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Panax ginseng C.A.Mey. (Araliaceae) cultivation suffers from the inability to cultivate the same fields continuously for long durations due to replant failure. The main cause of replant failure is considered to be the annual change in the soil microbial flora, especially the invasion and settlement of pathogenic microorganisms of soil-borne diseases. We analyzed the soil bacterial and fungal flora and inter-annual changes in their composition over 5 years in ginseng cultivation fields on Daikonshima Island, Shimane Prefecture of Western Japan by DNA metabarcoding using next-generation sequencing. Bacteria such as Sphingomonas sp., Bacillus sp., and Betaproteobacteria and the fungus Mortierella sp. were consistently detected throughout the cultivation period. The inter-annual compositional changes of the bacterial flora, especially two members of the family Burkholderiaceae, one member of the phylum Actinobacteria, one member of the genus Candidatus Koribacter, and one member of the genus Sphingomonas, corresponded to the cultivation period, whereas those of the fungal flora showed random changes, suggesting that the growth of ginseng may be greatly affected by changes in the bacterial flora. Therefore, a greater understanding of the bacterial flora could provide valuable information for the cultivation of ginseng. The absence of pathogenic microorganisms associated with soil-borne diseases, which have been reported as causative agents of the main diseases of ginseng, in all soil sampling sites throughout the entire cultivation period in this study proves, for the first time, that traditional cultivation management employing empirical methods and chemical control is an effective approach to control these pathogens. Therefore, the DNA metabarcoding of the bacterial flora could provide valuable information for cultivation management, specifically in detecting and controlling soil-borne pathogens responsible for ongoing cultivation damage in long-term cultivation of medicinal plants.

Significance In 1840, Justus von Liebig proposed the theory of mineral plant nutrition, through the invention of the Haber–Bosch process, leading to the industrialization of chemical fertilizer (inorganic nitrogen) to feed the human population. Because the excessive use of chemical fertilizer has led to numerous environmental problems, understanding the agroecosystem that contains plants, microbes, and soils is necessary for sustainable agriculture. We revealed the network structure of an agroecosystem established with different management practices and identified that organic nitrogen is a key component contributing to crop yield under the condition of soil solarization, even in the presence of inorganic nitrogen. Our finding provides a potential solution to make crop production more sustainable by utilizing organic nitrogen induced by soil solarization., Both inorganic fertilizer inputs and crop yields have increased globally, with the concurrent increase in the pollution of water bodies due to nitrogen leaching from soils. Designing agroecosystems that are environmentally friendly is urgently required. Since agroecosystems are highly complex and consist of entangled webs of interactions between plants, microbes, and soils, identifying critical components in crop production remain elusive. To understand the network structure in agroecosystems engineered by several farming methods, including environmentally friendly soil solarization, we utilized a multiomics approach on a field planted with Brassica rapa. We found that the soil solarization increased plant shoot biomass irrespective of the type of fertilizer applied. Our multiomics and integrated informatics revealed complex interactions in the agroecosystem showing multiple network modules represented by plant traits heterogeneously associated with soil metabolites, minerals, and microbes. Unexpectedly, we identified soil organic nitrogen induced by soil solarization as one of the key components to increase crop yield. A germ-free plant in vitro assay and a pot experiment using arable soils confirmed that specific organic nitrogen, namely alanine and choline, directly increased plant biomass by acting as a nitrogen source and a biologically active compound. Thus, our study provides evidence at the agroecosystem level that organic nitrogen plays a key role in plant growth.

The amount of input DNA available to prepare next-generation sequencing (NGS) libraries is often limited, which can lead to GC content bias and enrichment of specific genomic regions with currently available protocols. In this study, we used breath capture technology to incorporate sequencing adapters into DNA to develop a novel cost-effective protocol for the preparation of genomic DNA libraries. We performed a benchmarking experiment comparing our protocol with common commercially available kits for genomic DNA library preparation with input DNA amount in the range of 1 to 50 ng. Our protocol can generate high-quality genomic sequence data with a marked improvement in coverage breadth and low GC bias, in contrast to standard protocols. Further, our protocol reduces sample handling time and reagent costs, and requires comparatively fewer enzymatic steps relative to other protocols, making it suitable for a range of genomics applications.

