Cucumber Fusarium wilt caused by Fusarium oxysporum f. sp. cucumerinum (FOC) severely threatens global cucumber production. Current control strategies face limitations in sustainability and efficacy, highlighting the need for new, cost-effective, and environmentally friendly alternatives. In this study, from a library of 675 fungal isolates, Trichoderma koningiopsis Snef2767, T. asperellum Snef2773, and T. virens Snef2774 exhibited broad-spectrum antagonism against FOC and F. oxysporum f. sp. melonis (FOM). Among them, Trichoderma koningiopsis Snef2767 exhibited the highest in vitro inhibition rates against FOC and FOM, which were 85.32% and 85.66%, respectively. In pot trials, root irrigation with culture filtrate of Snef2767, Snef2773, and Snef2774 reduced disease severity and enhanced plant growth, with biocontrol efficacies of 60.38%, 52.83%, and 39.63%, respectively. Two-year field trials confirmed consistent control efficacy of the three strains, with the control efficacy of Snef2767 being particularly remarkable (55.84%–56.11%). Mechanistically, all three strains inhibited FOC spore germination and mycelial growth via non-volatile metabolites and hyperparasitized FOC hyphae, inducing shrinkage and degradation. Furthermore, Snef2767 primed cucumber resistance by upregulating salicylic acid (SA) pathway genes and jasmonic acid (JA) pathway genes in cucumber roots, while also enhancing antioxidant enzymes. The high efficacy under field conditions and plant growth-promoting ability support the potential of Snef2767 as a sustainable biocontrol agent.

Root-knot nematodes (Meloidogyne spp.) cause catastrophic yield losses in global agriculture. This study identified itaconic acid (IA), through comparative metabolomic analysis (the study of small molecules in biological systems), as a key virulence-related metabolite produced by the fungus Trichoderma citrinoviride Snef1910. In vitro assays demonstrated potent nematicidal activity of IA, with an LC50 value of 243.4 mg/liter against J2s of M. incognita and 78.4% egg-hatching inhibition at 800 mg/liter, performance that is comparable with the structural analog trans-aconitic acid. Pot and greenhouse field trials demonstrated that IA has a significant impact against M. incognita while enhancing tomato growth. Seed priming with IA compensated for the damage of nematodes to the photosynthetic pigments and activated systemic resistance of tomatoes, and treatment with 100 mg/liter of IA enhanced catalase and ascorbate peroxidase activities, while concurrently inhibiting H2O2/MDA accumulation. Crucially, IA production by T. citrinoviride positions this strain as a self-fertilizing organism for production of a nematicide with plant-growth-promoting properties. This study highlights the dual functionality of IA in nematode management, offering a sustainable alternative to synthetic agrochemicals.

Peanut root-knot nematode (RKN) disease caused by Meloidogyne hapla is among the most destructive soil-borne diseases threatening peanut production worldwide. We isolated Trichoderma harzianum strain Snef3255 from peanut rhizosphere soil, which exhibited high virulence against second-stage juveniles, with a mortality rate of 86.08%. Field trials demonstrated that TC and TS provided control efficacies of 70.85% and 61.88%, respectively, while boosting peanut yields by 18.15% and 13.61%. We report a high-quality chromosome-level genome assembly of T. harzianum Snef3255 using Oxford Nanopore, Illumina and high-throughput chromatin conformation capture (Hi-C) sequencing data. This assembly comprised seven chromosomes (40,811,729 bp) with a BUSCO completeness of 99.31%. A total of 13,611 protein-coding genes were predicted. Systematic comparative genomic analysis of T. harzianum Snef3255 with other Trichoderma species was performed, and putative functional gene clusters were investigated. Furthermore, we identified two candidate secreted proteins, ThCP1 and ThLysM1, and screened their interacting proteins in peanut, respectively. This study demonstrated the potential of Snef3255 as a biocontrol agent for peanut RKN disease and provided a genomic basis for understanding Trichoderma–peanut interactions.

