Max Cv Research Articles

Biochar improved nodulation and nitrogen metabolism of soybean under salt stress

To investigate salt stress and biochar application effects on nodulation and nitrogen metabolism of soybeans (Glycine max cv. M7), an experiment was conducted under the control condition. The treatments comprised three biochar rates (non, 50 and 100 g kg−1 soil) and three salinities (0, 5 and 10 dS m−1 NaCl), with four replications of treatments. Salt stress diminished the number of nodules and their weights in the soybean roots. Nitrogen content and metabolism decreased in nodules, roots and shoots, while reducing the activity of glutamate dehydrogenase (GDH), glutamine synthetase (GS), glutamine oxoglutarate aminotransferase (GOGAT) and nitrate reductase (NR). Also, salinity brought down root and shoot weight, total plant biomass, chlorophyll content, leaf area (LA) and rubisco activity in the soybean. On the other hand, application of biochar improved nodulation, nitrogen content, rubisco activity, GDH, GS, GOGAT and NR activities in different parts of the soybean and nodules under salt stress, and consequently improved chlorophyll content, LA, root and shoot weight. Both the 50 and 100 g kg−1 biochar rates showed similar effects in improving nitrogen metabolism and plant performance under salt stress. Generally, biochar increased nodulation and nitrogen metabolism of the soybean under saline conditions.

Analysis of Herbivore Stress- and Phytohormone-Mediated Urease Expression in Soybean (Glycine max)

Ureases catalyze the hydrolysis of urea into ammonia and carbon dioxide and, thus, are involved in the metabolism and bioavailability of nitrogen. Ureases occur in plants, fungi, and bacteria. In plants, besides their enzymatic activity, ureases as proteins play a role in defense against insect and phytopathogenic fungi. Little is known about the regulation of urease in plants under stress and whether or not phytohormones may be involved. In this study, we addressed the regulation of ubiquitous urease (ubSBU) gene expression after phytohormone applications, insect herbivory, and mechanical damage in soybean (Glycine max cv. Williams 82). Stress-related phytohormones were applied. In addition, Spodoptera littoralis feeding and mechanical damage by MecWorm were performed. Ureolytic activity and transcripts for ubSBU and UreG were quantified. Roots and leaves showed the highest levels of ubSBU transcripts. The results show a significant increase of ubSBU transcripts upon jasmonic acid application and after herbivory, but downregulation after MecWorm treatment. UreG transcripts were downregulated after MecWorm, S. littoralis, and application of gibberellic acid, but upregulated by jasmonic acid. However, the ureolytic activities in leaves were influenced neither by phytohormones nor by herbivory and MecWorm. We conclude that the enzymatic activity of ureases is constitutive and basal levels of the enzyme are sufficient to perform the ureolytic activities in defense against insects and fungi. The defense role of ureases, which does not require the ureolytic activity, may underlie their differential regulation in response to different stress stimuli.

Response of soybean plants to the application of synthetic and biodegradable Fe chelates and Fe complexes

The growing concern over the environmental risk of synthetic chelate application promotes the search for alternatives in Fe fertilization, such as biodegradable chelating agents and natural complexing agents. In this work, plant responses to the application of several Fe treatments (chelates and complexes) was analyzed to study their potential use in Fe fertilization under calcareous conditions. Thus, the root ferric chelate reductase (FCR) activity of soybean (Glycine max cv. Klaxon) plants was determined, and the effectiveness of the Fe chelates and complexes assessed in a pot experiment, by SPAD and fluorescence induction measurements, and the determination of Fe distribution in plant and soil. Additionally, 57Fe Mössbauer spectroscopy was conducted to identify the Fe forms present in the soybean roots. The highest FCR activity was observed for the chelates EDDS/Fe3+ and IDHA/Fe3+; while no activity was observed when using complexes as Fe substrates. In contrast to the FCR data, the pot experiment confirmed that the o,oEDDHA/Fe3+ is the most effective treatment, and the complexes LS/Fe3+ and GA/Fe3+ are able to alleviate Fe chlorosis, also indicated by SPAD data and the maximal quantum efficiency of photosystem II reaction centers as vitality parameters, and the enhanced plant uptake of Fe from natural sources.

