Induction of plant disease resistance by mixed oligosaccharide elicitors prepared from plant cell wall and crustacean shells.

Basal plant immune responses are activated by the recognition of conserved microbe-associated molecular patterns (MAMPs), or breakdown molecules released from the plants after damage by pathogen penetration, so-called damage-associated molecular patterns (DAMPs). While chitin-oligosaccharide (CHOS), a primary component of fungal cell walls, is most known as MAMP, plant cell wall-derived oligosaccharides, cello-oligosaccharides (COS) from cellulose, and xylo-oligosaccharide (XOS) from hemicellulose are representative DAMPs. In this study, elicitor activities of COS prepared from cotton linters, XOS prepared from corn cobs, and chitin-oligosaccharide (CHOS) from crustacean shells were comparatively investigated. In Arabidopsis, COS, XOS, or CHOS treatment triggered typical defense responses such as reactive oxygen species (ROS) production, phosphorylation of MAP kinases, callose deposition, and activation of the defense-related transcription factor WRKY33 promoter. When COS, XOS, and CHOS were used at concentrations with similar activity in inducing ROS production and callose depositions, CHOS was particularly potent in activating the MAPK kinases and WRKY33 promoters. Among the COS and XOS with different degrees of polymerization, cellotriose and xylotetraose showed the highest activity for the activation of WRKY33 promoter. Gene ontology enrichment analysis of RNAseq data revealed that simultaneous treatment of COS, XOS, and CHOS (oligo-mix) effectively activates plant disease resistance. In practice, treatment with the oligo-mix enhanced the resistance of tomato to powdery mildew, but plant growth was not inhibited but rather tended to be promoted, providing evidence that treatment with the oligo-mix has beneficial effects on improving disease resistance in plants, making them a promising class of compounds for practical application.

Neoxanthin is undetectable in human blood after ingestion of fresh young spinach leaf.

In a previous study, we demonstrated that the carotenoid neoxanthin was contained in young leafy vegetables including spinach and showed a fat accumulation inhibitory effect in vitro. To evaluate the bioavailability of neoxanthin, a raw young spinach leaf (100 g day-1 for 4 weeks) intake test was performed on 14 participants (36.5 ± 8.0 years; male:female ratio = 9:5). Neoxanthin, neochrome, ß-carotene, and lutein concentration in the spinach and blood of participants (before and after the test) was measured using high performance liquid chromatography. Neither neoxanthin nor neochrome was detected in the blood samples, whereas ß-carotene and lutein concentration significantly increased (1.4- and 1.9-fold, respectively) during testing. Neoxanthin bioavailability in humans is low; thus, it is unlikely to have a fat accumulation inhibitory effect in vivo, contrary to the result in vitro. Ingesting the leafy vegetables raw can help maintain high neoxanthin levels, but it is not beneficial for neoxanthin bioavailability.

Botrytis cinerea tolerates phytoalexins produced by Solanaceae and Fabaceae plants through an efflux transporter BcatrB and metabolizing enzymes.

Botrytis cinerea, a plant pathogenic fungus with a wide host range, has reduced sensitivity to fungicides as well as phytoalexins, threatening cultivation of economically important fruits and vegetable crops worldwide. B. cinerea tolerates a wide array of phytoalexins, through efflux and/or enzymatic detoxification. Previously, we provided evidence that a distinctive set of genes were induced in B. cinerea when treated with different phytoalexins such as rishitin (produced by tomato and potato), capsidiol (tobacco and bell pepper) and resveratrol (grape and blueberry). In this study, we focused on the functional analyses of B. cinerea genes implicated in rishitin tolerance. LC/MS profiling revealed that B. cinerea can metabolize/detoxify rishitin into at least 4 oxidized forms. Heterologous expression of Bcin08g04910 and Bcin16g01490, two B. cinerea oxidoreductases upregulated by rishitin, in a plant symbiotic fungus Epichloë festucae revealed that these rishitin-induced enzymes are involved in the oxidation of rishitin. Expression of BcatrB, encoding an exporter of structurally unrelated phytoalexins and fungicides, was significantly upregulated by rishitin but not by capsidiol and was thus expected to be involved in the rishitin tolerance. Conidia of BcatrB KO (ΔbcatrB) showed enhanced sensitivity to rishitin, but not to capsidiol, despite their structural similarity. ΔbcatrB showed reduced virulence on tomato, but maintained full virulence on bell pepper, indicating that B. cinerea activates BcatrB by recognizing appropriate phytoalexins to utilize it in tolerance. Surveying 26 plant species across 13 families revealed that the BcatrB promoter is mainly activated during the infection of B. cinerea in plants belonging to the Solanaceae, Fabaceae and Brassicaceae. The BcatrB promoter was also activated by in vitro treatments of phytoalexins produced by members of these plant families, namely rishitin (Solanaceae), medicarpin and glyceollin (Fabaceae), as well as camalexin and brassinin (Brassicaceae). Consistently, ΔbcatrB showed reduced virulence on red clover, which produces medicarpin. These results suggest that B. cinerea distinguishes phytoalexins and induces differential expression of appropriate genes during the infection. Likewise, BcatrB plays a critical role in the strategy employed by B. cinerea to bypass the plant innate immune responses in a wide variety of important crops belonging to the Solanaceae, Brassicaceae and Fabaceae.

