter soil amendments, such as agricultural by-products, has been implemented with the aim of increasing soil fertility, while minimizing the environmental impact of agriculture. Sheep wool residues (SWR) have shown beneficial effects on plant nutrition and soil properties, while only few works assessed their impact on soil microbial communities. The main aim of this work was to investigate the possible valorization of two SWR types (scoured residues, white wool, WW, and carbonized scoured residues, black wool, BW) as organic soil amendments, in pot-grown olive trees, by evaluating their impact on soil bacterial communities and mycorrhizal symbionts. The two SWR types did not negatively impact on the diversity and composition of soil bacterial communities, as revealed by PCR-denaturating gradient gel electrophoresis (PCR-DGGE) of partial 16S rRNA gene, and on the activity of native arbuscular mycorrhizal fungi (AMF), while positively affecting plant growth. Only the highest doses of one SWR type (2% BW) caused a decrease in bacterial diversity and native AMF ability to colonize olive roots. DGGE bands sequencing allowed the identification of the major bacterial taxa. Sequences corresponding to Ohtaekwangia spp., Beta proteobacterium, Blastocatella sp., Ramlibacter monticola and Massilia frigida/rubra, Dongia sp. and Chloroflexi were mainly represented in SWR-amended soils, while those represented by Chryseolinea soli and Acidobacteria were abundant in control soil. Overall, this work showed that SWR may be valorized as organic soil amendments, as soil bacteria and AMF, representing key factors of biological soil fertility, were not negatively affected, while the activity of bacterial genera and species known for their ability to decompose complex compounds was boosted. Further studies will investigate the biodegradation efficiency of the diverse bacterial taxa developing in SWR-amended soils.Graphic

Sex Difference of Effect of Sophora flavescens on Gut Microbiota in Rats.

Objective By observing the sex difference of the gut microbiota in rats and the influence of Sophora flavescens (S. flavescens) on the gut microbiota in rats of different genders, it is hoped that it can provide reference materials for the rational use of S. flavescens in clinical practice. Method Taking samples of the jejunum (containing intestinal contents) and feces of 8-week-old rats, and detecting the composition of gut microbiota of females and males by 16S rRNA sequencing technology; At the same time, 8-week-old rats were gavaged with different doses of S. flavescens decoction, and the duodenum, jejunum, ileum, and colon (including the intestinal contents) samples were collected at 1, 2, and 3 weeks, using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technology and real-time fluorescent quantitative PCR (qRT-PCR) technology to observe the changes in the structure and the quantitative changes of 4 major intestinal dominant bacteria Enterococcus, Bacteriodes, Lactobacillus, and Clostridium in each intestinal segment, respectively. Result (1) The gut microbiota of normal rats without administration also had obvious gender differences; (2) S. flavescens significantly affects the composition of gut microbiota, and in different intestinal segments, this effect was different between genders under different dosages and different continuous administration times. Conclusion The effect of S. flavescens on the gut microbiota of rats had gender differences.

Evidence of functional and structural changes in the microbial community beneath a succulent invasive plant in coastal dunes

Abstract Coastal dunes represent priority habitats for conservation due to the provision of valuable ecosystem services such as land protection, water supply or biodiversity conservation. Soil microbial communities are of crucial importance to maintain plant diversity due to harsh environmental conditions, water limitation and nutrient scarcity. Invasive alien plants represent a major threat to ecosystem conservation. Here, we explored different impacts of Carpobrotus edulis, a succulent plant invading coastal areas worldwide, on the function and structure of bacterial communities. Sand represents a challenging substrate due to low organic matter content and limited microbial activity. We optimized bacterial extraction for functional evaluation before assessing ecosystem impacts produced by C. edulis. We compared 12 extracting procedures combining different soil storage, sample amount and extracting solutions on the functional activity of sand communities through the community-level physiological profile. We further explored the function (using Biolog Ecoplates) and structure [using polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE)] of bacterial communities from dunes invaded by C. edulis. Saline solution consistently increased bacterial cells detected by cytometry (P ≤ 0.001). Principal component analysis suggested a limited temporal framework (0–24 h) in which community function can be explored without significant alterations in C substrate consumption. Changes under C. edulis invasion exhibited a different pattern of C substrate utilization comparing native and non-native zones (interspecific), but also between native zones (intraspecific), suggesting that functional impacts are site-dependent. Complementary, results obtained from PCR-DGGE indicated that the bacterial community structure of native dunes significantly differed from dunes invaded by C. edulis.