Aureobasidium pullulans produced \u03b2-glucan is effective to enhance Kurosengoku soybean extract induced Thrombospondin-1 expression

Black yeast, Aureobasidium pullulans is extracellularly produced β-(1,3), (1,6)-D-glucan (β-glucan) under certain conditions. In this study, using Glycine max cv. Kurosengoku (Kurosengoku soybeans), the production of β-glucan through fermentation of A. pullulans was evaluated, and the effects of A. pullulans cultured fluid (AP-CF) containing β-glucan made with Kurosengoku soybeans (kAP-CF) on a human monocyte derived cell line, Mono Mac 6 cells were investigated. Concentration of β-glucan in kAP-CF reached the same level as normal AP-CF. An anti-angiogenic protein, Thrombospondin-1 (THBS1) was effectively induced after the stimulation with kAP-CF for comparison with AP-CF. The THBS1 is also induced after stimulation with hot water extract of Kurosengoku soybeans (KS-E), while the combined stimulation of β-glucan with KS-E more effectively induced THBS1 than that with KS-E alone. These results suggest effects of A. pullulans-produced β-glucan on the enhancement of Kurosengoku soybean-induced THBS1 expression.

Physiological responses of three soybean species (Glycine soja, G. gracilis, and G. max cv. Melrose) to salinity stress.

Understanding the mechanism for salt tolerance in wild soybean (Glycine soja) can help researchers improve that trait in cultivated soybean lines. We analyzed the effects of excess NaCl on the growth, physiology, and ion distribution in three soybean species: wild G. soja (W8), semi-wild G. gracilis (SW18), and the cultivated salt-sensitive G. max (cv. Melrose). These comparisons revealed that, under salt stress, shoot and root lengths and biomass (either shoot or root dry weights) were significantly higher for the W8 genotype than for the other two. Most of the morphological parameters for roots from the W8 plants were also increased, including total length, specific root length, and surface area. However, the average root diameter for W8 was significantly lower than that of either SW18 or 'Melrose' soybeans. In response to salinity, photosynthesis was suppressed to a greater extent in 'Melrose' than in W8. The relatively higher tolerance shown by W8 plants was also associated with lower levels of malondialdehyde, hydrogen peroxide, and relative electrical conductivity, but higher activities by superoxide dismutase and peroxidase, as well as more free proline and glycine betaine. In addition, the W8 plants contained less Na+ and Cl-, but more K+, and they had a higher K+/Na+ ratio in their leaves and roots when compared with either SW18 or 'Melrose' plants. Therefore, the W8 genotype performs better in terms of seedling growth, photosynthetic characteristics, and physiological indexes. These findings provide guidance for developing new soybean cultivars with improved tolerance to salt stress. Our data also contribute to the knowledge base for plant salt tolerance as a tool for increasing the yields of other crops in high-salinity soils.

Pathogenic races of Phytophthora sojae in Glycine max cv. Tanbakuro producing areas in Hyogo and selection of parental soybeans for breeding new resistant cultivars

Pathogenic races of Phytophthora sojae in Glycine max cv. Tanbakuro producing areas in Hyogo and selection of parental soybeans for breeding new resistant cultivars

Enhancing soybean photosynthetic CO2 assimilation using a cyanobacterial membrane protein, ictB

Soybean C3 photosynthesis can suffer a severe loss in efficiency due to photorespiration and the lack of a carbon concentrating mechanism (CCM) such as those present in other plant species or cyanobacteria. Transgenic soybean (Glycine max cv. Thorne) plants constitutively expressing cyanobacterial ictB (inorganic carbon transporter B) gene were generated using Agrobacterium-mediated transformation. Although more recent data suggest that ictB does not actively transport HCO3-/CO2, there is nevertheless mounting evidence that transformation with this gene can increase higher plant photosynthesis. The hypothesis that expression of the ictB gene would improve photosynthesis, biomass production and seed yield in soybean was tested, in two independent replicated greenhouse and field trials. Results showed significant increases in photosynthetic CO2 uptake (Anet) and dry mass in transgenic relative to wild type (WT) control plants in both the greenhouse and field trials. Transgenic plants also showed increased photosynthetic rates and biomass production during a drought mimic study. The findings presented herein demonstrate that ictB, as a single-gene, contributes to enhancement in various yield parameters in a major commodity crop and point to the significant role that biotechnological approaches to increasing photosynthetic efficiency can play in helping to meet increased global demands for food.