Draft Genome Sequences of Neofusicoccum parvum Strains Isolated from Mango and Rice-Paper Plant (Tetrapanax papyrifer).

Neofusicoccum parvum is a polyxenous phytopathogenic fungus that infects important fruits, such as grapes and mangoes. Here, we report the genome sequences of N. parvum strains that were isolated from mango in Okinawa, Japan (strain PPO83), and an invasive weed (rice-paper plant [Tetrapanax papyrifer]) in Nagoya, Japan (strain NSSI1).

Inter-Day Variation in the Fasting Plasma Lipopolysaccharide Concentration in the Morning Is Associated with Inter-Day Variation in Appetite in Japanese Males: A Short-Term Cohort Study.

Injection of lipopolysaccharide (LPS), a product of gut bacteria, into the blood increases blood triglycerides and cortisol, an appetite-stimulating hormone. Meanwhile, small amounts of LPS derived from gut bacteria are thought to enter the bloodstream from the gut in daily basis. This study aimed to investigate the effect of LPS influx on appetite or lipid metabolism in humans in everyday life. We measured the fasting plasma LPS concentration before breakfast and the corresponding days' appetite and fat-burning markers for 10 days in four Japanese males (28-31 years) and analyzed the correlation of their inter-day variation. The LPS concentration was negatively correlated with fullness, and positively correlated with the carbohydrate intake. Against our hypothesis, the LPS concentration was positively correlated with the fasting breath acetone concentration, a fat-burning marker. There was a positive correlation between the LPS concentration and fasting body mass index (BMI), but the inter-day variation in BMI was slight. The results suggest that the LPS influx in everyday life is at least associated with appetite in the day.

Draft Genome Sequences of Prototrophic and Biotin-Auxotrophic Fusarium langsethiae Strains Isolated from an Oat Grain in the Northern Region of Russia.

Fusarium langsethiae is a suspected plant-pathogenic fungus causing cereal contamination with trichothecene mycotoxins. Here, we report the genome sequences of two F. langsethiae strains, MFG217701 (a prototroph) and MFG217702 (a biotin auxotroph), isolated from a grain of oat harvested in Russia.

Botrytis cinerea identifies host plants via the recognition of antifungal capsidiol to induce expression of a specific detoxification gene.

The gray mold pathogen Botrytis cinerea has a broad host range, causing disease in >400 plant species, but it is not known how this pathogen evolved this polyxenous nature. Botrytis cinerea can metabolize a wide range of phytoalexins, including the stilbenoid resveratrol in grape, and the sesquiterpenoids capsidiol in tobacco and rishitin in potato and tomato. In this study, we analyzed the metabolism of sesquiterpenoid phytoalexins by B. cinerea. Capsidiol was dehydrogenated to capsenone, which was then further oxidized, while rishitin was directly oxidized to epoxy- or hydroxyrishitins, indicating that B. cinerea has separate mechanisms to detoxify structurally similar sesquiterpenoid phytoalexins. RNA-seq analysis revealed that a distinct set of genes were induced in B. cinerea when treated with capsidiol or rishitin, suggesting that B. cinerea can distinguish structurally similar phytoalexins to activate appropriate detoxification mechanisms. The gene most highly upregulated by capsidiol treatment encoded a dehydrogenase, designated Bccpdh. Heterologous expression of Bccpdh in a capsidiol-sensitive plant symbiotic fungus, Epichloë festucae, resulted in an acquired tolerance of capsidiol and the ability to metabolize capsidiol to capsenone, while B. cinerea Δbccpdh mutants became relatively sensitive to capsidiol. The Δbccpdh mutant showed reduced virulence on the capsidiol producing Nicotiana and Capsicum species but remained fully pathogenic on potato and tomato. Homologs of Bccpdh are found in taxonomically distant Ascomycota fungi but not in related Leotiomycetes species, suggesting that B. cinerea acquired the ancestral Bccpdh by horizontal gene transfer, thereby extending the pathogenic host range of this polyxenous pathogen to capsidiol-producing plant species.

