Induced suppression of soft rot disease in tobacco by combined application of Bacillus subtilis strain B4 and chemical elicitor BTH

In this study, the utility of the combined application of Benzo-(1,2,3)-thidiazole-7-carbothioic acid S-methyl ester (BTH), a systemic acquired resistance (SAR) inducer, and the plant growth-promoting rhizobacteria (PGPR), Bacillus subtilis strain B4 (B4), was investigated for the suppression of soft rot disease caused by Pectobacterium carotovorum SCC1 (SCC1) in tobacco seedlings. Soft rot disease was completely suppressed in tobacco with combined application of B4 strain and 0.1 mM BTH when compared to individual application upon pathogen challenge. In addition, the population of bacterial cells of B4 strain was found to be greater when cultured in growth media in the presence of BTH, compared to the growth of B4 strain in the absence of BTH. Spectrophotometer analysis revealed that there was an increased broad range of compounds in the culture filtrate of B4 strain when grown with BTH, compared to the culture filtrate of B4 strain alone. The combined application of B4 strain and 0.1 mM BTH induced the increased expression of PR1a::GUS on tobacco and elicited systemic resistance against SCC1 when compared to individual application. However, there was low expression of PR1a::GUS in the water-treated control. Hence, the integrated use of BTH and B4 strain might be one of the strategies for biological control of soft rot disease through induced systemic resistance (ISR) in tobacco plants.     

Fusarial wilt control and growth promotion of pigeon pea through bioactive metabolites produced by two plant growth promoting rhizobacteria

The bioactive metabolites produced by two plant growth promoting rhizobacteria strains, a Pseudomonas aeruginosa strain RRLJ 04 and a Bacillus cereus strain BS 03, which showed growth promotion and disease control in pigeon pea against Fusarium udum, were isolated and screened for their efficacy to control fusarial wilt of pigeon pea under gnotobiotic and nursery condition. Bioactive metabolites viz., BM 1 and BM 2 from RRLJ 04 and BM 3 from BS 03 also showed in vitro antibiosis against F. udum. Seeds treated with 50 μl seed^?1 of BM 1, 30 μl seed^?1 of BM 2 and 70 μl seed^?1 of BM 3 and grown in pathogen infested soil showed suppression of wilt disease besides growth enhancement. Per cent disease control was 90 % with BM 2 application as compared to 87 and 83 %, respectively in BM 1 and BM 3 after 90 days of growth. BM 2 treated plants were more resistant to the pathogen as compared to the other fractions tested. Mycelial dry weight was found to be reduced on treatment with the bioactive metabolites. Formation of chlamydospore-like structures was observed in the pathogen mycelium treated with BM 3. The analytical studies confirmed that two of these metabolites are phenazine derivatives.     

Induction of systemic resistance in rice to bacterial blight by 1,2,3-benzothiadiazole 7-carbothioic acid-S-methyl ester (BTH) treatments

Use of BTH to evaluate the disease severity and induction of systemic resistance in rice to bacterial blight caused by Xanthomonas oryzae pv. oryzae is investigated. A new batch of 25 isolates of Xanthomonas oryzae pv. oryzae was obtained from infected rice lead tissues collected from Pattambi, Kerala, south India. Their identification was confirmed by the plant inoculation test on to IR24 rice plants which produced characteristic bacterial blight lesions. Among the 25 of X.o. pv. oryzae, four of the isolates were also virulent to IRBB21 rice plants (a near isogenic line of IR24) which carry the Xa-21 gene for BB resistance. The results confirm that there are pathogen strains in India which can overcome Xa-21. Development of BB lesions developed in IR24 (BB susceptible) plants after they were treated with BTH applications either as seed treatment or as foliar spray at 0.1, 0.5, 0.1 and 2.0 mM concentrations showed that even at 2.0 mM concentrations, IR24 plants were still susceptible to the pathogen. There was very little or marginal effect of BTH on the induction of resistance to BB in IR24 rice plants. When the same concentrations of BTH were applied to IRBB21 (Xa-21) rice plants, they showed pronounced triggering of systemic resistance to BB pathogen even at 0.1 mM concentration of BTH applied either as seed treatment or as foliar spry. Disease severity index was reduced to 5 (against a score of 9 in untreated) and there was 85–86% reduction in BB incidence in plants that received 0.1 mM BTH. These results provide evidence that BTH-induced systemic resistance complements the R-gene resistance in IRBB21 plants but not in IR24 rice plants.     