Background Biological control is widely recognized for its environmental benefits and has gained increasing attention. The peptide flg22 derived from biocontrol bacteria Pseudomonas fragi Sneb1990 (flg22(Pf)) exhibits significant efficacy against Meloidogyne incognita, but its mechanism is still unknown. Results In this study, we cloned the full-length flagellin gene from P. fragi Sneb1990 and found that it shares 32.02% sequence identity with the flagellin from non-pathogenic Pseudomonas syringae pv. tomato DC3000 (Pst DC300). Compared to flg22 derived from Pst DC3000 (flg22(Ps)), flg22(Pf) contains an amino acid substitution at position 19. Growth inhibition assays in Arabidopsis seedlings confirmed that flg22(Pf) activates immunity in an FLS2-dependent manner. The Nicotiana benthamiana leaf inoculation experiments indicated that flg22(Pf) significantly induces the expression of immune-related genes PTI5 and WRKY7, thereby enhancing resistance against Pst DC3000 infection. Furthermore, tomato treatment with flg22(Pf) promoted H2O2 production, ROS accumulation, callose deposition, and lignin accumulation. Consequently, this induction of defense responses resulted in suppressed nematode infestation. Conclusion Collectively, our results reveal that flg22(Pf) from P. fragi Sneb1990 elicits a multi-layered immune response similar to flg22(Ps), leading to an enhanced early immune response in tomato against M. incognita infestation. This study provides a novel plant immune-based strategy for sustainable M. incognita control.

Soybean, an essential oil crop in China, has witnessed accelerated seed transfer domestically and abroad in recent years. Seed carriage has emerged as a major route for the dissemination of soybean diseases. In this study, 14 soybean cultivars from three northeastern provinces were collected and examined for seed-borne microorganisms using traditional detection technology and high-throughput sequencing technology. Through traditional detection techniques, a total of six genera of bacteria and seventeen genera of fungi were isolated from the test varieties. The quantity and types of microorganisms on the seed surface were greater than those on the seed coat and within the seed, while the seed coat and internal seed contained fewer microorganisms. The dominant fungal genera were Cladosporium, Fusarium, Aspergillus, and Alternaria, accounting for 21.23%, 17.45%, 15.57%, and 11.56% of the genera, respectively. The dominant bacterial genera were Pseudomonas, Sphingomonas, and Pantoea, accounting for 37.46%, 17.29%, and 15.27% of the genera, respectively. The dominant genera obtained through traditional seed-carrying assay techniques were also dominant in high-throughput sequencing. However, some dominant genera obtained through high-throughput sequencing were not isolated by traditional methods. High-throughput sequencing analysis revealed that soybean seeds from Jilin Province had the highest abundance of seed-borne fungi, followed by seeds from Liaoning Province and Heilongjiang Province. Jilin Province also had the highest abundance of seed-borne bacteria, followed by Heilongjiang Province and Liaoning Province. The isolation and identification of microorganisms on soybean seeds provide a scientific basis for seed quarantine treatment and disease control, which is of great significance for soybean production in China.

SWEET (Sugars Will Eventually be Exported Transporter) proteins facilitate the movement of sugars through cell membranes and are essential for loading sucrose into phloem. Beyond its role in sugar transport, the SWEET protein also modulates plant resistance to various biotic and abiotic stresses. Among sugar transporter genes, GmSWEET20 is a positive regulatory factor involved in soybean cyst nematode (Heterodera glycines) resistance. In this study, susceptible soybean cultivars (Williams 82) were used to conduct a transcriptome analysis to characterize the responses to nematode infection, in which multiple sugar transporter genes were highly expressed. The RT-qPCR analysis confirmed a significant increase in the expression of GmSWEET20 in soybean roots with H. glycines infection. Heterologous expression tests indicated that the protein encoded by GmSWEET20 does not transport hexose in yeast. Further analysis showed that soybean lines overexpressing GmSWEET20 exhibited increased resistance to H. glycines compared with the control. A yeast one-hybrid assay was employed to discover that LOC114390362 binds to the GmSWEET20 promoter. Transient expression in tobacco leaf cells revealed nucleus and cytosolic localization of LOC114390362. LOC114390362-overexpressing soybean lines showed a reduced number of nematode infections. Overall, the results indicate that the binding of LOC114390362 to the GmSWEET20 promoter plays a positive role in regulating soybean resistance to H. glycines. The GmSWEET20 gene has great potential to improve resistance to plant-parasitic nematodes in soybean and other plants.

