Glycine max (L.) Merr. (Soybean) metabolome responses to potassium availability.

Potassium (K+) has vital physiological and metabolic functions in plants and its availability can impact tolerance to biotic and abiotic stress conditions. Limited studies have investigated the effect of K+ fertilization on soybean metabolism. Using integrated omics, ionomics and metabolomics, we investigated the field-grown Glycine max (soybean) response, after four K+ soil fertilization rates. Soybean leaf and pod tissue (valves and immature seeds) extracts were analysed by ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-HRMS) and inductively coupled plasma optical emission spectroscopy (ICP-OES). Multivariate analyses (PCA-X&Y e O2PLS-DA) showed that 51 compounds of 19 metabolic pathways were regulated in response to K+ availability. Under very low potassium availability, soybean plants accumulated of Ca2+, Mg2+, Fe2+, Cu2+, and B in young and old leaves. Potassium fertilization upregulated carbohydrate, galactolipid, and flavonol glycoside biosynthesis in leaves and pod valves, while K+ deficient pod tissues showed increasing amino acids, oligosaccharides, benzoic acid derivatives, and isoflavones contents. Severely K+ deficient soils elicited isoflavones, coumestans, pterocarpans, and soyasaponins in trifoliate leaves, likely associated to oxidative and photodynamic stress status. Additionally, results demonstrate that L-asparagine content is higher in potassium deficient tissues, suggesting this compound as a biomarker of K+ deficiency in soybean plants. These results demonstrate that potassium soil fertilization did not linearly contribute to changes in specialised constitutive metabolites of soybean. Altogether, this work provides a reference for improving the understanding of soybean metabolism as dependent on K+ availability.

Crotalaria spectabilis as a source of pyrrolizidine alkaloids and phenolic compounds: HPLC-MS/MS dereplication and monocrotaline quantification of seed and leaf extracts.

INTRODUCTION: Crotalaria spectabilis is an important species used as a pre-plant cover for soybean crops to control the proliferation of endoparasitic nematodes. Species from the Crotalaria genus are known for presenting pyrrolizidine alkaloids (PAs) in their composition, however, C. spectabilis is still considered chemically under-explored. OBJECTIVE: The goal of this manuscript is the development and validation of a method for PAs and flavonoids identification and quantification of C. spectabilis seeds and leaves, a toxic plant used for nematode proliferation control in soil, especially in soybean crops. MATERIALS AND METHODS: Seeds and leaves extracts were analysed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the identification of the compounds. RESULTS: PAs and phenolic compounds could be identified in both samples based on the MS/MS fragmentation pattern. Molecular formulas of the annotated compounds were confirmed by ultra-high-performace liquid chromatography-quadrupole time-of-flight (UHPLC-QToF), and monocrotaline could also be confirmed by standard comparison. The quantification of monocrotaline was performed by HPLC-MS/MS, resulting in 123 times higher monocrotaline content in seeds than in the leaves, which could explain its efficiency in combating nematode proliferation in soil. CONCLUSION: This was the first report of phenolic compounds in C. spectabilis. The current study highlights the importance of C. spectabilis for nematode control due to the presence of toxic PAs, and the employment of analytical techniques for identification and quantification of compounds present in the extracts.

Unraveling Asian Soybean Rust metabolomics using mass spectrometry and Molecular Networking approach.

Asian Soybean Rust (ASR), caused by the biotrophic fungus Phakopsora pachyrhizi, is a devastating disease with an estimated crop yield loss of up to 90%. Yet, there is a nerf of information on the metabolic response of soybean plants to the pathogen Untargeted metabolomics and Global Natural Products Social Molecular Networking platform approach was used to explore soybean metabolome modulation to P. pachyrhizi infection. Soybean plants susceptible to ASR was inoculated with P. pachyrhizi spore suspension and non-inoculated plants were used as controls. Leaves from both groups were collected 14 days post-inoculation and extracted using different extractor solvent mixtures. The extracts were analyzed on an ultra-high performance liquid chromatography system coupled to high-definition electrospray ionization-mass spectrometry. There was a significant production of defense secondary metabolites (phenylpropanoids, terpenoids and flavonoids) when P. pachyrhizi infected soybean plants, such as putatively identified liquiritigenin, coumestrol, formononetin, pisatin, medicarpin, biochanin A, glyoceollidin I, glyoceollidin II, glyoceollin I, glyoceolidin II, glyoceolidin III, glyoceolidin IV, glyoceolidin VI. Primary metabolites (amino acids, peptides and lipids) also were putatively identified. This is the first report using untargeted metabolomics and GNPS-Molecular Networking approach to explore ASR in soybean plants. Our data provide insights into the potential role of some metabolites in the plant resistance to ASR, which could result in the development of resistant genotypes of soybean to P. pachyrhizi, and effective and specific products against the pathogen.