Streptomyces sp. S160: a potential antagonist against chickpea charcoal root rot caused by Macrophomina phaseolina (Tassi) Goid

Biological control of charcoal root rot disease caused by Macrophomina phaseolina in chickpea was studied by using Streptomyces sp. S160. This biocontrol agent (BCA) inhibited the mycelial growth of M. phaseolina by 50 % in vitro and significantly reduced charcoal rot incidence in the greenhouse by 33.3 %. The greenhouse experiment revealed that seed treatment along with soil application supported the highest germination (88.6 %), vigor index (7326.91) and reduced root rot incidence (12.5 %) in comparison to seed treatment and soil application alone. BCA enhanced the growth and helped in inducing resistance against charcoal rot disease of chickpea caused by M. phaseolina by increasing activity of defense-related enzymes in chickpea plants, leading to the synthesis of defense chemicals in plants. BCA (Streptomyces sp. S160) was also characterized and identified by using polyphasic approaches including 16S rDNA sequencing.     

Benzo-thiadiazole-7-carbothioic Acid S-methyl Ester does not protect Melon Fruits against Fusarium pallidoroseum Infection but Induces Defence Responses in Melon Seedlings

The present study investigated the potential of benzo-thiadiazole-7-carbothioic acid S -methyl ester (BTH) to protect postharvest melons var. 'Orange Flesh' from the fruit rot caused by Fusarium pallidoroseum . It was noticed that melon fruits immersed in BTH and postinoculated with the fungus presented the same pattern of disease incidence/severity and activity of the defence-related enzymes superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, phenylalanine ammonia-lyase, and β-1,3-glucanase of controls, indicating that BTH was ineffective in protecting melons from the fruit rot disease. However, the preflowering application of BTH in melon seedlings induced stunted growth, probably related to enhanced lignification which is related to the plant cell wall reinforcement and increase of resistance against invading pathogens, and alterations of the activity of the studied defence-related enzymes in comparison with controls, suggesting that this strategy could probably be effective for the control of the postharvest rot of melon fruits through activation of systemic resistance.     

Application of benzothiadiazole and Trichoderma harzianum to control faba bean chocolate spot disease and their effect on some physiological and biochemical traits

Chocolate spot disease is the most prevalent and important disease in the major faba bean growing regions in the world. Different concentrations of the abiotic inducer (0.3 and 0.5 mM benzothiadiazole) and the biotic inducer (1 × 10⁷ and 2 × 10⁷ spore/ml Trichoderma harzianum) were used alone or in combination to study their efficiency against faba bean chocolate spot disease caused by Botrytis fabae and Botrytis cinerea and their effect on some chemical analyses (phenylalanine ammonia lyase activity, total flavonoids and peroxidase isozymes, pectin and lignin content and total chlorophyll content). Application of the tested inducers as foliar treatment significantly reduced the severity of chocolate spot disease as compared with untreated infected plants. The reduction in disease severity was associated with a gradual increase in phenylalanine ammonia lyase activity. Maximum increase was recorded at 72 h after inoculation with B. fabae and B. cinerea. In addition, the levels of flavonoids in induced infected leaves recorded a sharp increase at 24 h after inoculation with B. fabae or B. cinerea. Also, pectin and lignin contents in the cell wall of induced infected plants were significantly increased as compared with untreated infected plants. Beside the induction of resistance, the tested inducers markedly increased total chlorophyll content in treated infected plants as compared with untreated infected plants. Isozymes analysis revealed that new peroxidase bands were induced only in treated faba bean leaves in response to infection with B. fabae or B. cinerea.     

Timelapse scanning reveals spatial variation in tomato (Solanum lycopersicum L.) root elongation rates during partial waterlogging

Aims

Effects of soil amendments with crop residues on suppression of damping-off of sugar beet were examined by growing the seedlings in pasteurized, Rhizoctonia solani (AG2-2 IIIB)-infested soil at different temperatures. Dried residues of five dasheen or taro (Colocasia esculenta (L.) Schott) cultivars were compared with those of peanut (Arachys hypogaea L.) and Brassica rapa Olsson for their effects on disease suppression.

