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Cultivar Williams 82 has served as the reference genome for the soybean research community since 2008, but is known to have areas of genomic heterogeneity among different sub-lines. This work provides an updated assembly (version Wm82.a6) derived from a specific sub-line known as Wm82-ISU-01 (seeds available under USDA accession PI 704477). The genome was assembled using Pacific BioSciences HiFi reads and integrated into chromosomes using HiC. The 20 soybean chromosomes assembled into a genome of 1.01Gb, consisting of 36 contigs. The genome annotation identified 48 387 gene models, named in accordance with previous assembly versions Wm82.a2 and Wm82.a4. Comparisons of Wm82.a6 with other near-gapless assemblies of Williams 82 reveal large regions of genomic heterogeneity, including regions of differential introgression from the cultivar Kingwa within approximately 30 Mb and 25 Mb segments on chromosomes 03 and 07, respectively. Additionally, our analysis revealed a previously unknown large (>20 Mb) heterogeneous region in the pericentromeric region of chromosome 12, where Wm82.a6 matches the 'Williams' haplotype while the other two near-gapless assemblies do not match the haplotype of either parent of Williams 82. In addition to the Wm82.a6 assembly, we also assembled the genome of 'Fiskeby III,' a rich resource for abiotic stress resistance genes. A genome comparison of Wm82.a6 with Fiskeby III revealed the nucleotide and structural polymorphisms between the two genomes within a QTL region for iron deficiency chlorosis resistance. The Wm82.a6 and Fiskeby III genomes described here will enhance comparative and functional genomics capacities and applications in the soybean community.

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This study reports the use of the Arabidopsis KASII promoter (AtKASII) to develop an efficient CRISPR/Cas9 system for soybean genome editing. When this promoter was paired with Arabidopsis U6 promoters to drive Cas9 and single guide RNA expression, respectively, simultaneous editing of the three fatty acid desaturase genes GmFAD2-1A, GmFAD2-1B, and GmFAD3A occurred in more than 60% of transgenic soybean lines at T2 generation, and all the triple mutants possessed desirable high-oleic traits. In sharp contrast, not a single line underwent simultaneous editing of the three target genes when AtKASII was replaced by the widely used AtEC1.2 promoter. Furthermore, our study showed that the stable and inheritable mutations in the high-oleic lines did not alter the overall contents of oil and protein or amino acid composition while increasing the oleic acid content up to 87.6% from approximately 23.8% for wild-type seeds, concomitant with 34.4- and 3.7-fold reductions in linoleic and linolenic acid, respectively. Collectively, this study demonstrates that the AtKASII promoter is highly promising for optimization of the CRISPR/Cas9 system for genome editing in soybean and possibly beyond.

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Isoflavones, secondary metabolites with numerous health benefits, are predominantly found in legume seeds, especially soybean; however, their contents in domesticated soybean seeds are highly variable. Wild soybeans are known for higher seed isoflavone contents than cultivars. Here we used experimental and modelling approaches on wild soybean (W05) and cultivated soybean (C08) to delineate factors influencing isoflavone accumulation. We found imported nutrients were converted into storage compounds, with isoflavone accumulation in W05 seeds being faster than in C08 ones. The isoflavone accumulation during seed development was simulated using context-specific cotyledon metabolic models of four developmental stages on cultivar C08, and the metabolic burden imposed by increasing biomass was evaluated. Trade-off analyses between biomass and isoflavone suggest that high biomass requirement in cultivars could limit the reallocation of resources for secondary metabolite production. Isoflavone production in mature seeds was also influenced by biomass compositions. Seeds with higher carbohydrate contents favour isoflavone production, while those with highest protein and oil contents had lowest isoflavone contents. Although seeds could synthesize isoflavones on their own, the predicted fluxes from biosynthesis alone were lower than the empirical levels. Shadow price analyses indicated that isoflavone accumulation depended on both intrinsic biosynthesis and direct contribution from the plant.