Correction: The number of measurements needed to obtain high reliability for traits related to enzymatic activities and photosynthetic compounds in soybean plants infected with Phakopsora pachyrhizi.

[This corrects the article DOI: 10.1371/journal.pone.0192189.].

The number of measurements needed to obtain high reliability for traits related to enzymatic activities and photosynthetic compounds in soybean plants infected with Phakopsora pachyrhizi.

Asian rust affects the physiology of soybean plants and causes losses in yield. Repeatability coefficients may help breeders to know how many measurements are needed to obtain a suitable reliability for a target trait. Therefore, the objectives of this study were to determine the repeatability coefficients of 14 traits in soybean plants inoculated with Phakopsora pachyrhizi and to establish the minimum number of measurements needed to predict the breeding value with high accuracy. Experiments were performed in a 3x2 factorial arrangement with three treatments and two inoculations in a random block design. Repeatability coefficients, coefficients of determination and number of measurements needed to obtain a certain reliability were estimated using ANOVA, principal component analysis based on the covariance matrix and the correlation matrix, structural analysis and mixed model. It was observed that the principal component analysis based on the covariance matrix out-performed other methods for almost all traits. Significant differences were observed for all traits except internal CO2 concentration for the treatment effects. For the measurement effects, all traits were significantly different. In addition, significant differences were found for all Treatment x Measurement interaction traits except coumestrol, chitinase and chlorophyll content. Six measurements were suitable to obtain a coefficient of determination higher than 0.7 for all traits based on principal component analysis. The information obtained from this research will help breeders and physiologists determine exactly how many measurements are needed to evaluate each trait in soybean plants infected by P. pachyrhizi with a desirable reliability.

Efeitos pós-ingestivos de flavonóides no epitélio do intestino médio de larvas de Anticarsia gemmatalis (Hübner 1818) (Lepidoptera: Erebidae) / Post-ingestive effects of flavonoids in the midgut epithelium of Anticarsia gemmatalis (Hübner 1818) (Lepidoptera: Erebidae) larvae

An alternative control to soybean caterpillar has been the use of insect resistant plants that present phenolic flavonoids. The midgut is the main access pathway of food and chemical substances ingested. The present study examined morphological alterations in the midgut epithelium of the soybean caterpillar, after the ingestion of soybean genotypes containing the flavonoids rutin and genistin. The caterpillars and genotypes (BRS 257 - control, BR 16, Dowling, PI 229358, IAC 100 and PI 227687) were obtained from the insect rearing facility at the Embrapa Soja. The midguts were collected, fixed in Karnovsky, processed and analyzed under a light microscope. All treatments caused alterations in the midgut epithelial cells. These alterations in the columnar cells were more assiduous than the on other cell types, showing an increase of cytoplasmic protrusions and vacuoles. The goblet cells showed few changes for all genotypes tested, while the regenerative cells presented alterations mainly for the Dowling and PI 227687 treatments. The peritrophic membrane was absent for genotypes IAC100 and PI 227687. We conclude that the Dowling and PI 227687 genotypes were effective and promoted great morphological alterations in the midgut of the soybean caterpillars, being able to be very effective for the control of this plague.

Um controle alternativo para a lagarta da soja tem sido o uso de plantas resistentes a insetos que contém flavonoides fenólicos. O intestino médio é a principal via de acesso do alimento e substâncias químicas ingeridas. O presente estudo examinou as alterações morfológicas no epitélio do intestino médio da lagarta da soja, após a ingestão de genótipos de soja contendo os flavonoides rutina e genistina. As lagartas e os genótipos (BRS 257 - controle, BR 16, Dowling, PI 229358, IAC 100 e PI 227687) foram obtidos do laboratório de criação de insetos da Embrapa Soja. Os intestinos médios foram coletados, fixados em Karnovsky, processados e analisados ao microscópio de luz. Todos os tratamentos causaram alterações nas células epiteliais do intestino médio. As alterações foram mais assíduas nas células colunares do que nos demais tipos celulares. Essas apresentaram grande quantidade de protrusões citoplasmáticas e de vacúolos. As células caliciformes apresentaram poucas alterações para todos os genótipos testados, enquanto as células regenerativas apresentaram alterações principalmente nos tratamentos Dowling e PI 227687. A membrana peritrófica estava ausente para os genótipos IAC100 e PI 227687. Conclui-se que os genótipos Dowling e PI 227687 foram bastante efetivos e promoveram grandes alterações morfológicas no intestino médio das lagartas da soja, podendo ser bastante eficaz para o controle desta praga.