Methods and Results

When the seedlings were grown at 17/12 °C (day/night), all residues equally suppressed the disease when amended into the soil just before sowing. At 22/17 or 32/27 °C, damping-off developed in non-treated soil within 10 days, and differential suppressive effects by the residues became apparent by 21 days. When non-pasteurized, non-treated soil was infested with the pathogen, seedling survival was markedly better than in the same but pasteurized, infested soil. Yet, the effect was not different within the entire temperature ranges. Growth of both R. solani and the seedlings peaked near 25 °C and leveled off at higher temperatures.

Conclusions

These results suggest that damping-off was suppressed by antagonistic soil microorganisms, and individual residues elevated their effects differently. Under cool conditions, the antagonists dominated the pathogen to suppress the disease. Under warmer conditions, pathogenesis overcame antagonism depending on the residue, resulting in differential effects of disease suppression.     

Incorporation into the transplant soil plug of the plant protective agent Paenibacillus alvei strain K165 confers protection to melon against Fusarium wilt

Fusarium oxysporum f.sp. melonis (FOM) is a plant pathogen affecting melon production worldwide. An environmental friendly disease management strategy is the use of biocontrol agents (BCAs). Towards this direction, two BCA release strategies, seed coating and amendment of the transplant soil plug with the BCA strain Paenibacillus alvei K165 at various ratios, were evaluated against FOM in planta and in vitro. A reduction in Fusarium wilt symptom development was observed in melon plants, after mixing the transplant soil plug with K165 (10⁷ cfu g^?1 powder) at a ratio of 10 % (v/v). The monitoring of K165 rhizosphere population in the different treatments revealed a possible existence of a threshold population level that has to be attained before suppression of disease occurs. The data of the present study suggest that K165 plant protective activity against FOM can be possibly attributed to antibiosis and the triggering of Chit1 and Pal1 gene expression.     

Fungal elicitor protein PebC1 from Botrytis cinerea improves disease resistance in Arabidopsis thaliana

We previously identified a novel protein elicitor, PebC1, from Botrytis cinerea and described its enhancement of plant growth, drought tolerance and disease resistance in tomato. Here, we have investigated the defense-associated molecular responses in Arabidopsis thaliana after treatment with recombinant PebC1. PebC1 was expressed in Escherichia coli. Recombinant protein treatments improved plant resistance to Botrytis infection and maintained plant defenses for more than 21 days. The purified protein at 10 μg ml^?1 activated extracellular medium alkalization (pH) and reactive oxygen species and nitric oxide generation and also induced defense gene expression. Arabidopsis mutants that are insensitive to salicylic acid had increased resistance to Botrytis infection after PebC1 treatment but PebC1 did not affect the resistance of mutants with jasmonic acid and ethylene transduction pathways. The results suggest that PebC1 can function as an activator of plant disease resistance and can promote disease resistance to Botrytis in A. thaliana through the ethylene signal transduction pathway.     

Phosphorus and nitrogen allocation in Allium ursinum on an alluvial floodplain (Eastern France). Is there an effect of flooding history?

The change in phosphorus and nitrogen content in a common geophyte spring species, Allium ursinum, is studied in alluvial forests in relation to three flooding histories related to river regulation: (1) annually flooded, (2) unflooded for 30 years, and (3) unflooded for 200 years. Flood suppression leads to a reduction of available P soil content as well as decreasing the biomass and the amount of phosphorus in plants, but has no significant effect on N plant content. Plant N:P ratio increases with the suppression of floods and is primarily driven by soil N:P ratios, in turn markedly linked to the total nitrogen in the soil. We highlighted a lower nutrient accumulation in leaves during plant growth in unflooded forests. Overall, our results suggest that P was the main limiting factor in unflooded forests while nitrogen was the main limiting factor in annually flooded forests. Flood suppression strongly affects the morphology and nutrient uptake by Allium ursinum and thus nutrient cycling in riverine forests.     