Proteomics Changes in Filling Seeds of Vegetable Soybean

Vegetable soybean is an important economic and nutritious crop. In this study, 48 differentially expressed proteins were identified from filling seeds of soybean (Glycine max) cv. Mindou 6 by using two-dimensional electrophoresis (2-DE) combined with liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Among them, 25% were related to protein destination and storage, 42% to energy and metabolism, 15% to disease/defense, 6% to transporters, 4% to secondary metabolism, 4% to transcription, 2% to protein synthesis, and 2% to cell growth/division. Along with the maturity of seeds, the number of unchanged abundance proteins decreased, while that of both upregulated and downregulated proteins increased. Both downregulated expression of caffeic acid O-methyltransferase (COMT) and upregulated expression of sucrose-binding protein (SBP) 2 precursor may contribute to increase in digestibility, nutritional value, and eating quality of vegetative seeds at suitable picking period. The pattern of unchanged proteins during the whole seed-filling stage may be also beneficial to the quality of vegetable soybean.

Characterization of Biochars and Their Use as an Amendment to Acid Soils

Biochar, because of its porous nature, calcium carbonate (CaCO3) equivalent, surface functional groups, and other properties, could serve as an acid soil amendment. To investigate the liming potential of biochars, laboratory characterization and greenhouse and field experiments were conducted in Hawaii and West Java, Indonesia, respectively. Six wood-derived biochars were characterized and amended to a Hawaiian acid soil (pH 4.6, exchangeable aluminum [Al] 1.8 cmolc kg) at 2% and 4% alone or in combination with 2 cmolc kg of lime and then planted with Desmodium intortum (a forage legume sensitive to Al) twice in a greenhouse trial. To the Indonesian acid soils (pH 3.9–4.0, exchangeable Al 8–14 cmolc kg), a rice husk and a lac tree biochars at 4% and 8% alone or in combination with lime at 4 and 8 cmolc kg and compost at 0.1 and 0.2% were applied and then planted with soybean (Glycine max) cv. Anjasmoro twice in field trials. Biochar effects on soil properties and plant growth were measured. The results indicated that the six biochars varied in pH, ash content, CaCO3 equivalent, total basic cations, cation exchange capacity, and other properties (pore size, surface functional groups). Soil pH was increased, soil exchangeable Al was reduced, and plant nutrients were enriched to different degrees upon additions of biochars. Total dry weights of Desmodium were increased 2- to 4-fold over the control or lime treatment upon applications of biochar. Similar effects on soils and soybean were obtained for the Indonesian field trials. It was concluded that CaCO3 equivalent and total basic cations were among the most important properties of biochar responsible for improving acid soil productivity and plant growth.

The involvement of peroxidases in soybean seedlings’ defense against infestation of cowpea aphid

Changes in the level of hydrogen peroxide (H2O2) and activity of peroxidases towards phenolic substrates (EC 1.11.1.7) such as pyrogallol (PPX), syringaldazine (SPX) and guaiacol (GPX), and cytosolic ascorbate peroxidase (cAPX, EC 1.11.1.11) in response to infestation of cowpea aphid (Aphis craccivora Koch) were analyzed in soybean (Glycine max (L.) Merr. cv. “Nam Dan”) at the V3 stage (first two trifoliate leaves fully developed, third trifoliate leaf unrolled) for 96 h post-infestation (hpi). Influence of A. craccivora at a varied population size (10, 20 and 30 individuals per each soybean plant) caused a burst of H2O2 generation in the aphid-infested leaves at 12 hpi. Paralleling the H2O2 accumulation, peroxidase activity in all the infested plants remarkably increased and was significantly higher than that observed in controls (uninfested plants). The cascade of enzymes induced was continuously overlapped by the early enhancement of SPX within 6–24 hpi, an expression of cAPX (12–48 hpi) followed by an accumulation of GPX (24–72 hpi) and PPX (24–96 hpi). The differential induction of SPX, GPX, PPX and cAPX resulted in a rapid reduction of H2O2 content in aphid-infested leaves, and the activity of peroxidase was closely correlated with the intensity of A. craccivora infestation around the defined points of time at which the activity of each enzyme reached the maximum level. The increase in activity of peroxidases matched their function as controlling accumulation of H2O2 and detoxifying this reactive oxygen product when soybean plants were challenged with cowpea aphid. Furthermore, peroxidases could directly deter cowpea aphid feeding through other functions such as the anti-nutritive and/or toxicological defenses and/or limiting the penetration of aphid stylets into plant tissues via participating to strengthen and reinforce the cell wall barrier. These results indicated that peroxidases may be some elements of the defense system that increased the resistance of G. max cv. “Nam Dan” to infestation of A. craccivora.