Omnipresence of Partitiviruses in Rice Aggregate Sheath Spot Symptom-Associated Fungal Isolates from Paddies in Thailand.

Partitiviruses are one of the most prevalent double-stranded RNA viruses that have been identified mostly in filamentous fungi and plants. Partitiviruses generally infect host fungi asymptomatically but infrequently exert significant effect(s) on morphology and virulence, thus being considered a potential source of biological control agents against pathogenic fungi. In this study, we performed a screening for mycoviruses of a collection of Thai isolates of rice fungal pathogen Rhizoctonia oryzae-sativae, a causal agent of rice aggregated sheath spot disease. As a result, 36% of tested isolates carried potentially viral double-stranded RNAs with sizes ranging from 2 to 3 kbp. By conventional cDNA library construction and RNA-seq, we determined six new alphapartitiviruses that infected three isolates: tentatively named Rhizoctonia oryzae-sativae partitivirus 1 to 6 (RosPV1-6). Furthermore, RT-PCR detection of each virus revealed their omnipresent nature in different R. oryzae-sativae isolates. Although virus-curing of basidiomycetous fungi is generally difficult, our repeated attempts successfully obtained virus-free (for RosPV1, RosPV2, and uncharacterized partitiviruses), isogenic strain of R. oryzae-sativae TSS190442. The virus-cured strain showed slightly faster colony growth on the synthetic media and severe symptom development on the rice sheath compared to its virus-infected counterpart. Overall, this study shed light on the distribution of partitiviruses in R. oryzae-sativae in a paddy environment and exemplified a virus-curing protocol that may be applicable for other basidiomycetous fungi.

Geographic Distribution, Genetic Variability and Biological Properties of Rice Orange Leaf Phytoplasma in Southeast Asia.

Rice orange leaf phytoplasma (ROLP) causes clear orange to yellowish leaf discoloration and severe stunting in rice seedlings. The ecological and biological characteristics of ROLP are largely unknown because the disease has not widely caused serious problems in rice cultivated areas, thereby leading to the low accumulation of research data. However, in the past decade, the disease became a threat to rice production, particularly in South China and India; it has also been recognised in other Asian countries, such as Vietnam, Thailand and the Philippines. Here, we observed the occurrence of ROLP in paddies of the Southeast Asian counties (Cambodia, Vietnam and the Philippines) and found that the isolates in the Philippines and Vietnam were monophyletic, while those in India, Thailand and Cambodia were more diverse, suggesting their potential origins. In Cambodia, it was revealed that following polymerase chain reaction (PCR) detection, the known ROLP-insect vectors, N. virescens Distant and Recilia dorsalis Motchulsky, were ROLP-positive, indicating their roles in pathogen dispersal. Moreover, fluorescent and scanning electron microscopy revealed the intensive accumulation of the phytoplasma in phloem tissues and massive accumulation of storage starch in vascular bundle sheath and parenchyma. Altogether, this study illustrated the genetic variability of global ROLP isolates and the pathogen's biological impact on rice tissue.

AP2/ERF Transcription Factor NbERF-IX-33 Is Involved in the Regulation of Phytoalexin Production for the Resistance of Nicotiana benthamiana to Phytophthora infestans.

Plants recognize molecular patterns unique to a certain group of microbes to induce effective resistance mechanisms. Elicitins are secretory proteins produced by plant pathogenic oomycete genera including Phytophthora and Pythium. Treatment of INF1 (an elicitin produced by P. infestans) induces a series of defense responses in Nicotiana species, including reactive oxygen species (ROS) production, transient induction of ethylene production, hypersensitive cell death and accumulation of the sesquiterpenoid phytoalexin capsidiol. In this study, we analyzed the expression profiles of N. benthamiana genes after INF1 treatment by RNAseq analysis. Based on their expression patterns, N. benthamiana genes were categorized into 20 clusters and 4,761 (8.3%) out of 57,140 genes were assigned to the clusters for INF1-induced genes. All genes encoding enzymes dedicated to capsidiol production, 5-epi-aristolochene (EA) synthase (NbEAS, 10 copies) and EA dehydrogenase (NbEAH, 6 copies), and some genes for ethylene production, such as 1-aminocyclopropane 1-carboxylate (ACC) synthase (NbACS) and ACC oxidase (NbACO), were significantly upregulated by INF1 treatment. Analysis of NbEAS1 and NbEAS4 promoters revealed that AGACGCC (GCC box-like motif) is the essential cis-element required for INF1-induced expression of NbEAS genes. Given that the GCC box is known to be targeted by ERF (ethylene-responsive factor) transcription factors, we created a complete list of N. benthamiana genes encoding AP2/ERF family transcription factors, and identified 45 out of 337 AP2/ERF genes in the clusters for INF1-induced genes. Among INF1-induced NbERF genes, silencing of NbERF-IX-33 compromised resistance against P. infestans and INF1-induced production of capsidiol. Recombinant NbERF-IX-33 protein can bind to the promoter sequence of NbEAS4, suggesting that NbERF-IX-33 is a transcription factor directly regulating the expression of genes for phytoalexin production.