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Soil salinization is one of the major factors limiting agricultural production. Utilizing beneficial microorganisms like Piriformospora indica (P. indica) to enhance plant tolerance to abiotic stresses is a highly effective method, but the influence of P. indica on the growth of soybean in natural saline-alkaline soil remains unclear. Therefore, we investigated the effects of non-inoculation, P. indica inoculation, and fertilization on the growth, antioxidant defense, osmotic adjustment, and photosynthetic gas exchange parameters of soybean under two different levels of saline-alkaline stress in non-sterilized natural saline-alkaline soil. The study found that: 1) P. indica inoculation significantly promoted soybean growth, increasing plant height, root length, and biomass. Under mildly saline-alkaline stress, the increases were 11.5%, 16.0%, and 14.8%, respectively, compared to non-inoculated treatment. Under higher stress, P. indica inoculation achieved the same level of biomass increase as fertilization, while fertilization only significantly improved stem diameter. 2) Under saline-alkaline stress, P. indica inoculation significantly increased antioxidant enzyme activities and reduced malondialdehyde (MDA) content. Under mildly stress, MDA content was reduced by 47.1% and 43.3% compared to non-inoculated and fertilized treatments, respectively. Under moderate stress, the MDA content in the inoculated group was reduced by 29.9% and 36.6% compared to non-inoculated and fertilized treatments, respectively. Fertilization only had a positive effect on peroxidase (POD) activity. 3) P. indica inoculation induced plants to produce more osmotic adjustment substances. Under mildly stress, proline, soluble sugars, and soluble proteins were increased by 345.7%, 104.4%, and 6.9%, respectively, compared to non-inoculated treatment. Under higher stress, the increases were 75.4%, 179.7%, and 12.6%, respectively. Fertilization had no significant positive effect on proline content. 4) With increasing stress, soybean photosynthetic capacity in the P. indica-inoculated treatment was significantly higher than in the non-inoculated treatment, with net photosynthetic rate increased by 14.8% and 37.0% under different stress levels. These results indicate that P. indica can enhance soybean's adaptive ability to saline-alkaline stress by regulating ROS scavenging capacity, osmotic adjustment substance content, and photosynthetic capacity, thereby promoting plant growth. This suggests that P. indica has great potential in improving soybean productivity in natural saline-alkaline soils.

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Various species of rhizobium establish compatible symbiotic relationships with soybean (Glycine max) leading to the formation of nitrogen-fixing nodules in roots. The formation of functional nodules is mediated through complex developmental and transcriptional reprogramming that involves the activity of thousands of plant genes. However, host transcriptome that differentiate between functional or non-functional nodules remain largely unexplored. In this study, we investigated differential compatibilities between rhizobium strains (Bradyrhizobium diazoefficiens USDA110 Bradyrhizobium sp. strain LVM105) and cultivated and wild soybeans. The nodulation assays revealed that both USDA110 and LVM105 strains effectively nodulate G. soja but only USDA110 can form symbiotic relationships with Williams 82. LVM105 formed pseudonodules on Williams 82 that consist of a central nodule-like mass that are devoid of any rhizobia. RNA-seq data revealed that USDA110 and LVM105 induce distinct transcriptome programing in functional mature nodules formed on G. soja roots, where genes involved in nucleosome assembly, DNA replication, regulation of cell cycle, and defense responses play key roles. Transcriptome comparison also suggested that activation of genes associated with cell wall biogenesis and organization and defense responses together with downregulation of genes involved in the biosynthesis of isoprenoids and antioxidant stress are associated with the formation of non-functional nodules on Williams 82 roots. Moreover, our analysis implies that increased activity of genes involved in oxygen binding, amino acid transport, and nitrate transport differentiates between fully-developed nodules in cultivated versus wild soybeans.