English

The purpose of this research was to assess the crop rotation affects on agronomic traits of soybean in optimal (OCS) and dry cropping seasons (DCS). This research was carried out in 2010/2011 (OCS) and 2011/2012 (DCS) in a Rhodic Hapludox soil. The experimental design was set up in a randomized complete block and the treatments were arranged in a factorial 9 x 2, consisting of 9 crop rotations systems and two cropping seasons, with four replications. The soybean (Glycine max cv. BMX-Potencia RR) was sowed on October 20 th 2010 and October 10 th 2011 (spring-summer season) 20 days after the fall-winter cover crops desiccation. It was assessed in the R8 reproductive stage (Full maturation) the plant height, the height of the first pod insertion, number of pod per plants, number of branches per plant, number of seed per pod, 1000-grain weight and grain yield. It was observed that in drought stress (DCS), the number of branches decreased, this reduction was 36% in relation to OCS. For the traits as plant height, first pod height, 1000-grain weight and yield, it was showed small values in DCS in contrast to OCS. It is feasible to introduce these cover crops in fall-winter season to make part of a crop rotation system. The number of pod per plant showed higher values under the crop rotation of corn/rapeseed/soybean, which showed highly associated with the soybean grain yield. This research brought options for cover crops system to be viable in no-till system with soybean in spring-summer season.

Aerodynamic consequences of wing morphing during emulated take-off and gliding in birds.

Birds morph their wings during a single wingbeat, across flight speeds and among flight modes. Such morphing may allow them to maximize aerodynamic performance, but this assumption remains largely untested. We tested the aerodynamic performance of swept and extended wing postures of 13 raptor species in three families (Accipitridae, Falconidae and Strigidae) using a propeller model to emulate mid-downstroke of flapping during take-off and a wind tunnel to emulate gliding. Based on previous research, we hypothesized that (1) during flapping, wing posture would not affect maximum ratios of vertical and horizontal force coefficients (CV:CH), and that (2) extended wings would have higher maximum CV:CH when gliding. Contrary to each hypothesis, during flapping, extended wings had, on average, 31% higher maximum CV:CH ratios and 23% higher CV than swept wings across all biologically relevant attack angles (α), and, during gliding, maximum CV:CH ratios were similar for the two postures. Swept wings had 11% higher CV than extended wings in gliding flight, suggesting flow conditions around these flexed raptor wings may be different from those in previous studies of swifts (Apodidae). Phylogenetic affiliation was a poor predictor of wing performance, due in part to high intrafamilial variation. Mass was only significantly correlated with extended wing performance during gliding. We conclude that wing shape has a greater effect on force per unit wing area during flapping at low advance ratio, such as take-off, than during gliding.

Simulation of CO2 enrichment and climate change impacts on soybean production

Abstract The potential doubling of atmospheric CO2 concentration and associated changes in temperature and precipitation are crucial issues for agricultural productivity. The CROPGRO-Soybean model in decision support system for agro-technology transfer v4.5 to simulate soybean (Glycine max cv. Pioneer 93B15) grown in an elevated CO2 environment was calibrated and validated. Crop growth and yield data were obtained from a series of experiments conducted in central Illinois at the soybean free air CO2 enrichment facility from 2002 to 2006. The model was applied to simulate the possible impacts of climate change on soybean yield in the region for the future years of 2080-2100, centred on 2090. The model reproduced the measured soybean growth and yield well under ambient and elevated CO2 conditions. For the period from 2081 to 2100, soybean yield was projected to decrease due to elevated temperature but to increase due to elevated precipitation and CO2 concentration, achieving counterbalance. The adverse impacts of the warming conditions on soybean yield can be mitigated by late planting within an optimum planting range (day of year 145 to 152) as a management option, as well as by controlling genetic responses to thermal days in the reproductive stage.

EFEITOS DE DIFERENTES CONCENTRAÇÕES DE COBALTO ASSOCIADAS A MOLIBDÊNIO NO DESENVOLVIMENTO RADICULAR E PRODUÇÃO DA SOJA ‘BR-133’

EFEITOS DE DIFERENTES CONCENTRAÇÕES DE COBALTO ASSOCIADAS A MOLIBDÊNIO NO DESENVOLVIMENTO RADICULAR E PRODUÇÃO DA SOJA ‘BR-133’

NopC Is a Rhizobium-Specific Type 3 Secretion System Effector Secreted by Sinorhizobium (Ensifer) fredii HH103.