Root-knot nematodes (Meloidogyne incognita) present a significant threat to global agriculture, and the development of multi-drug resistance in these nematodes exacerbates this problem. Benzothiazole, a heterocyclic compound has been reported as a potential nematicide, however, its mode of action is not fully understood. This study aims to elucidate the nematicidal mechanism of benzothiazole against M. incognita. In the toxicity assays, benzothiazole exhibited rapid and effective nematocidal activity, significantly compromising egg masses and inhibiting egg hatching while killing newly hatched second-stage juveniles (J2s) of M. incognita. Microscopic observations revealed that after 48 h of incubation, a marked reduction in protein and carbohydrate levels within the J2s was observed. Notably, benzothiazole at a concentration of 14 mmol/L significantly inhibits glutathione S-transferase (GST) enzyme activity, leading to the accumulation of reactive oxygen species (ROS), ultimately resulting in rapid nematode death. Molecular docking and dynamics simulations demonstrated that benzothiazole forms a stable complex with GST, thereby disrupting its antioxidant function. Furthermore, in pot experiments, benzothiazole effectively reduced the gall formations of M. incognita on tomato roots. Overall, this novel inhibitory mechanism of glutathione S-transferase (GST) differs from that of the neurotoxicant abamectin, which targets glutamate-gated chloride channels (GluCl). This mechanism holds significant promise for the development of environmentally friendly nematicides. It offers a potential solution to the growing problem of multidrug resistance in root-knot nematodes and could help mitigate the substantial economic losses caused by these pests in global agriculture.

The soybean cyst nematode (SCN; Heterodera glycines) is one of the most devastating pathogens for soybean production. The second stage juvenile (J2) invades the host root, develops and form white females which then become brown cysts enter the soil. The brown cyst wall plays a key role in protecting inside eggs from adverse environmental conditions. However, the function of cyst wall tanning (sclerotization and pigmentation) in nematodes is not clear. A browning-related gene discovered from the whole-genome sequencing was cloned and characterized in this study, the gene was confirmed to be the laccase gene and was named HgLac. HgLac mRNA and HgLac protein was detected in the epidermis of juveniles using in situ hybridization and immunolocalization techniques. The HgLac expression level was greater in fourth-stage juveniles (J4s) than in the other stages. Knockdown of HgLac by in vitro RNA interference (RNAi) significantly decreased the infectivity, development and reproduction of J2s but had no effect on cyst wall tanning. Further research revealed that HgLac expression in nematodes was significantly suppressed by 35.41-59.17 % through in planta RNAi, 52.96-58.19 % females could not tan successfully, and the female wall was very soft and fragile, with a low egg hatching rate (1.33 %), which was significantly lower than that of normal females (68.85 %). These results indicate that HgLac plays a key role in cyst wall tanning and suppressing the development and reproduction of the SCN, which provides new ideas for the use of this gene as a target to control SCN.

Soybean cyst nematodes (SCNs) are a significant disease that causes yield loss and reducing seed quality in soybeans (Glycine max). Developing SCN-resistant soybean varieties can minimize the need for insecticide use and reduce yield loss. Cinnamate-4-hydroxylase (C4H) and laccase (Lac) are key enzymes in the lignin synthesis pathway. In this study, SCN stress significantly promoted lignin accumulation in soybean roots and upregulated the expression of lignin signaling pathway genes GmC4H (Glyma.02G236500), GmLac55 (Glyma.13G076900), and GmLac85 (Glyma.20G051900). Using Agrobacterium rhizogenes-mediated transformation, the pNI900 expression vector was introduced into the soybean cultivar Williams 82 to generate GmLac55-overexpressing plants. The overexpression of GmLac55 enhanced soybean roots resistance to SCN and inhibited the further development of J2 larvae. Our study presents a strategy for increasing SCN resistance in soybean through Agrobacterium-mediated targeted mutagenesis of the GmLac55 gene.