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Soybean is one of the most important crops for producing high quality oil and protein. Mineral nutrient deficiencies are frequently observed in soybeans. However, there are few studies to understand the absorption process of mineral nutrients in soybeans. Here, we investigated the functions of soybean (Glycine max L.) IRT1.1 (IRON-REGULATED TRANSPORTER 1.1) in the transportation of mineral elements. Heterologous expression of GmIRT1.1 in yeast mutants revealed that GmIRT1.1 compensated for the growth defects of Δfet3fet4 and Δsmf1 mutants under iron (Fe) and manganese (Mn) deficiency conditions, respectively, and enhanced the sensitivity of the Δycf1 mutant to cadmium (Cd) toxicity. Expression analysis revealed that GmIRT1.1 was only significantly induced by Fe deficiency and was primarily expressed in roots. Furthermore, the GmIRT1.1 overexpression lines enhanced Arabidopsis tolerance to Fe deficiency, leading to increased accumulation of Fe in the roots and shoots. Additionally, the transgenic lines increased the sensitivity to Mn and Cd toxicity. Subcellular localization analysis revealed that GmIRT1.1 was localized on the plasma membrane. Moreover, the results obtained from the soybean hairy roots system indicated that the localization of GmIRT1.1 was dependent on the regulation of Fe homeostasis in plant. Consequently, these results suggested that GmIRT1.1 was responsible for the transportation of Fe, Mn and Cd.

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Phytic acid (IP6) and its degradation products lower myo-inositol phosphates exert different impacts on nutrient bioavailability and product quality characteristics. However, information regarding the occurrence of IP6 and its degradation products is scarce. In this work, simultaneous determination of IP6 and its degradation products in soybeans was developed, with emphasis on analysis by UPLC-MS/MS and a BEH Amide column both with hybrid surface technology. The retention and analyte/metal surface interactions issues were effectively addressed without ion-pairing reagents addition or derivatization. This method was applied to analyze soybeans from China. Total contents were 0.44-13.2 mg/g, and IP6 and its degradation product myo-inositol pentakisphosphate (IP5) were the predominant analytes, accounting for over 99%. Accession type significantly affected IP5 content, and landraces had significantly higher IP5 than cultivars. Geographically, the lowest IP6 was concentrated in the Huanghuaihai region. Significant correlations existed between IP6 and longitude, altitude, and annual cumulative sunshine hours. This study provides comprehensive insights into the IP6 and its degradation product profile in soybeans, which will benefit breeding soybeans based on specific requirements.

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Non-CG DNA methylation, a plant-specific epigenetic mark mainly regulated by chromomethylase (CMT), is known to play important roles in Arabidopsis thaliana. However, whether and to what extent non-CG DNA methylation modulates agronomic traits in crops remain to be explored. Here, we describe the consequences of non-CG DNA hypomethylation on development, seed composition, and yield in soybean (Glycine max). We created a Gmcmt mutant line lacking function of all four CMT genes. This line exhibited substantial hypomethylation of non-CG (CHG and CHH) sites. Non-CG hypomethylation enhanced chromatin accessibility and promoted or repressed the expression of hundreds of functionally relevant genes, including upregulation of GOLDEN-LIKE 10 (GmGLK10), which led to enhanced photosynthesis and, unexpectedly, improved nitrogen fixation efficiency. The Gmcmt line produced larger seeds with increased protein content. This study provides insights into the mechanisms of non-CG methylation-based epigenetic regulation of soybean development and suggests viable epigenetic strategies for improving soybean yield and nutritional value.

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BACKGROUND: Soybean is a worldwide-cultivated crop due to its applications in the food, feed, and biodiesel industries. Genome editing in soybean began with ZFN and TALEN technologies; however, CRISPR/Cas has emerged and shortly became the preferable approach for soybean genome manipulation since it is more precise, easy to handle, and cost-effective. Recent reports have focused on the conventional Cas9 nuclease, Cas9 nickase (nCas9) derived base editors, and Cas12a (formally Cpf1) as the most commonly used genome editors in soybean. Nonetheless, several challenges in the complex plant genetic engineering pipeline need to be overcome to effectively edit the genome of an elite soybean cultivar. These challenges include (1) optimizing CRISPR cassette design (i.e., gRNA and Cas promoters, gRNA design and testing, number of gRNAs, and binary vector), (2) improving transformation frequency, (3) increasing the editing efficiency ratio of targeted plant cells, and (4) improving soybean crop production. AIM OF REVIEW: This review provides an overview of soybean genome editing using CRISPR/Cas technology, discusses current challenges, and highlights theoretical (insights) and practical suggestions to overcome the existing bottlenecks. KEY SCIENTIFIC CONCEPTS OF REVIEW: The CRISPR/Cas system was discovered as part of the bacterial innate immune system. It has been used as a biotechnological tool for genome editing and efficiently applied in soybean to unveil gene function, improve agronomic traits such as yield and nutritional grain quality, and enhance biotic and abiotic stress tolerance. To date, the efficiency of gRNAs has been validated using protoplasts and hairy root assays, while stable plant transformation relies on Agrobacterium-mediated and particle bombardment methods. Nevertheless, most steps of the CRISPR/Cas workflow require optimizations to achieve a more effective genome editing in soybean plants.