Safety and efficacy of manual vacuum suction compared with conventional dilatation and sharp curettage and electric vacuum aspiration in surgical treatment of miscarriage: a randomized controlled trial.

BACKGROUND: The World Health Organization does not recommend dilatation and sharp curettage (D&C) for the surgical treatment of miscarriage during the first trimester because this may cause Asherman's syndrome due to endometrial damage; therefore, suction remains the primary treatment option. While manual vacuum aspiration (MVA) has been widely used since the 1990s outside Japan, the use of an MVA device (Women's MVA system) was approved in Japan in October 2015. Here, we examined the efficacy of the MVA kit in women surgically treated for miscarriage. METHODS: This retrospective cohort study was conducted between 2014 and 2018 at the International University of Health and Welfare Hospital in Japan. Women who underwent surgical treatment for miscarriage within 12 weeks of pregnancy were identified and enrolled in the study. A total of 404 women were included who underwent the following procedures: 121 D&C, 123 electric vacuum aspiration (EVA), and 160 MVA. For each participant, the duration of surgery, amount of bleeding, amount of anesthetic used, incomplete abortion requiring repeat procedures, and intraoperative/postoperative complications were evaluated. RESULTS: The duration of surgery was 13.7 ± 7.2, 11.2 ± 4.2, and 6.9 ± 4.3 min in the D&C, EVA, and MVA groups, respectively (p = 1.00). The amount of anesthetic used was not significantly different among all groups. Bleeding of ≥ 100 mL was confirmed in three (2.4%), one (0.8%), and one (0.6%) patient(s) in the D&C, EVA, and MVA groups, respectively (p = 0.50). Incomplete abortion was identified in three (2.4%), two (1.6%), and one (0.6%) patient(s) in the D&C, EVA, and MVA groups, respectively (p = 0.61). However, severe intraoperative/postoperative complications were not observed in any group. CONCLUSIONS: Surgical treatment for miscarriage performed using the MVA kit has safety and efficacy similar to those of conventional methods, such as D&C and EVA.

In Vitro Assessment of Biocontrol Effects on Fusarium Head Blight and Deoxynivalenol (DON) Accumulation by DON-Degrading Bacteria.

Fusarium head blight (FHB) of cereals is a severe disease caused by the Fusarium graminearum species complex. It leads to the accumulation of the mycotoxin deoxynivalenol (DON) in grains and other plant tissues and causes substantial economic losses throughout the world. DON is one of the most troublesome mycotoxins because it is a virulence factor to host plants, including wheat, and exhibits toxicity to plants and animals. To control both FHB and DON accumulation, a biological control approach using DON-degrading bacteria (DDBs) is promising. Here, we performed a disease control assay using an in vitro petri dish test composed of germinated wheat seeds inoculated with F. graminearum (Fg) and DDBs. Determination of both grown leaf lengths and hyphal lesion lengths as a measure of disease severity showed that the inoculation of seeds with the DDBs Devosia sp. strain NKJ1 and Nocardioides spp. strains SS3 or SS4 were protective against the leaf growth inhibition caused by Fg. Furthermore, it was as effective against DON accumulation. The inoculation with strains SS3 or SS4 also reduced the inhibitory effect on leaves treated with 10 µg mL-1 DON solution (without Fg). These results indicate that the DDBs partially suppress the disease by degrading DON.

Cutaneous Carotenoid Level Measured by Multiple Spatially Resolved Reflection Spectroscopy Sensors Correlates with Vegetable Intake and Is Increased by Continual Intake of Vegetable Juice.

