Function and Characterization of Fungal Communities in Chestnut Soils (Castanea crenata) of Kansai Region, Japan

Chestnut (Castanea crenata) is an important fruit crop in Japan, grown under three cultivation systems in Kansai region, which succumb to fungal root disease pathogens. The fungal community in soils of chestnut in these cultivation systems were characterized along with the potential of soil bacterial species as biological control agent against these root-invading fungi. Bacteria from the chestnut soil rhizosphere were identified and their ability to suppress diseases in vitro was evaluated. Bacteria DAC17225011 and DAC17225014 showed 99% similarity to Bacillus aryabhattai and Pseudomonas frederiksbergensis, respectively, which could suppress the growth of Armilaria mellea and Phytophtora cambivora, respectively, in in vitro conditions. The assay in vivo indicated the positive effect of these bacteria on the reduction of disease infection spots in chestnut roots; however, no visible symptoms were detected aboveground. For microbial community analysis, chestnut soil was sampled from four locations (Wachi, Ayabe, Fukuchiyama and Sasayama) considering three management systems, conventional, organic and wild. The amplicon from the ITS region (The genomic library of the fungal detection in soils) was sequenced by Illumina MiSeq 250bp and used to analyze the fungal community in the sampled soil. Nectriaceae, which contains pathogenic fungi, was very common in all samples, but lower in wild areas. Ceratobasidiacea was also higher in conventional areas. For the symbiotic families, Hypocraceae and Russulaceae were typical in wild soils, whereas Amanitaceae was found in organic soils. The fungal community was clearly distinct in the wild system, differing from conventional and organic systems.

Chromosome-level genome assembly of Japanese chestnut (Castanea crenata Sieb. et Zucc.) reveals conserved chromosomal segments in woody rosids

Japanese chestnut (Castanea crenata Sieb. et Zucc.), unlike other Castanea species, is resistant to most diseases and wasps. However, genomic data of Japanese chestnut that could be used to determine its biotic stress resistance mechanisms have not been reported to date. In this study, we employed long-read sequencing and genetic mapping to generate genome sequences of Japanese chestnut at the chromosome level. Long reads (47.7 Gb; 71.6× genome coverage) were assembled into 781 contigs, with a total length of 721.2 Mb and a contig N50 length of 1.6 Mb. Genome sequences were anchored to the chestnut genetic map, comprising 14,973 single nucleotide polymorphisms (SNPs) and covering 1,807.8 cM map distance, to establish a chromosome-level genome assembly (683.8 Mb), with 69,980 potential protein-encoding genes and 425.5 Mb repetitive sequences. Furthermore, comparative genome structure analysis revealed that Japanese chestnut shares conserved chromosomal segments with woody plants, but not with herbaceous plants, of rosids. Overall, the genome sequence data of Japanese chestnut generated in this study is expected to enhance not only its genetics and genomics but also the evolutionary genomics of woody rosids.

Expression of Castanea crenata Allene Oxide Synthase in Arabidopsis Improves the Defense to Phytophthora cinnamomi

Allene oxide synthase (AOS) is a key enzyme of the jasmonic acid (JA) signaling pathway. The AOS gene was previously found to be upregulated in an Asian chestnut species resistant to infection by the oomycete Phytophthora cinnamomi (Castanea crenata), while lower expression values were detected in the susceptible European chestnut (Castanea sativa). Here, we report a genetic and functional characterization of the C. crenata AOS (CcAOS) upon its heterologous gene expression in a susceptible ecotype of Arabidopsis thaliana, which contains a single AOS gene. It was found that Arabidopsis plants expressing CcAOS delay pathogen progression and exhibit more vigorous growth in its presence. They also show upregulation of jasmonic acid and salicylic acid-related genes. As in its native species, heterologous CcAOS localized to plastids, as revealed by confocal imaging of the CcAOS-eGFP fusion protein in transgenic Arabidopsis roots. This observation was confirmed upon transient expression in Nicotiana benthamiana leaf epidermal cells. To further confirm a specific role of CcAOS in the defense mechanism against the pathogen, we performed crosses between transgenic CcAOS plants and an infertile Arabidopsis AOS knockout mutant line. It was found that plants expressing CcAOS exhibit normal growth, remain infertile but are significantly more tolerant to the pathogen than wild type plants. Together, our results indicate that CcAOS is an important player in plant defense responses against oomycete infection and that its expression in susceptible varieties may be a valuable tool to mitigate biotic stress responses.

Expression of Castanea crenata Allene Oxide Synthase in Arabidopsis Improves the Defense to Phytophthora cinnamomi

ABSTRACTAllene oxide synthase (AOS) is a key enzyme of the jasmonic acid (JA) signaling pathway. The AOS gene was previously found to be upregulated in an Asian chestnut species resistant to infection by the oomycete Phytophthora cinnamomi (Castanea crenata), while lower expression values were detected in the susceptible European chestnut (Castanea sativa). Here, we report a genetic and functional characterization of the C. crenata AOS (CcAOS) upon its heterologous gene expression in a susceptible ecotype of Arabidopsis thaliana, which contains a single AOS gene. It was found that Arabidopsis plants expressing CcAOS delay pathogen progression and exhibit more vigorous growth in its presence. They also show upregulation of jasmonic acid and salicylic acid-related genes. As in its native species, heterologous CcAOS localized to plastids, as revealed by confocal imaging of the CcAOS-eGFP fusion protein in transgenic Arabidopsis roots. This observation was confirmed upon transient expression in Nicotiana benthamiana leaf epidermal cells. To further confirm a specific role of CcAOS in the defense mechanism against the pathogen, we performed crosses between transgenic CcAOS plants and an infertile Arabidopsis AOS knockout mutant line. It was found that plants expressing CcAOS exhibit normal growth, remain infertile but are significantly more tolerant to the pathogen than wild type plants.Together, our results indicate that CcAOS is an important player in plant defense responses against oomycete infection and that its expression in susceptible varieties may be a valuable tool to mitigate biotic stress responses.One-sentence summaryHeterologous expression of the Castanea crenata allene oxide synthase gene in Arabidopsis thaliana improves the defense response to the pathogen Phytophthora cinnamomi.