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Aluminum (Al) toxicity adversely impacts soybean (Glycine max) growth in acidic soil. Reversible protein phosphorylation plays an important role in adapting to adverse environmental conditions by regulating multiple physiological processes including signal transduction, energy coupling and metabolism adjustment in higher plant. This study aimed to reveal the Al-responsive phosphoproteins to understand their putative function and involvement in the regulation of Al resistance in soybean root. We used immobilized metal affinity chromatography to enrich the key phosphoproteins from soybean root apices at 0, 4, or 24 h Al exposure. These phosphoproteins were detected using liquid chromatography-tandem mass spectrometry measurement, verified by parallel reaction monitoring (PRM), and functionally characterized via overexpression in soybean hairy roots. A total of 638 and 686 phosphoproteins were identified as differentially enriched between the 4-h and 0-h, and the 24-h and 0-h Al treatment comparison groups, respectively. Typically, the phosphoproteins involved in biological processes including cell wall modification, and RNA and protein metabolic regulation displayed patterns of decreasing enrichment (clusters 3, 5 and 6), however, the phosphoproteins involved in the transport and metabolic processes of various substrates, and signal transduction pathways showed increased enrichment after 24 h of Al treatment. The enrichment of phosphoproteins in organelle organization bottomed after 4 h of Al treatment (cluster 1). Next, we selected 26 phosphoproteins from the phosphoproteomic profiles, assessed their enrichment status using PRM, and detected enrichment patterns similar to those observed via phosphoproteomic analysis. Among them, 15 phosphoproteins were found to reduce the accumulation of Al and callose in Al-stressed soybean root apices when their corresponding genes were individually overexpressed in soybean hairy roots. In summary, the findings of this study facilitated a comprehensive understanding of the protein phosphorylation events involved in Al resistance responses and revealed some critical phosphoproteins that enhance Al resistance in soybean roots.

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Hydrogen sulphide (H2S) is required for optimal establishment of soybean (Glycine max)-Sinorhizobium fredii symbiotic interaction, yet its role in regulating the nitrogen fixation-senescence transition remains poorly understood. A S. fredii cystathionine γ-lyase (CSE) mutant deficient in H2S synthesis showed early nodule senescence characterized by reduced nitrogenase activity, structural changes in nodule cells, and accelerated bacteroid death. In parallel, the CSE mutant facilitated the generation of reactive oxygen species (ROS) and elicited antioxidant responses. We observed that H2S-mediated persulfidation of cysteine C31/C80 in ascorbate peroxidase (APX) and C32 in APX2 modulated enzyme activity, thereby participating in hydrogen peroxide (H2O2) detoxification and delaying nodule senescence. Comparative transcriptomic analysis revealed a significant up-regulation of GmMYB128, an MYB transcription factor (TF), in the CSE mutant nodules. Functional analysis through overexpression and RNAi lines of GmMYB128 demonstrated its role as a positive regulator in nodule senescence. MYB128-OE inoculated with the CSE mutant strain exhibited a reduction in nitrogenase activity and a significant increase in DD15 expression, both of which were mitigated by NaHS addition. Changes at the protein level encompassed the activation of plant defenses alongside turnover in carbohydrates and amino acids. Our results suggest that H2S plays an important role in maintaining efficient symbiosis and preventing premature senescence of soybean nodules.