Although vegetables are beneficial for human health, in many countries, the recommended vegetable intake is not reached. To assess vegetable intake, it is important to understand vegetable consumption. Therefore, we conducted a cross-sectional and intervention study of 26 healthy individuals (50% women; 37.0 ± 8.9 years) and estimated vegetable intake on the basis of the cutaneous carotenoid level (CCL) with a noninvasive skin carotenoid sensor, considering that vegetable juice intake can increase CCL. Participants consumed vegetable juice containing 350 g of vegetables daily for 4 weeks. Blood carotenoid levels and CCL were measured for 12 weeks. Cross-sectional analysis showed a significant positive correlation between CCL and vegetable intake (r = 0.489). Vegetable juice consumption significantly increased CCL and the blood levels of α-carotene, ß-carotene, and lycopene (p < 0.05). The correlation coefficient between the blood level and CCL for lycopene was smaller (r = 0.001) compared to that between the blood level and CCL for α-carotene (r = 0.523) and ß-carotene (r = 0.460), likely because of bioavailability differences. In conclusion, noninvasive skin carotenoid measurements are effective for determining vegetable intake, and vegetable juice significantly increases CCL.

Draft Genome Sequence of Deoxynivalenol-Degrading Actinomycete Nocardioides sp. Strain LS1, Isolated from Wheat Leaves in Japan.

Actinomycete Nocardioides sp. strain LS1, isolated from wheat leaf, is a bacterium that degrades and assimilates the mycotoxin deoxynivalenol (DON) as the carbon source. This is the first study of the genome sequence of the DON-degrading genus Nocardioides, and it facilitates the study of genes encoding the DON-degrading pathway.

Nicotiana benthamiana RanBP1-1 Is Involved in the Induction of Disease Resistance via Regulation of Nuclear-Cytoplasmic Transport of Small GTPase Ran.

Plant cells enhance the tolerances to abiotic and biotic stresses via recognition of the stress, activation and nuclear import of signaling factors, up-regulation of defense genes, nuclear export of mRNA and translation of defense proteins. Nuclear pore-mediated transports should play critical roles in these processes, however, the regulatory mechanisms of nuclear-cytoplasmic transport during stress responses are largely unknown. In this study, a regulator of nuclear export of RNA and proteins, NbRanBP1-1 (Ran-binding protein1-1), was identified as an essential gene for the resistance of Nicotiana benthamiana to potato blight pathogen Phytophthora infestans. NbRanBP1-1-silenced plants showed delayed accumulation of capsidiol, a sesquiterpenoid phytoalexin, in response to elicitor treatment, and reduced resistance to P. infestans. Abnormal accumulation of mRNA was observed in NbRanBP1-1-silenced plants, indicating that NbRanBP1-1 is involved in the nuclear export of mRNA. In NbRanBP1-1-silenced plants, elicitor-induced expression of defense genes, NbEAS and NbWIPK, was not affected in the early stage of defense induction, but the accumulation of NbWIPK protein was reduced. Nuclear export of the small G-protein NbRan1a was activated during the induction of plant defense, whereas this process was compromised in NbRanBP1-1-silenced plants. Silencing of genes encoding the nuclear pore proteins, Nup75 and Nup160, also caused abnormal nuclear accumulation of mRNA, defects in the nuclear export of NbRan1a, and reduced production of capsidiol, resulting in decreased resistance to P. infestans. These results suggest that nuclear export of NbRan is a key event for defense induction in N. benthamiana, and both RanBP1-1 and nucleoporins play important roles in the process.

EIN2-mediated signaling is involved in pre-invasion defense in Nicotiana benthamiana against potato late blight pathogen, Phytophthora infestans.

Nicotiana benthamiana ABCG1 and ABCG2 are ABC transporters which are probably involved in the export of capsidiol, the major phytoalexin of Nicotiana species. While capsidiol export by these transporters plays an essential role in post-invasion defense against Phytophthora infestans, they also export unidentified antimicrobial compound(s) involved in pre-invasion defense. In this study, promoter activity of NbABCG2 (Pabcg2a) was analyzed using a GFP marker. Expression of GFP under the control of Pabcg2a was significantly increased by co-expression with the INF1 elicitor from P. infestans. Disruption of the ethylene-responsive GCC box in Pabcg2a compromised INF1-induced activation of Pabcg2a. Consistently, penetration by P. infestans was increased by gene-silencing of NbEIN2, the key ethylene-signaling component, suggesting the involvement of ethylene for pre-invasion defense of N. benthamiana.

The effects of tomato juice on male infertility.