Background The rapid and accurate identification of a minimal-size core set of representative microbial species plays an important role in the clustering of microbial community data and interpretation of clustering results. However, the huge dimensionality of microbial metagenomics datasets is a major challenge for the existing methods such as Dirichlet multinomial mixture (DMM) models. In the approach of the existing methods, the computational burden of identifying a small number of representative species from a large number of observed species remains a challenge. Results We propose a novel approach to improve the performance of the widely used DMM approach by combining three ideas: (i) we propose an indicator variable to identify representative operational taxonomic units that substantially contribute to the differentiation among clusters; (ii) to address the computational burden of high-dimensional microbiome data, we propose a stochastic variational inference, which approximates the posterior distribution using a controllable distribution called variational distribution, and stochastic optimization algorithms for fast computation; and (iii) we extend the finite DMM model to an infinite case by considering Dirichlet process mixtures and estimating the number of clusters as a variational parameter. Using the proposed method, stochastic variational variable selection (SVVS), we analyzed the root microbiome data collected in our soybean field experiment, the human gut microbiome data from three published datasets of large-scale case-control studies and the healthy human microbiome data from the Human Microbiome Project. Conclusions SVVS demonstrates a better performance and significantly faster computation than those of the existing methods in all cases of testing datasets. In particular, SVVS is the only method that can analyze massive high-dimensional microbial data with more than 50,000 microbial species and 1000 samples. Furthermore, a core set of representative microbial species is identified using SVVS that can improve the interpretability of Bayesian mixture models for a wide range of microbiome studies.

Microbiota are a major component of agroecosystems. Root microbiota, which inhabit the inside and surface of plant roots, play a significant role in plant growth and health. As next-generation sequencing technology allows the capture of microbial profiles without culturing the microbes, profiling of plant microbiota has become a staple tool in plant science and agriculture. Here, we have developed a novel high-throughput method based on a two-step PCR amplification protocol, involving DNA extraction using magnetic beads and PCR purification using exonuclease, for 16S rRNA gene amplicon sequencing of plant root microbiota. This method reduces sample handling and captures microbial diversity comparable to that obtained by the standard method. We found that using a buffer with magnetic beads enabled efficient extraction of microbial DNA directly from plant roots. In addition, we demonstrated that purification using exonuclease before the second PCR step enabled the capture of higher degrees of microbial diversity, thus allowing for the detection of minor bacteria compared with the purification using magnetic beads in this step. Our method offers a simple and high-throughput solution for maintaining the quality of plant root microbial community profiling.

The amount of input DNA available to prepare next-generation sequencing (NGS) libraries is often limited, which can lead to GC content bias and enrichment of specific genomic regions with currently available protocols. In this study, we used breath capture technology to incorporate sequencing adapters into DNA to develop a novel cost-effective protocol for the preparation of genomic DNA libraries. We performed a benchmarking experiment comparing our protocol with common commercially available kits for genomic DNA library preparation with input DNA amount in the range of 1 to 50 ng. Our protocol can generate high-quality genomic sequence data with a marked improvement in coverage breadth and low GC bias, in contrast to standard protocols. Further, our protocol reduces sample handling time and reagent costs, and requires comparatively fewer enzymatic steps relative to other protocols, making it suitable for a range of genomics applications.

Agrobacterium-mediated transformation represents a key innovation for plant breeding and is routinely used in research and applied biology. However, for several species, the efficacy of transformation is limited. In this study, we discovered that oxicam-type nonsteroidal anti-inflammatory drugs (NSAIDs), including tenoxicam (TNX), enhance the efficiency of Agrobacterium-mediated transient transformation in the model species Arabidopsis thaliana via leaf infiltration and can be successfully applied in analyses of the subcellular localisation of fluorescent fusion proteins. TNX acts as an inhibitor of plant immune responses and lacks similar transient transformation efficiency in a dde2/ein2/pad4/sid2 quadruple mutant background, thereby indicating that TNX increases the efficiency of Agrobacterium infection via a transient shutdown of the immune system mediated by jasmonic acid, ethylene, and salicylic acid signalling. In addition, we found that TNX enhances the efficiency of stable transformation in crops of agricultural and economic importance, such as Jatropha and maize, indicating that TNX can enhance the integration of exogenous DNA into the plant genome via an increased introduction of DNA into plant cells. Given that treatment with oxicam compounds is simple, cost effective, and has broad utility, we anticipate that this discovery will contribute to accelerating genome-editing technologies in plants.