Root-knot nematodes (RKNs, especially Meloidogyne incognita) are a growing threat to greenhouse tomatoes in China. However, control methods in the greenhouses in China are limited. The use of chemical pesticides such as fluopyram and thiazole phosphate to control M. incognita is expensive and harmful to the environment. The Chinese herb Asarum sieboldii is commonly used, and it shows potential as a plant-derived pesticide. We evaluated the effectiveness of A. sieboldii in regulating the soil nematode community and M. incognita in tomato greenhouses. Combining morphological identification with high-throughput sequencing techniques, we demonstrated that A. sieboldii root extract significantly reduced the relative abundance of plant-parasitic nematodes (PPNs) while promoting the ecological and functional recovery of bacterivorous (BF) and fungivorous (FF) nematodes. The aqueous extract exhibited high toxicity against M. incognita J2s and reduced the number of root galls by 69.5% per gram of root, decreased the number of egg masses by 58.4%, and enhanced shoot fresh weight by 53.6%. Through column chromatography and HPLC-MS analysis, mono-ethyl fumarate (MEF) and methyl gallate (MG) were identified as key active compounds, demonstrating LC50 values of 10.20 mg/L and 49.47 mg/L with root-knot suppression rates reaching 64.8% and 61.3% under greenhouse conditions. This study presents the first evidence that A. sieboldii aqueous extract effectively controls M. incognita without compromising the ecological balance of soil, providing a theoretical foundation for developing environmentally friendly botanical nematicides.

IntroductionHeterodera glycines is one of the most important pathogens of soybean production worldwide. Biological control provides a strategy for sustainable and environmentally friendly nematode management.MethodsIn this study, solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS) was used to reveal the volatile nematicidal compounds of Microbacterium maritypicum Sneb159 and the mode of action was further elucidated via whole genome sequencing.ResultsThe present study demonstrated that M. maritypicum Sneb159 fermentation broth showed strong nematicidal activities against H. glycines. The filtrate rather than bacterial cells played a role in the nematicidal character, and reduced the invasion number as well as suppressed the development of juveniles in soybean. The analysis of chemotaxis showed that M. maritypicum Sneb159 has a repellent effect on H. glycines in pot experiments. The volatiles produced by M. maritypicum Sneb159 are toxic to H. glycines for both juveniles and eggs. The seven compounds were analyzed using SPME-GC-MS. In the bioassays, dimethyl disulfide and dimethyl trisulfide showed both direct contact and fumigated effect on juveniles and eggs. The complete genome sequence of the bacterium M. maritypicum Sneb159 was completed using the PacBio sequencing platform. The genome comprised 3895529 bp and a 68.63% G + C content. Three secondary metabolites gene clusters were predicted by the antiSMASH system.DiscussionThe findings reveal multifunction of M. maritypicum Sneb159 towards H. glycines, and has the potential to be developed as a safe nematicidal agent.

Soybean cyst nematode (SCN; Heterodera glycines Ichinohe) is a plant-parasitic nematode that causes substantial yield losses in soybean production. Light signalling is a critical environmental factor that influences photomorphogenesis and carbohydrate metabolism. However, its transcriptional regulation under pathogen-induced stress remains unclear. In this study, the biological function and regulatory mechanism of TGACG-motif binding factor 3/4 (GmSTF3/4), a shoot-to-root mobile protein in soybean (Glycine max), were investigated during H. glycines infection. Evidence was provided that light signalling modulated soybean susceptibility to cyst nematode, marked by light-enhanced nematode infection and upregulation of photoreceptor gene expression post-infection. GmSTF3/4 interacted with CONSTITUTIVE PHOTOMORPHOGENIC1a (GmCOP1a) and mediated its degradation. The nematode was identified to accelerate the shoot-to-root translocation of GmSTF3/4. Phenotypic analysis revealed that GmSTF3/4 promoted nematode development, whereas GmCOP1a exerted an antagonistic effect. Furthermore, the integrated analyses of Cleavage Under Targets and Tagmentation (CUT&Tag) and RNA sequencing (RNA-seq) indicated that GmSTF3/4 bound to the promoters of multiple sugar transporter genes, including GmSWEET8, GmSWEET10b, GmSWEET13d, GmSUC8, and GmERD6-like. The subcellular localization confirmed their plasma membrane targeting, and functional validation in yeast demonstrated the sucrose transport activity of four of these genes. Transient expression assays of five candidate genes during nematode infection supported the positive regulatory role of GmSWEET10b in facilitating nematode infection and development, a result further supported by the Gmsweet10b mutant. Collectively, this study revealed that GmSTF3/4 enhanced soybean susceptibility to cyst nematodes by transcriptionally activating sugar transporter genes, offering a new avenue for SCN-resistance research.