Effect of wheel traffic and green manure treatments on forage yield and crown rot in alfalfa (Medicago sativa)

Harvesting alfalfa damages crowns and increases the opportunity for entry of pathogens. Incorporation of green manures into soil increases population density of streptomycetes with broad pathogen antagonist activity. This study aimed to measure the impact of wheel traffic on forage yield and plant health and the effect of green manures to reduce disease. Buckwheat and sorghum-sundangrass were incorporated into soil 3 weeks before seeding alfalfa. Total bacteria, streptomycete, and pathogen antagonist densities were measured prior to planting green manures and alfalfa. Wheel traffic was applied 2 days after each forage harvest. Wheel traffic reduced forage yield 12 % to 17 % depending on year and location, significantly reduced plant counts, and increased crown rot compared to the no traffic control. Cultivar had a significant effect on yield, plant counts, and crown rot. Streptomycete density and pathogen antagonists increased when fall-sown green manure crops were incorporated in spring. Forage yields were significantly higher in plots with greater antagonist density when traffic was applied. Green manure treatments did not affect plant counts or crown rot. Mechanical wheel traffic reduces forage yield and increases disease. Green manure crops may provide benefits in alfalfa production systems by increasing pathogen antagonists.     

A Time-Course Proteomic Analysis of Rice Triggered by Plant Activator BTH

Benzothiadiazole (BTH) is an artificial inducer of systemic acquired resistance. Due to rice being an important food crop and model plant, we investigated its response to BTH using label-free proteomics technology coupled with bioinformatics. Protein expression levels were verified using the multi-reaction monitoring mode and semi-quantitative RT-PCR. BTH treatment can up- or down-regulate many proteins produced by the rice host at all four periods, with the numbers of proteins being 6/24, 9/10, 14/10, and 8/20, respectively. Compared with mock treatments (phosphate buffered saline with 0.1 % dimethylsulfoxide and 0.5 % Tween-20), some proteins related to plant resistance were only detected after BTH treatments, such as ascorbate peroxidase (POD) 3, chitinase A, thioredoxin-dependent POD 2, beta-1,3-glucanase 2, POD superfamily protein, major facilitator superfamily (MFS) protein, copper/zinc-superoxide dismutase (SOD) 1, pathogenesis-related protein (PR) 1. Other proteins showing up-regulation after BHT treatment included PR-5, glyceraldehyde-3-phosphate dehydrogenase C, plasma-membrane associated cation-binding protein 1, and oxidoreductase family proteins. These results indicated that BTH was involved with inducing rice resistance. Some up-regulated proteins were also involved in other metabolic processes. The activity and expression level of POD, phenylalanine ammonia-lyase (PAL), and SOD, lipoxygenase (LOX), beta-1,3-glucanases, and chitinases were determined using the enzyme activity assay and semi-quantitative RT-PCR. These results indicated that BTH can enhance the activity of beta-1,3-glucanases, LOX, PAL, and POD. BTH can also induce up-regulation of the copper/zinc-SOD, ascorbate POD, glutathione POD 1, Chitinase, and LOX1 genes.     

A Novel Pyrimidin-Like Plant Activator Stimulates Plant Disease Resistance and Promotes Growth

Plant activators are chemicals that induce plant defense responses to a broad spectrum of pathogens. Here, we identified a new potential plant activator, 5-(cyclopropylmethyl)-6-methyl-2-(2-pyridyl)pyrimidin-4-ol, named PPA (pyrimidin-type plant activator). Compared with benzothiadiazole S-methyl ester (BTH), a functional analog of salicylic acid (SA), PPA was fully soluble in water and increased fresh weight of rice (Oryza sativa) and Arabidopsis plants at low concentrations. In addition, PPA also promoted lateral root development. Microarray data and real-time PCR revealed that PPA-treated leaves not challenged with pathogen showed up-regulation of genes related to reactive oxygen species (ROS), defenses and SA. During bacterial infection, Arabidopsis plants pretreated with PPA showed dramatically decreased disease symptoms and an earlier and stronger ROS burst, compared with plants pretreated with BTH. Microscopy revealed that H₂O₂ accumulated in the cytosol, plasma membrane and cell wall around intracellular bacteria, and also on the bacterial cell wall, indicating that H₂O₂ was directly involved in killing bacteria. The increase in ROS-related gene expression also supported this observation. Our results indicate that PPA enhances plant defenses against pathogen invasion through the plant redox system, and as a water-soluble compound that can promote plant growth, has broad potential applications in agriculture.     