BACKGROUND AND OBJECTIVES: This study aimed to investigate the effects of tomato juice consumption on seminal plasma lycopene levels and sperm parameters in infertile men. METHODS AND STUDY DESIGN: Subjects were male infertility patients with poor sperm concentration (<20×10 6/mL) and/or motility (<50%). Following a fourweek observation period, subjects were randomly assigned among three groups: a tomato juice group, an antioxidant group, and a control group. The subjects in the tomato juice group and the antioxidant group daily consumed one can of tomato juice (containing 30 mg of lycopene) or one antioxidant capsule (containing vitamin C 600 mg, vitamin E 200 mg, and glutathione 300 mg), respectively, for 12 weeks (feeding period). Seminal plasma lycopene levels and sperm parameters were measured every 6 weeks during the feeding period. RESULTS: Forty-four patients completed the study (control group: 12, antioxidant group: 15, tomato juice group: 17). In the tomato juice group, plasma lycopene level was significantly increased at the 12th week of the feeding period. Moreover, a decrease in seminal plasma white blood cells and an increase in sperm motility in the tomato juice group were statistically significant at the 12th and 6th weeks, respectively, compared to the control group. In the antioxidant capsule group, no significant improvement was observed in semen parameters. CONCLUSIONS: In conclusion, regular consumption of tomato juice seems to improve sperm motility in infertile patients. This is the first report to show that commercially available food, such as tomato juice, might be beneficial for male infertility.

Simultaneous bioethanol distillery wastewater treatment and xylanase production by the phyllosphere yeast Pseudozyma antarctica GB-4(0).

Bioethanol production using lignocellulosic biomass generates lignocellulosic bioethanol distillery wastewater (LBDW) that contains a large amount of xylose, making it a potential inexpensive source of xylose for biomaterials production. The main goal of this study was the production of useful enzymes from LBDW during treatment of this wastewater. In this study, we found that xylose strongly induced two yeast strains, Pseudozyma antarctica T-34 and GB-4(0), to produce novel xylanases, PaXynT and PaXynG, respectively. The nucleotide sequence of PaXynT [accession No. DF196774 (GAC73192.1)], obtained from the genome database of strain T-34 using its N-terminal amino acid sequence, was 91% identical to that of PaXynG (accession No. AB901085), and the deduced amino acid sequence is 98% identical. The specific activities of the purified PaXynT and PaXynG were about 52 U/mg. The optimal pH and temperature for both enzymes' activities were 5.2 and 50°C, respectively. They hydrolyzed xylan to xylose and neither had ß-xylosidase (EC 3.2.1.37) activity, indicating that they are endo-ß-xylanases (EC 3.2.1.8). With these results, we expect that PaXyns can be employed in saccharizing lignocellulosic biomass materials for the production of useful products just like other endoxylanases. After 72 h of LBDW fed-batch cultivation using a jar-fermentor, strain GB-4(0) produced 17.3 U/ml (corresponding to about 0.3 g/l) of PaXynG and removed 63% of dissolved organic carbon and 87% of dissolved total phosphorus from LBDW. These results demonstrate the potential of P. antarctica for xylanase production during LBDW treatment.

Analysis of variations in band positions for normalization in across-gel denaturing gradient gel electrophoresis.

Variation in band position between gels is a well-known problem in denaturing gradient gel electrophoresis (DGGE). However, few reports have evaluated the degree of variation in detail. In this study, we investigated the variation in band positions of DNA samples extracted from soil, normalized using reference positions within marker lanes for DGGE in three organismal (bacterial, fungal, and nematode) conditions. For sample lanes, marker DNA (as a control) and sample DNA were used. The test for normality of distribution showed that the position data of a large percentage of bands were normally distributed but not for certain bands. For the normally-distributed data, their variations [standard deviation of marker bands (SDM) and standard deviation of sample bands (SDS), respectively] were assessed. For all organismal conditions, the degree of within-gel variation were similar between SDMs and SDSs, while between-gel variations in SDSs were larger than those in SDMs. Due to the large effect of between-gel variations, the total variations in SDSs were more varied between sample bands, and the mean variations of all sample bands were higher than those in the markers. We found that the total variation in the fungal and nematode SDSs decreased when the intervals between marker bands were narrowed, suggesting that band interval is important for reducing total variation in normalized band positions. For the non-normally distributed data, the distribution was examined in detail. This study provided detailed information on the variation of band positions, which could help to optimize markers for reducing band position variation, and could aid in the accurate identification of bands in across-gel DGGE analyses.