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Soybean (Glycine max) is the world's most cultivated legume; currently, most of its varieties are Bt. Spodoptera spp. (Lepidoptera: Noctuidae) are important pests of soybean. An artificial neural network (ANN) is an artificial intelligence tool that can be used in the study of spatiotemporal dynamics of pest populations. Thus, this work aims to determine ANN to identify population regulation factors of Spodoptera spp. and predict its density in Bt soybean. For two years, the density of Spodoptera spp. caterpillars, predators, and parasitoids, climate data, and plant age was evaluated in commercial soybean fields. The selected ANN was the one with the weather data from 25 days before the pest's density evaluation. ANN forecasting and pest densities in soybean fields presented a correlation of 0.863. It was found that higher densities of the pest occurred in dry seasons, with less wind, higher atmospheric pressure and with increasing plant age. Pest density increased with the increase in temperature until this curve reached its maximum value. ANN forecasting and pest densities in soybean fields in different years, seasons, and stages of plant development were similar. Therefore, this ANN is promising to be implemented into integrated pest management programs in soybean fields.

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Arsenic (As) contamination of agricultural soils poses a serious threat to crop productivity and food safety. Zinc oxide nanoparticles (ZnONPs) have emerged as a potential amendment for mitigating the adverse effects of As stress in plants. Soybean crop is mostly grown on marginalized land and is known for high accumulation of As in roots than others tissue. Therefore, this study aimed to elucidate the underlying mechanisms of ZnONPs in ameliorating arsenic toxicity in soybean. Our results demonstrated that ZnOB significantly improved the growth performance of soybean plants exposed to arsenic. This improvement was accompanied by a decrease (55%) in As accumulation and an increase in photosynthetic efficiency. ZnOB also modulated hormonal balance, with a significant increase in auxin (149%), abscisic acid (118%), gibberellin (160%) and jasmonic acid content (92%) under As(V) stress assuring that ZnONPs may enhance root growth and development by regulating hormonal signaling. We then conducted a transcriptomic analysis to understand further the molecular mechanisms underlying the NPs-induced As(V) tolerance. This analysis identified genes differentially expressed in response to ZnONPs supplementation, including those involved in auxin, abscisic acid, gibberellin, and jasmonic acid biosynthesis and signaling pathways. Weighted gene co-expression network analysis identified 37 potential hub genes encoding stress responders, transporters, and signal transducers across six modules potentially facilitated the efflux of arsenic from cells, reducing its toxicity. Our study provides valuable insights into the molecular mechanisms associated with metalloid tolerance in soybean and offers new avenues for improving As tolerance in contaminated soils.

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The growth of cover crops can contribute to the increase in phosphorus content at depth by root decomposition. The aim of this work was to verify the effect of cover crops on soil phosphorus availability and use by successive plants, and the accumulation of soil P in a no-tillage system conducted for 14 years. This research was carried out during the 2016/2017 and 2017/2018 crop seasons, whose treatments have been installed and maintained since 2003. The experimental design was a randomized block design, and the plots consisted of spring crops: pearl millet, forage sorghum, sunn hemp, and additionally, a fallow/chiseling area. The evaluation of available P was determined by P fractionation. In general, in the two years of evaluation, the accumulation of P in the shoot dry matter was higher in sunn hemp growth, on average 25% higher than pearl millet in 2016 and 40% higher than sorghum in 2017. The highest contents of labile inorganic P were in the sorghum-soybean and fallow/chiseling-soybean successions, with values higher than 50 mg kg-1 of P in the 0-0.1 m soil layer. However, in the other layers analyzed, the cover crops obtained higher availability of labile inorganic P. The systems using cover crops recovered 100% of the P fertilized in soybean.