Inoculation of seed-borne fungus in the rhizosphere of Festuca arundinacea promotes hydrocarbon removal and pyrene accumulation in roots

Background and aims

The selective inoculation of specific hydrocarbon-degrading microbes into the plant rhizosphere offers a useful means for remediating hydrocarbon-contaminated soils. The effect of inoculating a seed-borne filamentous fungus (Lewia sp.) on hydrocarbon removal by Festuca arundinacea and its growth was studied on perlite (model soil) and soil, both spiked with hydrocarbons.

Methods

A hydrocarbon mixture (1,500 mg kg^?1) of two polycyclic aromatic hydrocarbons (PAH), phenanthrene and pyrene, blended with hexadecane (1.0:0.5:0.5 weight) was used. Greenhouse experiments were carried out for 45 days. Inoculated and non-inoculated plants were grown in dark cylindrical glass pots containing perlite or soil.

Results

Inoculation with Lewia sp. stimulated (100 %) root growth in spiked perlite. Inoculated plants showed higher phenanthrene removal (100 %) compared to non-inoculated plants in perlite and soil. Pyrene removal by inoculated plants was 37-fold higher than that by non-inoculated plants in perlite; in soil, pyrene removal by inoculated plants (97.9 %) differed significantly from that of non-inoculated plants (91.4 %). Accumulation of pyrene in roots (530.9 mg kg^?1 of dry roots) was promoted in perlite.

Conclusions

Our results demonstrate that Lewia sp. (endophytic fungus) improved the efficiency of PAH removal by F. arundinacea, on both perlite and soil, stimulating pyrene accumulation in roots.     

Elevated amino sugar nitrogen concentrations in soils: a potential method for assessing N fertility enhancement by actinorhizal plants

No single soil nitrogen (N) assay seems to be adequate to predict N fertility. An amino sugar-nitrogen assay has been proposed as a method to estimate soil N fertility and determine N fertilization requirements of agronomic crops. Such chemical methods of estimating potentially-available soil N are appealing because of their rapidity and simplicity. In this case study an amino sugar-N assay was used in established plantings of actinorhizal woody plants to determine whether they increased soil amino sugar N concentrations in soil and by how much. Amino sugar N is derived primarily from bacterial and fungal cell walls, but not plant cells, and probably reflects N-stimulation of microbial activity in soils. It was hypothesized that actinorhizal plants would increase soil amino sugar N. Mine spoil Entisols supporting Frankia-nodulated autumn olive shrubs (Elaeagnus umbellata) had 104 mg kg^-1 of amino sugar-N while European alder (Alnus glutinosa) trees on the same soil type had amino sugar N concentrations of 98 mg kg^-1 beneath their canopies. Soil adjacent to, and between, the two plantations and lacking N-fixing plants had 54 mg kg^-1 N of soil amino sugars. At another location a cluster of European alder trees increased soil amino sugar concentrations in a fertile Mollisol by a similar amount, 45 mg kg^-1, but by a lesser proportion. This Mollisol had a high amino sugar N background level of 336.5 mg kg^-1 indicating high N fertility. Results indicated that there was no correspondence between amino sugar N concentrations and total N concentrations, nor was there correspondence between amino sugar N concentrations and total soil organic matter concentration on mine spoils. Actinorhizal plants apparently increase the amino sugar N concentration in soils and an amino sugar N assay has potential for development as an additional means of indexing N fertility enhancement by N-fixing trees.     

A Benzothiadiazole Primes Parsley Cells for Augmented Elicitation of Defense Responses

Systemic acquired resistance is an important component of the disease-resistance arsenal of plants, and is associated with an enhanced potency for activating local defense responses upon pathogen attack. Here we demonstrate that pretreatment with benzothiadiazole (BTH), a synthetic activator of acquired resistance in plants, augmented the sensitivity for low-dose elicitation of coumarin phytoalexin secretion by cultured parsley (Petroselinum crispum L.) cells. Enhanced coumarin secretion was associated with potentiated activation of genes encoding Phe ammonia-lyase (PAL). The augmentation of PAL gene induction was proportional to the length of pretreatment with BTH, indicating time-dependent priming of the cells. In contrast to the PAL genes, those for anionic peroxidase were directly induced by BTH in the absence of elicitor, thus confirming a dual role for BTH in the activation of plant defenses. Strikingly, the ability of various chemicals to enhance plant disease resistance correlated with their capability to potentiate parsley PAL gene elicitation, emphasizing an important role for defense response potentiation in acquired plant disease resistance.     