Sinorhizobium (Ensifer) fredii HH103 is a broad host-range nitrogen-fixing bacterium able to nodulate many legumes, including soybean. In several rhizobia, root nodulation is influenced by proteins secreted through the type 3 secretion system (T3SS). This specialized secretion apparatus is a common virulence mechanism of many plant and animal pathogenic bacteria that delivers proteins, called effectors, directly into the eukaryotic host cells where they interfere with signal transduction pathways and promote infection by suppressing host defenses. In rhizobia, secreted proteins, called nodulation outer proteins (Nops), are involved in host-range determination and symbiotic efficiency. S. fredii HH103 secretes at least eight Nops through the T3SS. Interestingly, there are Rhizobium-specific Nops, such as NopC, which do not have homologues in pathogenic bacteria. In this work we studied the S. fredii HH103 nopC gene and confirmed that its expression was regulated in a flavonoid-, NodD1- and TtsI-dependent manner. Besides, in vivo bioluminescent studies indicated that the S. fredii HH103 T3SS was expressed in young soybean nodules and adenylate cyclase assays confirmed that NopC was delivered directly into soybean root cells by means of the T3SS machinery. Finally, nodulation assays showed that NopC exerted a positive effect on symbiosis with Glycine max cv. Williams 82 and Vigna unguiculata. All these results indicate that NopC can be considered a Rhizobium-specific effector secreted by S. fredii HH103.

Effect of soaking and fermentation on content of phenolic compounds of soybean (Glycine max cv. Merit) and mung beans (Vigna radiata [L] Wilczek)

Mung beans (Vigna radiata [L] Wilczek) purchased from a Spanish company as “green soybeans”, showed a different phenolic composition than yellow soybeans (Glycine max cv. Merit). Isoflavones were predominant in yellow soybeans, whereas they were completely absent in the green seeds on which flavanones were predominant. In order to enhance their health benefits, both types of bean were subjected to technological processes, such as soaking and fermentation. Soaking increased malonyl glucoside isoflavone extraction in yellow beans and produced an increase in apigenin derivatives in the green beans. Lactobacillus plantarum CECT 748 T fermentation produced an increase in the bioactivity of both beans since a conversion of glycosylated isoflavones into bioactive aglycones and an increase of the bioactive vitexin was observed in yellow and green beans, respectively. In spite of potential consumer confusion, since soybean and “green soybean” are different legumes, the health benefits of both beans were enhanced by lactic fermentation.

Late-season catch crops reduce nitrate leaching risk after grazed green manures but release N slower than wheat demand

Late season catch crops could be an effective tool to conserve highly available N after grazing of green manures. An experiment was established to investigate the productivity and N capturing abilities of barley (Hordeum vulgare cv. Cowboy) and oilseed radish (Raphanus sativus L.) crops seeded after a grazed green manure. Green manure was a mix of forage pea (Pisum sativum cv. 40–10), soybean (Gylcine max cv. Prudence) and oat (Avena sativa cv. Legget). The experiment was repeated twice in Carman, Manitoba in 2010 and 2011. Catch crops were seeded in late summer either no-till or after soil cultivation of the grazed plots. Wheat (Triticum aestivum cv. Waskada) was seeded in the second year as a test crop for both experiments. Catch crop productivity and N uptake was influenced by season (greater in the wetter year) and catch crop type (barley and radish produced 1990 and 1490kgha−1, respectively) but not tillage system. The catch crops had their greatest overall effect on soil NO3-N content in 2010 under conditions of high autumn precipitation when N leaching was more severe. Here, the catch crops significantly reduced NO3-N at all depths. Under drier conditions in 2011, catch crops only reduced NO3-N in the top 30cm. There was average 57 and 12kgha−1 more soil NO3-N in plots with no catch crops than plots where catch crops were grown in 2010 and 2011, respectively. Wheat N uptake at maturity was reduced around 25% when grown after catch crops. Similarly, wheat grain yield was 12–31% less after catch crops than no catch crops. This study showed that catch crops can be used to capture excess nutrients after grazing, but N release from the selected catch crops in the following year was not in synchrony with the wheat N demand.