The Gretchen Hagen 3 genes maintain endogenous hormone homeostasis by conjugating excess hormones with amino acids. Herein, we identified the members of the GH3 family in soybeans and analyzed their phylogeny, gene duplication, structure, domains, conserved motifs, cis-elements in promoter regions for stress responses, and functional characteristics. We found that GH3 genes are induced by pathogens in Group-II. Furthermore, eight out of 16 Group-II genes responded to cyst nematode infection. Overexpression of eight GmGH3 genes can enhance soybean resistance to the cyst nematode. In addition, our metabolomic analysis showed that overexpression of them affected the content of salicylic acid, jasmonic acid, indole-3-acetic acid, and gibberellic acid. Overexpression of GmGH3 in soybean affects the expression of genes involved in plant hormone biosynthesis. This provides valuable insights into the complex molecular mechanisms underlying the interaction between soybeans and cyst nematodes.

Ubiquitination-related genes are pivotal in modulating plant responses to biotic stress. The U-box E3 ubiquitin ligase gene GmPUB33A has been identified as a negative regulator of resistance to soybean cyst nematode (SCN). To elucidate the underlying molecular mechanisms, we conducted high-throughput sequencing and transcriptome analyses on GmPUB33A-overexpression and -RNAi transgenic hairy roots. Three differentially expressed genes (DEGs) were silenced using siRNA for functional validation. Compared with empty vector control, 11,088 DEGs were identified. Gene Ontology (GO) and KEGG analyses revealed these DEGs were significantly enriched in defense responses, oxidative stress, phenylpropanoid biosynthesis, and MAPK signaling. Functional analysis revealed that silencing Glyma.07G061500 and Glyma.19G258700 reduced the number of nematodes by 32.62% and 19.34%, respectively. Furthermore, β-glucuronidase (GUS) activity and reactive oxygen species (ROS) assays demonstrated their involvement in SCN parasitism and ROS accumulation. These findings provide novel insights into GmPUB33A-mediated SCN susceptibility in soybean.

Soybean cyst nematode (SCN) development depends on syncytium formation, which requires cell-wall degradation and fusion. Lignin, the main barrier in cell walls, is critical for SCN resistance. 4-Coumarate: CoA ligase (4CL) drives the phenylpropanoid pathway by converting p-coumaric acid to p-coumaroyl-CoA, supplying lignin precursors. Here, resistant cv. Huipizhiheidou accumulated more lignin than susceptible Williams 82 after SCN inoculation. SCN stress induced distinct Gm4CL-family expression profiles across cultivars; Gm4CL3 and Gm4CL4 were markedly upregulated in Huipizhiheidou. Transient expression of Gm4CL3 in tobacco thickened leaf cell walls, implying enhanced wall reinforcement against SCN. Thus, 4CLs, especially Gm4CL3, may promote lignin deposition and secondary wall thickening to strengthen soybean SCN resistance.

BACKGROUND:Root-knot nematodes (Meloidogyne incognita) pose a persistent threat to global agriculture. The widespread use of chemical nematicides for their controlling has raised environmental safety and human health concerns, highlighting the urgent need for sustainable and eco-friendly alternatives. Asarum sieboldii, a medicinal plant with documented bioactive properties, is a potential source of eco-friendly biocontrol agents. In this study, the nematicidal efficacy of A. sieboldii root volatile organic compounds (VOCs) against M. incognita was evaluated, and the mechanisms underlying the reduction in the number of root galls and egg masses by VOCs was elucidated. RESULTS:VOCs from A. siebedii roots exhibited significant nematicidal activity, causing 76.31% and 96.89% mortality of second-stage M. incognita juveniles after 24 and 48 h, respectively, and effectively inhibited egg hatching by 72.94%. Activated carbon adsorption experiments confirmed VOCs as the primary bioactive agents. VOCs reduced the number of root galls and egg masses in tomato. We identified and screened three effective VOCs, namely 3,5-dimethoxytoluene, 4-methoxyacetophenone and β-pinene, that exhibited direct-contact and fumigant activity, and outperformed the commercial nematicide dazomet, reducing root galls on tomato by 65.5%, 55.3% and 44.4%, respectively, while enhancing the growth. These compounds triggered the accumulation of reactive oxygen species in nematodes, leading to oxidative stress and concurrent inhibition of peroxidase and catalase activities. CONCLUSIONS:Three compounds from A. sieboldii exhibited fumigant activity against M. incognita and promoted tomato growth, highlighting their dual advantage over traditional chemical nematicides. These findings should contribute to the development of plant-based, environmentally-friendly strategies for integrated pest management. © 2025 Society of Chemical Industry.