Lepidopteran larva consumption of soybean foliage: basis for developing multiple-species economic thresholds for pest management decisions.

BACKGROUND: Defoliation by Anticarsia gemmatalis (Hübner), Pseudoplusia includens (Walker), Spodoptera eridania (Cramer), S. cosmioides (Walker) and S. frugiperda (JE Smith) (Lepidoptera: Noctuidae) was evaluated in four soybean genotypes. A multiple-species economic threshold (ET), based upon the species' feeding capacity, is proposed with the aim of improving growers' management decisions on when to initiate control measures for the species complex. RESULTS: Consumption by A. gemmatalis, S. cosmioides or S. eridania on different genotypes was similar. The highest consumption of P. includens was 92.7 cm(2) on Codetec 219RR; that of S. frugiperda was 118 cm(2) on Codetec 219RR and 115.1 cm(2) on MSoy 8787RR. The insect injury equivalent for S. cosmoides, calculated on the basis of insect consumption, was double the standard consumption by A. gemmatalis, and statistically different from the other species tested, which were similar to each other. CONCLUSIONS: As S. cosmioides always defoliated nearly twice the leaf area of the other species, the injury equivalent would be 2 for this lepidopteran species and 1 for the other species. The recommended multiple-species ET to trigger the beginning of insect control would then be 20 insect equivalents per linear metre.

Are chemical compounds important for soybean resistance to Anticarsia gemmatalis?

The identification and quantification of flavonoids (rutin and genistin) present in extracts of soybean genotypes, and their effects on the biology and physiology of Anticarsia gemmatalis Hübner (Lep.: Noctuidae) were studied. Analysis of covariance and bicoordinate utilization plots were used to remove the effect of feeding time from pupal weight and consumption as well as to separate pre- and postingestive effects of treatment on A. gemmatalis growth. Genotypes PI 274454, PI 227687, and "IAC-100" extracts in general, caused higher mortality, negatively influenced initial larval and pupal weight, and elongated larval cycle. Larvae fed on the "IAC-100" extract diet ingested larger amounts of food per unit of time, but were less efficient in its conversion to biomass. Leaf extracts of PI 227687 had the largest concentration of rutin (quercitin 3-O-rhamnosylglucoside), followed by PI 274454, and "IAC-100"; PI 74454 also had the highest genistin (genistein 7-O-glucoside) content. The susceptible cultivar "BR-16" showed only a kaempferol-based flavonoid in its chemical profile, indicating that after successive crosses, secondary compounds responsible for plant defenses were eliminated. Genotypes PI 274454, PI 227687, and "IAC-100" showed accentuated resistance characteristics and were considered inadequate sources for the development of A. gemmatalis. Considering rutin and genistin concentration in these genotypes, it is suggested that flavonoids are important factors conferring resistance to A. gemmatalis.

Flavonoid increase in soybean as a response to Nezara viridula injury and its effect on insect-feeding preference.

The ability of the stink bug (Nezara viridula) to induce and/or increase production of chemical defenses, i.e., flavonoids, in immature seeds of five genotypes of soybean (BR-16, IAC-100, PI 227687, PI 229358, and PI 274454) was investigated under greenhouse and laboratory conditions. Samples from pods of each genotype damaged by stink bug were analyzed for flavonoid content with high performance liquid chromatography. A dual-choice test was conducted to evaluate the feeding preference of N. viridula comparing BR-16 pods treated with extracts of PI 227687 seeds (with and without stink-bug injury), with water-treated pods. Seeds of PI 227687 damaged by N. viridula presented the highest concentration (352 microg/g) of daidzin (4'-hydroxyisoflavone-7-glucoside). The same trend was observed with genistin (4',5,7-trihydroxyisoflavone-7-glucoside): PI 227687 contained 142.4 microg/g, PI 274454,31,6 microg/g, and PI 229358,38.9 microg/g. Seeds damaged by stink bugs had higher isoflavone contents (daidzin and genistin), compared to controls. However, after being damaged, PI 274454 and PI 229358 produced less genistin than the other genotypes and no differences in concentration between damaged and nondamaged plants of this genotypes were observed. The numbers of observations of the insect feeding and the numbers of stylet sheaths left in water-treated BR-16 pods were greater than in those treated with PI 227687 extracts. The insects fed for longer periods on BR-16 pods treated with extract of PI 227687 without injury compared to those that were treated with extract of PI 227687 previously injured by stink bugs. Extracts of PI 227687 pods (damaged or not) were deterrent to adults of N. viridula, and insect injury increased concentrations of daidzin and genistin in PI 227687 seeds. The deterrence seemed to be more pronounced after pods had suffered stink-bug injury.