Salecan Enhances the Activities of β-1,3-Glucanase and Decreases the Biomass of Soil-Borne Fungi

<div><p>Salecan, a linear extracellular polysaccharide consisting of β-1,3-D-glucan, has potential applications in the food, pharmaceutical and cosmetic industries. The objective of this study was to evaluate the effects of salecan on soil microbial communities in a vegetable patch. Compositional shifts in the genetic structure of indigenous soil bacterial and fungal communities were monitored using culture-dependent dilution plating, culture-independent PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR. After 60 days, soil microorganism counts showed no significant variation in bacterial density and a marked decrease in the numbers of fungi. The DGGE profiles revealed that salecan changed the composition of the microbial community in soil by increasing the amount of <i>Bacillus</i> strains and decreasing the amount of <i>Fusarium</i> strains. Quantitative PCR confirmed that the populations of the soil-borne fungi <i>Fusarium oxysporum</i> and <i>Trichoderma</i> spp. were decreased approximately 6- and 2-fold, respectively, in soil containing salecan. This decrease in the amount of fungi can be explained by salecan inducing an increase in the activities of β-1,3-glucanase in the soil. These results suggest the promising application of salecan for biological control of pathogens of soil-borne fungi.</p></div

PCR primers and targeted microorganisms used in this study.

<p>^a Primers with a 40-bp GC clamp at the 5’ end.</p><p>^b Q-PCR primers.</p><p>PCR primers and targeted microorganisms used in this study.</p

Changes in soil bacteria community structure.

<p>(a) Bacterial colony-forming units assessed using the culture plating method. All values are the means ± SD (n = 3). (b) Similarity dendrogram of the bacterial banding patterns of soil samples. The scale for 0.44–1.00 was the similarity coefficient among the samples. DGGE fingerprints of 16S rDNA sequences amplified using the primer pair 357fGC/518r from soil-extracted community DNA. Lanes 1, 2 and 3 show the results of soil samples without salecan treatment (C); lanes 4, 5 and 6 show the results of 0.02% salecan-treated samples; and lanes 7, 8 and 9 show the results of 0.2% salecan-treated samples. Seven changed DGGE bands (B1, B2, B3, B4, B5, B6, B7) are marked by arrows. (c) The phylogenetic relationship between the 16S rRNA gene sequences of the soil samples and the related organisms from the GenBank database. The dendrogram was generated using the neighbor-joining method, with 1000 bootstrap resamplings. The scale bar represented 0.02, estimated nucleotide changes per sequence position.</p

Quantification of soil fungi.

<p>The relative abundances of the two fungal groups in soil samples treated with different concentrations of salecan, as estimated using qPCR assays. The results show DNA from <i>Fusarium oxysporum</i> (a) and <i>Trichoderma</i> spp. (b). All values are the means ± SD (n = 3). **<i>P</i> < 0.01 vs 0% group.</p

The chemical structure of the repeated unit of salecan.

<p>The chemical structure of the repeated unit of salecan.</p

β-1,3-Glucanase activity in soil samples treated with different amounts of salecan.

<p>The soil β-1,3-glucanase activity was defined as the amount of enzyme required to release 1 mg of glucose per day per gram soil (mg/d/g). All values are the means ± SD (n = 3). *<i>P</i> < 0.05 vs 0% group.</p

Changes in the soil fungus community structure.

<p>(a) Fungal colony-forming units assessed using the culture plating method. All values are the means ± SD (n = 3). *<i>P</i> < 0.05 vs 0% group. (b) Similarity dendrogram of the fungal banding patterns of the soil samples. The scale for 0.49–1.00 was the similarity coefficient among the samples. DGGE fingerprints of the 18S rDNA sequences amplified using the primer pair Fung-GC/NS1 from soil-extracted community DNA. Lanes 1, 2 and 3 show the results of soil samples treated without salecan (C); lanes 4, 5 and 6 show the results of 0.02% salecan-treated samples; and lanes 7, 8 and 9 show the results of 0.2% salecan-treated samples. Five changed DGGE bands (F1, F2, F3, F4, F5) are marked by vertical black lines. (c) The phylogenetic relationship between the 18S rRNA gene sequences from the soil samples and related organisms from the GenBank database. The dendrogram was generated using the neighbor-joining method with 1000 bootstrap resamplings. The scale bar represented 0.5, estimated nucleotide changes per sequence position.</p

Primer sequences for real-time RT-PCR analysis.

<p>Primer sequences for real-time RT-PCR analysis.</p

Daily changes of SAH concentration and SAM/SAH ratio in WT liver.

<p>One-way ANOVA showed that both (A) SAH and (B) SAM/SAH ratio displayed a clear diurnal variation (<i>p</i> < 0.01). Data represent means ± S.E.M. (n = 4). *<i>p</i> < 0.05, **<i>p</i> < 0.01 for LSD post hoc test compared with ZT17.</p

Circadian rhythmic parameters of 5-methylcytosine content, SAH concentration, SAM/SAH ratio and DNMT gene transcriptions in livers of WT and <i>Per1^-/-Per2^-/-</i> DKO mice.

<p>Data represent means ± S.E.M. (n = 3–4).</p><p>*<i>p</i> < 0.05,</p><p>**<i>p</i> < 0.01 compared with WT.</p><p>Circadian rhythmic parameters of 5-methylcytosine content, SAH concentration, SAM/SAH ratio and DNMT gene transcriptions in livers of WT and <i>Per1^-/-Per2^-/-</i> DKO mice.</p