Multifunctional Exopolysaccharides from Pseudomonas aeruginosa PF23 Involved in Plant Growth Stimulation,Biocontrol and Stress Amelioration in Sunflower Under Saline Conditions

Isolate PF23 selected from among 110 fluorescent pseudomonads, identified as Pseudomonas aeruginosa, displayed salinity tolerance and exopolysaccharides (EPS) production up to 2,000 mM NaCl concentration. EPS-defective mutant PF23^EPS? of the isolate showed 86 % reduction in EPS production in comparison with wild strain. Defect in EPS production brought loss in salt tolerance capability. Purified EPS obtained from PF23 displayed multiple roles. At low concentration EPS functioned as biocontrol agent, at high concentration EPS behaved as osmoprotective or stress ameliorating metabolite and when introduced in saline soil, served as a plant growth promotor along with seed biopriming agent. Both in planta and in vivo studies were performed taking sunflower as a test crop and it was observed that PF23 showed plant growth promotion and significant biocontrol potential against dreadful phytopathogen Macrophomina phaseolina (under saline conditions). The mutant PF23^EPS? was ineffective under saline conditions both in growth enhancement as well as in disease suppression. The study reports a potent strain, Pseudomonas aeruginosa PF23, capable of enhancing production of sunflower crop in semiarid regions and minimizing the incidence of charcoal rot disease in sunflower.     

Lespedeza cuneata invasion alters soils facilitating its own growth

Lespedeza cuneata (sericea) is an Asian legume introduced to the US in the 1930s for erosion control and forage, but it can strongly reduce the abundance of native grassland plants. One possible explanation for this high invasive capacity is that L. cuneata is able to alter soil conditions to either improve its own growth, or reduce growth of native plants. To test for soil alteration following invasion, we collected soil from a previous 3-year field experiment in which L. cuneata was established in or excluded from randomly selected plots in a restored grassland. These soil history treatments were crossed with soil autoclaving—to disrupt microbial communities potentially important to plant interactions. For each treatment combination, a native plant, Sorghastrum nutans, was grown with L. cuneata or a conspecific in a 12-week greenhouse experiment. Although we found no evidence for competitive effects on the native species, L. cuneata biomass increased when grown in soil with a L. cuneata history as opposed to non-invaded soil (F _{1, 16} = 4.79, P = 0.04). Additionally, nodulation of L. cuneata increased in invaded compared to non-invaded soil (F _{1, 16} = 6.01, P = 0.026). These results indicate that, within three years of invasion, L. cuneata is able to alter soils to facilitate its own growth and suggest that at least part of the invasive success of L. cuneata is linked to altered soil conditions.     

Usefulness of 10 genomic regions in soybean associated with sudden death syndrome resistance

Sudden death syndrome (SDS) is an important soybean [Glycine max (L) Merrill] disease caused by the soilborne fungus Fusarium virguliforme. Currently, 14 quantitative trait loci (QTL) had been confirmed associated with resistance or tolerance to SDS. The objective of the study was to evaluate usefulness of 10 of these QTL in controlling disease expression. Six populations were developed providing a total of 321 F2-derived lines for the study. Recombinant inbred lines (RIL) used as parents were obtained from populations of ‘Essex’ × ‘Forrest’ (EF), ‘Flyer’ × ‘Hartwig’ (FH), and ‘Pyramid’ × ‘Douglas’ (PD). Disease resistance was evaluated in the greenhouse at three different planting times, each with four replications, using sorghum infested with F. virguliforme homogeneously mixed in the soil (Luckew et al., Crop Sci 52:2215–2223, 2012). Four disease assessment criteria—foliar disease incidence (DI), foliar leaf scorch disease severity (DS), area under the disease progress curve (AUDPC), and root rot severity—were used. QTL were identified in more than one of the disease assessment criteria, mainly associated with lines in the most resistant categories. Five QTL (qRfs4, qRfs5, qRfs7, qRfs12, and Rfs16) were associated with at least one of the disease assessments across multiple populations. Of the five, qRfs4 was associated with DI, AUDPC, and root rot severity, and Rfs16 with AUDPC and root rot severity. The findings suggest it may be possible for plant breeders to focus on stacking a subset of the previously identified QTL to improve resistance to SDS in soybean.