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Most soybean producers in the Cerrado biome use the direct seeding system, making it essential to cultivate cash or cover crops in the off-season, to promote soil protection, as well as increase organic matter, which is directly related to improvements in the chemical and physical characteristics of these soils. In this sense, this work was conducted in Jataí, state of Goias, Brazil, to evaluate the physical-chemical attributes of the soil and the performance of soybeans cultivated in different crop succession systems cultivated for 6 years in the region of Jataí, GO. The experimental design was randomized blocks with four plots and four replications; the crops that followed soybeans were arranged as follows: T1-corn (Zea mays); T2-pearl millet (Pennisetum glaucum); T3-Urochloa ruziziensis; and T4-corn + Urochloa ruziziensis. Soybean yield components and grain yield were evaluated in two harvests (2020/2021 and 2021/2022). Deformed and undisturbed soil samples were collected in 2022 to assess soil fertility and for physical analysis. The data were subjected to analysis of variance (F test) and the means were compared using the Tukey test at 5% probability. The soybean-millet succession system stood out for the chemical and physical attributes of the soil: calcium, magnesium, base saturation, hydrogen + aluminum, and total porosity. The crop succession system did not affect yield for the two years analyzed, but the accumulated grain yields were higher in the crop succession soybean/corn intercropped. The results highlight the importance of using cover crops in improving the physical and chemical qualities of the soil in the long term. However, in the Cerrado, there is a predominance of the soybean/corn succession system motivated by financial issues to the detriment of the qualitative aspects of the soil, in which the introduction of Urochloa ruziziensis in intercropping with corn would improve the chemical attributes of the soil and have a long-term impact on the accumulated grain production.

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Soybean meals (SBM) from different locations differ in their protein content, subsequently influencing their amino acid (AA) profile. In this study, standardized ileal digestibility (SID) of AA and growth or production performance were evaluated in pullets and hens fed SBM derived from soybean grown in Western Canada, primarily Manitoba (MB) labelled as A-, B- and C-SBM compared with that from Eastern Canada (Ontario, ON-SBM) and contained 38.3 ± 0.44, 38.6 ± 0.61, 39.4 ± 0.49, or 44.0 ± 0.87% CP, respectively. A N-free diet was used to determine basal ileal endogenous losses of AA. The study included the grower, developer, and layer phases (9-12, 13-16, and 44/59-64-wk old birds, respectively). Although a lower (P = 0.029) SID for cysteine was noted in the grower phase for the C-SBM compared with other SBM, the developer phase had higher (P < 0.05) SID for methionine, phenylalanine, cysteine (more by 4.4, 2.4 and 7.2% units, respectively) on average for SBM samples from MB compared with the ON-SBM. Regardless the source of the SBM, no difference in SID of AA was noted in the layer phase. Overall, in all phases the SID values of most AA in the SBM from MB were comparable with the ON-SBM, which may be linked to higher values of these AA per unit of protein content in the former source. In addition, the growth performance including feed intake, BW gain and feed conversion ratio in pullets, and egg production/quality in layers were similar between treatments. These findings show that the MB-SBM have a comparable feeding value with the ON-SBM, hence represent a suitable alternative protein source for poultry.

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Soybeans' isoflavone content increases with germination; nevertheless, their bioaccessibility in the gastrointestinal system is limited. This study evaluated the influence of germination time (1, 3, 5, and 7 days) and in vitro gastrointestinal conditions on the isoflavone profile of soybean sprouts. The total isoflavones (4.07 mg/g) and the malonyl genistin (1.37 mg/g) had the highest contents on day 5 in the gastric phase. The highest isoflavone bioaccessibility was observed in daidzein, genistein, and glycitin. An increase in antioxidant capacity was found during germination (day 7 > day 5 > day 3); however, the same trend was not observed during in vitro digestion. In summary, the results indicate that soybean sprouts germinated for 5 days may be more beneficial for consumption since they have the highest and most readily absorbed levels of isoflavones. These data suggest that soybean sprouts may be a functional food that provides bioavailable antioxidants.