PCR-DGGE Analysis Proves the Suppression of Rhizoctonia and Sclerotium Root Rot Due to Successive Inoculations

The soil-borne pathogens Rhizoctonia solani and Sclerotium rolfsii have emerged as major pathogens of radish (Raphanus sativus) worldwide. The induction of soil suppressive of radish root rot disease was evaluated in soil repeatedly inoculated with R. solani, nonpathogenic binucleate Rhizoctonia sp. AG-A W1 (BNR) and S. rolfsii. The repeated inoculations of soil with R. solani and BNR significantly suppressed the disease severity of R. solani and S. rolfsii compared to the control. In contrast, the repeated inoculation of soil with S. rolfsii significantly suppressed only the pathogen, S. rolfsii. The community structure was examined using PCR-DGGE (polymerase chain reaction denaturing gradient gel electrophoresis) method. The bands of Trichoderma sp. were observed in the first, second and third inoculations of the soil with BNR. Similarly, bands of Trichoderma sp. were observed in the second and third inoculations of the soil with S. rolfsii and R. solani. Compared to the control, disease severity was significantly reduced in the soil repeatedly inoculated with S. rolfsii and R. solani . In conclusion, Trichoderma species were accumulated in specific patterns depending on the applied fungal inoculum in the suppressive soil.

Analysis of rice microbial communities under different storage conditions using culture-dependent and -independent techniques

Rice is a staple food for over half of the world’s population, and fungal spoilage in stored rice may occur when the moisture content and temperature are conducive. Aspergillus sp. and Penicillium sp. are the most harmful toxigenic species that produce harmful mycotoxins. Molds pose a potential threat to public health and cause a huge economic loss. Therefore, it is of great importance to find out how molds multiply in rice. This study focused on the isolation and identification of fungi presented in rice and their evolution in rice with different moisture contents stored for varying periods of time and under different temperatures. Mold community was detected every month using the culture-dependent and -independent method of polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Significant differences were detected by the traditional culture method under different storage conditions. For potato dextrose agar (PDA) media, high temperature and moisture were suitable for the dominant strains including Penicillium aurantiogriseum and Penicillium oxalicum. In particular, P. oxalicum competitively inhibited the other fungi. For Rose Bengal medium, no difference was observed under different storage conditions, and only typical strains such as Aspergillus candidus and Alternaria were detected. PCR-DGGE identified some uncultured strains such as Trichoderma sp. and Cladosporium sp., the dominant strains and the flora diversity such as Aspergillus restrictus and Eurotium athecium. These results indicated that storage conditions greatly shape fungal growth. This study provides a foundation for the evolution of fungal flora in rice during storage in China and may help in developing biological control methods to prevent mold contamination in rice.

Effects of ferulic and p-hydroxybenzoic acids on Fusarium community structure and abundance in cucumber seedling rhizosphere

Soil microorganisms play an important role in agricultural ecosystem. However, there is little information about the effects of putative allelochemicals on specific soil microorganisms in vivo. Cucumber seedlings were treated with four concentrations of ferulic and p-hydroxybenzoic acids (0–1.0 μmol·g−1 soil) in soil. Effects of ferulic and p-hydroxybenzoic acids on rhizosphere Fusarium community structures and abundance were analyzed by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR techniques, respectively. The results showed that ferulic acid at concentrations of 0.25, 0.5, and 1.0 μmol·g−1 soil significantly reduced the number of bands of Fusarium, and ferulic acid at concentrations of 0.5 and 1.0 μmol·g−1 soil significantly reduced Shannon–Wiener and evenness index of Fusarium community. All concentrations of p-hydroxybenzoic acid changed the community structure of Fusarium, including decreasing the number of bands, Shannon–Wiener, and evenness index. Ferulic and p-hydroxybenzoic acids at all concentrations significantly promoted the abundance of Fusarium. Ferulic and p-hydroxybenzoic acids at 0.5 μmol·g−1 soil had the highest Fusarium abundance among all treatments. These results indicate that four concentrations of ferulic acid had various effects on Fusarium, which was different from p-hydroxybenzoic acid, and this may be related to the cucumber autotoxicity, giving us a further understanding of soil sickness.

Structure of intestinal microflora under different diets based on PCR-DGGE technology

This study aims to review the effects of conventional and organic diet on the diversity of rat intestinal microbiota and look at how intestinal microbiota composition changes the following moxibustion at the Piyu and Zusanli points. A total of twenty-four Sprague Dawley (SD) rats of the Specific-Pathogen-Free (SPF) grade were haphazardly assigned to one of 3 groups: conventional food + moxibustion, conventional food, and organic food. For 12 weeks, organic food was given to the organic category, and typical food was prepared for the conventional category of mices, and mices in the combination category received typical food for eight weeks and administered moxibustion at the Piyu and Zusanli points for another four weeks. Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) was used to assess alterations in intestinal flora. The content of intestinal microbiota differed significantly across the three groups, according to the similarity clustering analysis. In the combination group, the intestinal bacterial diversity index rose considerably (P < 0.05). Moxibustion at Zusanli and Piyu points enhanced Bacteroides stercoris and Barnesiella intestinihominis growth, according to DNA sequencing. It was concluded that moxibustion has a greater influence on intestinal bacterial diversity than an organic diet.