Parasitic plants infect other plants by forming haustoria, specialized multicellular organs consisting of several cell types, each of which has unique morphological features and physiological roles associated with parasitism. Understanding the spatial organization of cell types is, therefore, of great importance in elucidating the functions of haustoria. Here, we report a three-dimensional (3-D) reconstruction of haustoria from two Orobanchaceae species, the obligate parasite Striga hermonthica infecting rice (Oryza sativa) and the facultative parasite Phtheirospermum japonicum infecting Arabidopsis (Arabidopsis thaliana). In addition, field-emission scanning electron microscopy observation revealed the presence of various cell types in haustoria. Our images reveal the spatial arrangements of multiple cell types inside haustoria and their interaction with host roots. The 3-D internal structures of haustoria highlight differences between the two parasites, particularly at the xylem connection site with the host. Our study provides cellular and structural insights into haustoria of S. hermonthica and P. japonicum and lays the foundation for understanding haustorium function.

Parasitic plants infect other plants by forming haustoria, specialized multicellular organs consisting of several cell types each of which has unique morphological features and physiological roles associated with parasitism. Understanding the spatial organization of cell types is, therefore, of great importance in elucidating the functions of haustoria. Here, we report a three-dimensional (3-D) reconstruction of haustoria from two Orobanchaceae species, the obligate parasite Striga hermonthica infecting rice and the facultative parasite Phtheirospermum japonicum infecting Arabidopsis. Our images reveal the spatial arrangements of multiple cell types inside haustoria and their interaction with host roots. The 3-D internal structures of haustoria highlight differences between the two parasites, particularly at the xylem connection site with the host. Our study provides structural insights into how organs interact between hosts and parasitic plants.One-sentence summaryThree-dimensional image reconstruction was used to visualize the spatial organization of cell types in the haustoria of parasitic plants with special reference to their interaction with host roots.

Background Root-knot nematodes (RKN, Meloidogyne spp.) are harmful phytophagous pests of Solanum spp. Some RKN species are becoming worldwide problems because of their virulence to RKN-resistant Solanum species. A new Solanum species carrying broad-spectrum resistance to Meloidogyne spp. is required for the effective management of this pest. Here we sought to determine the host suitability of RKN to Solanum palinacanthum, a wild Solanum species, and to evaluate its potential effectiveness in RKN-management. Results We identified an RKN-resistant Solanum species, S. palinacanthum, by screening Solanum accessions. We tested its spectrum of resistance to common Meloidogyne spp. in Japan. in pot tests inoculated with second-stage juveniles, S. palinacanthum showed poor host suitability for Melidogyne incognita, M. arenaria genotypes A2-J and A2-O, M. javanica and M. hapla, indicating broad-spectrum resistance to RKN. The development of M. incognita within S. palinacanthum roots was significantly poorer than that in susceptible S. melongena and S. lycopersicum at 10 and 21 days after inoculation. Microplot tests confirmed that the number of second-stage juveniles in plots where S. palinacanthum grew and root galling of the root system were significantly lower than those of susceptible S. melongena, suggesting that the resistance could be used to manage RKN under field conditions. Conclusion S. palinacanthum showed poor host suitability to all Meloidogyne spp. tested in this study, and it thus has the potential to be used as a genetic resource with broad-spectrum RKN resistance, and it could be effective against multiple RKN species in a field. This article is protected by copyright. All rights reserved.

Most plants show remarkable developmental plasticity in the generation of diverse types of new organs upon external stimuli, allowing them to adapt to their environment. Haustorial formation in parasitic plants is an example of such developmental reprogramming, but its molecular mechanism is largely unknown. In this study, we performed field-omics using transcriptomics and metabolomics to profile the molecular switch occurring in haustorial formation of the root parasitic plant, Thesium chinense, collected from its natural habitat. RNA-sequencing with de novo assembly revealed that the transcripts of very long chain fatty acid (VLCFA) biosynthesis genes, auxin biosynthesis/signaling-related genes and lateral root developmental genes are highly abundant in the haustoria. Gene co-expression network analysis identified a network module linking VLCFAs and the auxin-responsive lateral root development pathway. GC-TOF-MS analysis consistently revealed a unique metabolome profile with many types of fatty acids in the T. chinense root system, including the accumulation of a 25-carbon long chain saturated fatty acid in the haustoria. Our field-omics data provide evidence supporting the hypothesis that the molecular developmental machinery used for lateral root formation in non-parasitic plants has been co-opted into the developmental reprogramming of haustorial formation in the linage of parasitic plants.