The effect of moisture content on physical, mechanical and rheological properties of soybean (Glycine maxcv. ATAEM-II) seed

Some physical, mechanical and rheological properties of soybean seed (Glycine max cv. ATAEM-II) were identified at moisture content of 8.02±(0.03), 12.08±(0.12), 16.07±(0.11), 20.11±(0.16) and 24.05±(0.09)% w.b. From initial moisture content with 8.02% to final moisture content with 24.05%, the average length, width, and thickness of seed increased by 11.93, 10.64 and 10.47%, respectively. Arithmetic and geometric mean diameters, surface area, terminal velocity, angle of repose, static friction angles and coefficients on aluminum, stainless steel, galvanized iron, rubber, glass and plywood plates increased with increasing moisture content whereas other all properties such as true and bulk densities, porosity, shpericity, aspect ratio, rupture force, hardness, elastic modulus, Poisson's ratio and energy decreased with increasing moisture content. The minimum friction was determined by glass, followed by aluminum, stainless steel, galvanized iron, plywood and rubber plates at all moisture contents. Elastic Modulus ranged between 118.83 and 100.91N mm−2 at initial and final moisture content, respectively. At the initial and final moisture content, the Poisson ratio changed between 0.527 and 0.262, respectively. The best germination ratio with 98.04% was identified at the 20.11% moisture content while the shortest germination duration with 12.96 days was determined at the 8.02% moisture content.

The effect of application of micromycetes on plant growth, as well as soybean and barley yields.

The possibility of application of micromycetes (strains Penicillium bilaiae Pb14, Aureobasidium pullulans YA05 and Rhodotorula mucilaginosa YR07) to increase yields of soybean (Glycine max cv Almaty) and barley (Hordeum vulgare cv Arna) was estimated. It was shown that the most positive effect on germination energy and seed germination after seed treatment with liquid culture, supernatant and filtrate, is achieved at 1:5 dilution. In studying the influence of cell-associated and extracellular biologically active compounds of micromycetes (liquid culture and supernatant) on biometric parameters of seedlings, the maximum stimulating effect was observed in the variants with liquid culture. These strains of micromycetes were used as a bases for various compositions of preparations - application of each strain separately and application of micromycetes mixes. In microfield experiments, the increase of soybean yield ranged from 4.5 to 9.4 quintal/ha, barley - from 2.9 to 5.9 quintal/ha. A significant increase in various parameters of structure of the yield was shown in all experimental variants when compared to the control. It was found that an increase in soybean and barley yields and yield components was higher in the variant with a mix of micromycetes when compared to the separate application of each strain. The most efficient mixture was based on the mix of fungal strains (culture filtrate of P. bilaiae Pb14 diluted 1:5 + liquid cultures of A. pullulans YA05 and Rh. mucilaginosa YR07 in a 1:5 dilution).

Effects of Epalrestat as Aldose Reductase Inhibitor (ARI), Sitagliptin as Incretin-Based Therapy (IBT), or Combined Epalrestat and Sitagliptin on Cardiac Vagal Neuropathy (CVN) in Patients with Type 2 Diabetic Mellitus

Background: Aldose reductase inhibitor (ARI) partially ameliorates cardiac vagal neuropathy (CVN) in patients with diabetes mellitus. Incretin-based therapy (IBT) has neuroprotective properties for neuropathy in mice and rat. Materials and Methods: Effects of epalrestat as ARI, sitaglipitin as IBT, or combined ARI and IBT on CVN were examined in 42 patients with CVN and type 2 diabetes mellitus. Subjects were divided into 3 groups; group A (n =12) was treated with add-on oral epalrestat; group B (n =18) was treated with addon oral sitaglipitin; and group C (n = 12) with CVN despite treatment with epalrestat (n=6) for 1 year in group A , sitagliptin (n=5) for 1 year in group B and subcutaneously injected exenatide (n = 1) as IBT for 1 year was treated with add-on combined epalrestat and sitaglipitin, although there were no placebo in all patients with CVN. CVN was defined as maximal coefficient of variance in electrocardiographic beatto- beat interval (max CV. R-R) of three measurements during deep breathing at rest on =2.00%. Since disease duration was =20 years in each group, patient had various chronic complications and various treatments. Results: Mean duration of treatment was 1 year. Max CV.R-R after treatment was significantly (P<0.001) increased after treatment in each group. Nevertheless, 8 (67%) and 5 patients (28%) still had CVN after treatment in groups A and B, respectively, whereas no patients had CVN after treatment in group C. There was significant difference (P <0.002) in magnitude increase of max CV. R-R after treatment between groups using ANOVA with multiple comparison test. No significant differences were observed in other variables before and after treatment between groups. Conclusion: Sitagliptin may be more effective than epalrestat for CVN in type 2 diabetic patients, and combined epalrestat and sitagliptin may be haven a synergistic effect.