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The plant microbiome and plant-associated bacteria are known to support plant health, but there are limited studies on seed and seedling microbiome to reveal how seed-associated bacteria may confer disease resistance. In this study, the application of antibiotics on soybean seedlings indicated that seed-associated bacteria were involved in the seed rot resistance against a soil-borne pathogen Calonectria ilicicola, but this resistance cannot be carried to withstand root rot. Using PacBio 16S rRNA gene full-length sequencing and microbiome analyses, 14 amplicon sequence variants (ASVs) including 2 ASVs matching to Bacillus altitudinis were found to be more abundant in the four most resistant varieties versus the four most susceptible varieties. Culture-dependent isolation obtained two B. altitudinis isolates that both exhibit antagonistic capability against six fungal pathogens. Application of B. altitudinis on the most resistant and susceptible soybean varieties revealed different colonization compatibility, and the seed rot resistance was restored in the five varieties showing higher bacterial colonization. Moreover, quantitative PCR confirmed the persistence of B. altitudinis on apical shoots till 21 days post-inoculation (dpi), but 9 dpi on roots of the resistant variety TN5. As for the susceptible variety HC, the persistence of B. altitudinis was only detected before 6 dpi on both shoots and roots. The short-term colonization of B. altitudinis on roots may explain the absence of root rot resistance. Collectively, this study advances the insight of B. altitudinis conferring soybean seed rot resistance and highlights the importance of considering bacterial compatibility with plant varieties and colonization persistence on plant tissues.

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From 2016 to 2021, nematode surveys in Florida strawberry fields revealed several species of foliar nematodes (Aphelenchoides spp.). Aphelenchoides besseyi sensu stricto was detected only in 2016 and 2017 on photosynthetic strawberry leaves/buds, but other not well characterized populations of Aphelenchoides sp. were found on declining/dessicated leaves. Morphological analyses showed that these samples of Aphelenchoides sp. consisted of A. bicaudatus, a species detected in Florida for the first time, and A. rutgersi, a species previously reported in Florida from the citrus rhizosphere. These two species differed from A. besseyi in the shape of their tail terminus: bifurcate in A. bicaudatus; mucronate with a ventral thin mucro in A. rutgersi; and stellate in A. besseyi. One population each of these species was used for morphological and molecular analyses after being reared on Monilinia fructicola. Body and tail length differences were observed among Florida A. bicaudatus and other populations from the Far East and South Africa. Phylogenetic analyses of the rRNA gene sequences showed that Florida A. bicaudatus grouped with those of species from South Korea, Taiwan, and the Netherlands and several other populations listed as Aphelenchoides sp. from Brazil, Costa Rica, and Japan, which were considered as representatives of A. bicaudatus in this study. Similarly, sequences of Florida A. rutgersi grouped with those from environmental samples in Japan and North Carolina, which were listed as Aphelenchoides sp. and were considered as representatives of A. rutgersi in this study. Photosynthetic strawberry leaf samples were free from both A. bicaudatus and A. rutgersi, indicating that these two species did not damage strawberry. They were associated with desiccated leaves and/or propagative stolons, usually infected by fungi, confirming that they are mycetophagous under field conditions in this study. Results of soybean leaf inoculation on moist filter paper containing A. bicaudatus specimens showed that this species could become phytophagous under artificial conditions. Nematodes penetrated the leaf epidermis and migrated into the mesophyll causing leaf tissue discoloration/necrosis, which remained localized within the infested area. Soybean leaf damage was almost negligible, and no nematode reproduction was observed in the inoculated soybean areas.

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The HKT transporter plays an important role for plants in response to salt stress, but the transport property of the soybean HKT transporters at the molecular level is still unclear. Here, using Xenopus oocyte as a heterologous expression system and two-electrode voltage-clamp technique, we identified four HKT transporters, GmHKT1;1, GmHKT1;2, GmHKT1;3, and GmHKT1;4, which all belong to type I subfamily, but having distinct ion transport properties. While GmHKT1;1, GmHKT1;2 and GmHKT1;3 function as Na+ transporters, GmHKT1;1 is less selective against K+ than the two other transporters. Astonishingly, GmHKT1;4, which lacks transmembrane segments and has no ion permeability, is significantly expressed, and its gene expression pattern is different from the other three GmHKTs under salt stress. Interestingly, GmHKT1;4 reduced the Na+/K+ currents mediated by GmHKT1;1. Further study showed that the transport ability of GmHKT1;1 regulated by GmHKT1;4 was related to the structural differences in the first intracellular domain and the fourth repeat domain. Overall, we have identified one unique GmHKT member, GmHKT1;4, which modulates the Na+ and K+ transport ability of GmHKT1;1 via direct interaction. Thus, we have revealed a new type of HKTs interaction model for altering their ion transport properties.