The legume-specific miR862b, acting via targets such as GmGRX1, is associated with soybean responses to Heterodera glycines. Exogenous ds-miR862b reduces SCN root infection while moderately and transiently suppressing seed germination and early seedling growth. Soybean cyst nematode (SCN) disease has emerged as a significant threat to soybean production globally, causing substantial yield losses. Identifying key resistance molecular determinants is essential for enhancing soybean resistance to SCN. Previous studies have shown that miR862b exhibits a strong response in the early stages of SCN infection across different soybean varieties; however, its role in the mechanism and function of SCN tolerance remains unclear. This study utilized bioinformatics prediction and qRT-PCR validation to elucidate the initial response of miR862b to SCN and screen its target genes, including GmGRX1 (Glyma.14G057300). Functional validation was conducted using K599-induced transgenic soybean roots, further indicating that overexpressing pre-miR862b positively regulates soybean resistance to SCN while significantly suppressing the expression of its target gene GmGRX1. Conversely, transgenic lines with tandem repeats silencing miR862b exhibited reduced SCN resistance, accompanied by upregulated expression levels of the target gene GmGRX1. In addition, treatment with artificially synthesized ds-miR862b in soybeans inhibited SCN infection in roots while also moderately suppressing seed germination and seedling development. In summary, these findings provide a theoretical basis for elucidating the function of miR862b and its target gene GmGRX1 in regulating soybean resistance to SCN.

The soybean cyst nematode (Heterodera glycines Ichinohe, SCN) poses a significant threat to soybean yield, often leading to total crop failure in heavily infested areas. Identifying key resistance genes is essential for enhancing soybean resistance to SCN. This study demonstrates that pre-treatment with a bacterial extract, CPT, can enhance SCN resistance in soybean roots by increasing lignin content and peroxidase (POD) activity. Further investigation revealed that the Class III POD gene GmPOD53L (Glyma.02G171600) is upregulated under SCN stress, correlating with increased peroxidase activity and lignin content. Overexpression of GmPOD53L significantly bolstered SCN resistance, as evidenced by reduced SCN numbers, slowed SCN development, heightened lignin deposition, and elevated POD activity. Conversely, silencing GmPOD53L had the opposite effect. These findings suggest that GmPOD53L positively regulates SCN stress by enhancing lignin content and POD activity, thereby inhibiting SCN invasion and development. This study identifies GmPOD53L as a candidate gene for soybean breeding programs aimed at improving SCN resistance and provides a theoretical foundation for the development of related bio-based seed coatings and SCN-resistant breeding efforts.

Soybean cyst nematode ( Heterodera glycines, soybean cyst nematode [SCN]) disease adversely affects the yield of soybean and leads to billions of dollars in losses every year. To control the disease, it is necessary to study the resistance genes of the plant and their mechanisms. Isoflavonoids are secondary metabolites of the phenylalanine pathway, and they are synthesized in soybean. They are essential in plant response to biotic and abiotic stresses. In this study, we reported that phenylalanine ammonia-lyase (PAL) genes GmPALs involved in isoflavonoid biosynthesis, can positively regulate soybean resistance to SCN. Our previous study demonstrated that the expression of GmPAL genes in the resistant cultivar Huipizhi (HPZ) heidou are strongly induced by SCN. PAL is the rate-limiting enzyme that catalyzes the first step of phenylpropanoid metabolism, and it responds to biotic or abiotic stresses. Here, we demonstrate that the resistance of soybeans against SCN is suppressed by PAL inhibitor l-α-(aminooxy)-β-phenylpropionic acid (L-AOPP) treatment. Overexpression of eight GmPAL genes caused diapause of nematodes in transgenic roots. In a petiole-feeding bioassay, we identified that two isoflavones, daidzein and genistein, could enhance resistance against SCN and suppress nematode development. This study thus reveals GmPAL-mediated resistance against SCN, information that has good application potential. The role of isoflavones in soybean resistance provides new information for the control of SCN. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .