A Near Gap-Free Haplotype-Resolved Genome Assembly of Zoysia japonica Uncovers Intra-Subgenomic Gene Expression and Regulatory Variation.

A single-base deletion in ZjSGR confers the STAY-GREEN phenotype and the enhanced stress tolerance in Zoysia japonica under dark and low temperature stresses

With the intensification of global climate change, the increasing frequency and severity of extreme weather events seriously affected agroecosystems and human health. Zoysia japonica Steud. (Z. japonica) is a warm season turfgrass with outstanding drought tolerance; therefore, gaining insight into the breeding and ecological restoration of drought-tolerant lawn grass species is of great significance. This study aimed to investigate the adaptive strategies of drought-resistant z047 and z388 by integrating transcriptome analysis and experimental physiological measurements in a drought field. Physiological experiments have demonstrated that z047 plants exhibited a stronger water retention capacity, lower cell membrane damage, and higher above-ground biomass. In addition, the relative water content and permanent wilting coefficient of z047 plants were superior to wild type plants. Our results verified that there were 108 and 208 significantly differentially expressed genes (DEGs) (fold change (FC) ≥ 4, p < 0.01) screened from z047 plants under drought stress for 7 and 14 days, respectively. Moreover, remarkable upregulation of MAPKKK17 and MAPKKK16 genes involved in the MAPK signalling pathway may be closely related to their drought tolerance. Collectively, this study reveals the molecular and physiological synergistic mechanism of drought tolerance in Z. japonica, thus providing a theoretical basis for molecular breeding of drought-tolerant plant cultivars and ecological restoration in arid areas.

Climate change has intensified the frequency and severity of urban droughts, exposing urban green spaces to abrupt and extreme water shortage that disrupts plant-microbe interactions and microbial multifunctionality. Understanding how rhizosphere and phyllosphere microbial communities respond to drought and how these shifts influence urban microbial functions is crucial for developing strategies to enhance the resilience of urban ecosystems under climate change. In this study, we conducted microcosm experiments simulating four drought intensities, integrating omics technologies with soil enzyme stoichiometry to investigate the effects of drought on microbial communities associated with Zoysia japonica (Steud) and urban microbial multifunctionality. Our results demonstrate that drought intensities significantly altered the compositions of bacterial and fungal communities in both the rhizosphere and phyllosphere. Moreover, drought enhanced microbial multifunctionality by significantly affecting 21 microbial functional potentials, including carbon fixation and denitrification. Although urban microbial multifunctionality largely returned to the control level after rehydration, five functions remained altered, including phyllosphere organic nitrogen mineralization and soil polyphenol oxidase activity. Biotic factors, particularly rhizosphere bacteria and fungi, directly influenced microbial multifunctionality during drought, whereas abiotic factors, such as electrical conductivity, dissolved organic carbon, and ammonium-nitrogen (NH4+-N), had indirect effects. After rehydration, abiotic factors, especially pH and NH4+-N, emerged as the main direct drivers. These findings underscore a shift from biotic to abiotic regulation of urban microbial multi-functionality across drought and rehydration, emphasizing the vital role of microbial communities in ecosystem resilience and the need to consider both biotic and abiotic factors in urban drought management.

Functional analysis in Arabidopsis revealed that ZjRVE8 -1 significantly enhances cold tolerance by activating the CBF pathway, identifying it as a promising gene for breeding cold hardy Zoysia japonica cultivars. REVEILLE (RVE) transcription factors regulate circadian rhythms and abiotic stress responses, but their roles in cold tolerance in the warm-season turfgrass Zoysia japonica remain uncharacterized. We identified 10 RVE family members through genome-wide analysis and performed comprehensive expression profiling under multiple abiotic stresses, revealing cold-responsive patterns. Based on this, five cold-induced RVE genes were selected for functional validation via heterologous overexpression in Arabidopsis thaliana. Overexpression of ZjRVE8-1 significantly enhanced freezing tolerance under both non-acclimated (NA) and cold-acclimated (CA) conditions by upregulating CBF1 and CBF3 expression. In contrast, overexpression of four tested RVE1-clade members failed to improve, and in one case reduced, freezing tolerance, correlating with their divergent cold-induced expression patterns. Promoter analysis identified a low-temperature responsive (LTR) element exclusively in ZjRVE8-1 of the cold-tolerant genotype, explaining its superior induction. This study uncovers ZjRVE8-1 as a novel regulator of cold adaptation via CBF activation and provides functional insights into RVE family divergence, offering candidate genes for breeding cold-hardy Z. japonica.

Water scarcity and distribution constitute a problem driven by population growth and industrial overexploitation. To secure water supply, desalination technologies for seawater and brackish water have been adopted, becoming critically important. Reverse osmosis is the highest-rated technology for this process and generates two output streams: permeate water and brine, the latter characterized by a high concentration of total dissolved solids (TDS). When untreated, brine is discharged into water bodies and soils, causing ecological damage. To mitigate this impact, the circular economy proposes reusing part of the brine in agriculture through halophyte plants, which offer the advantage of growing under high salt concentrations. The objective of the research was to document the salinity tolerance of halophyte species and the potential use of water rejected from the desalination process as irrigation water, with a circular economy approach. Brine reuse represents an opportunity to reduce waste and generate environmental, social, and economic benefits. Among the main halophyte species capable of tolerating brine above 30 000 mg L-1 are Suaeda salsa (L.) Pall., Salicornia bigelovii Torr., Rhizophora mangle L., and Chenopodium quinoa Willd. Salicornia europaea L. is classified as a halophyte species with medium tolerance (10 000–30 000 mg L-1). Species with low salinity tolerance (5000–10 000 mg L-1 TDS) include Atriplex nummularia Lindl., Zoysia japonica Steud., and Crithmum maritimum L. These plants also possess significant nutritional and pharmaceutical properties and can be used as livestock feed, human food, for oil extraction, soil remediation, and other applications.

Soil salinization poses a major threat to plant growth and ecosystem sustainability. Zoysia japonica, a salt-tolerant turfgrass, shows promise for saline-alkali soil remediation, yet its metabolic adaptation mechanisms remain poorly understood. Here, we applied non-targeted liquid chromatography/mass spectrometry (LC/MS) metabolomics to compare the responses of salt-tolerant (accession 68) and salt-sensitive (accession 9) genotypes of Z. japonica under salt stress. The sensitive genotype exhibited stronger metabolic disruption, with 843 differentially accumulated metabolites (largely down-regulated), compared with 595 in the tolerant genotype (predominantly up-regulated). We identified a coordinated tolerance mechanism primarily centered on lipid remodeling and energy maintenance. The tolerant genotype enhanced membrane stability through the accumulation of saturated glycerophospholipids and an increased phosphatidylcholine/phosphatidylethanolamine (PC/PE) ratio, while maintaining phosphatidic acid (PA) homeostasis which may facilitate SOS-dependent Na+ efflux. It also mitigated oxidative damage by stabilizing diacylglycerol (DAG), thereby potentially limiting protein kinase C (PKC) overactivation. Furthermore, sustained cardiolipin and riboflavin metabolism supported mitochondrial energy production in the tolerant genotype. Together, these findings provide new insights into the early metabolic basis of salt tolerance in Z. japonica, suggesting a potential crucial role for PA-mediated regulation of SOS-dependent sodium sequestration during the initial phase of stress, and implying potential targets for breeding stress-resilient turfgrasses.

Zoysia japonica ELONGATED HYPOCOTYL5 Interacts with STO to induce leaf narrowing and chloroplast thylakoid structural alterations in Agrostis stolonifera

The BRI1-EMS suppressor/Brassinazole-resistant (BES/BZR) transcription factors (TFs) act as regulators of the Brassinosteroid (BR) signaling pathway and play key roles in modulating plant growth, development, and abiotic stress tolerance. However, the function of BES/BZR TFs remains unknown in warm-season turfgrass species. In this study, ZjBZR2, a BES/BZR TF in Zoysia japonica was identified and shared the closest evolutionary relationship with OsBZR2 from Oryza sativa. ZjBZR2 was a nuclear-localized protein and had transcriptional activation activity. ZjBZR2 was predominantly expressed in roots, stems, and lamina joints, and could be significantly induced by BR treatment and osmotic stresses including PEG and salinity. ZjBZR2-overexpressing rice lines increased leaf angle compared with wild-type plants. Furthermore, overexpression of ZjBZR2 enhanced osmotic stress (PEG and salt) tolerance which is associated with the upregulation of stress-responsive and ROS-scavenging genes. These findings provide the first functional characterization of ZjBZR2 in rice and offer excellent genetic resources for the improvement of turfgrass cultivars.

Soccer is one of the most popular sports in the world including Indonesia. However, the development of soccer in Indonesia has been delayed, one of the causes is the lack of soccer sports facilities. The management of soccer stadiums in Indonesia still pays little attention to proper treatment to grow turfgrass in accordance with international standards from FIFA. Another type of grass that can be used as an alternative to soccer stadium turfgrass in Indonesia is Japanese grass (Zoysia japonica), this type of grass is chosen because it has pointed leaves so it will not be quickly damaged by soccer shoe spool. Therefore, this study aims to determine the characteristics of Zoysia japonica with various management, especially on the use of shade and the effect of auxin hormone administration which can be used as a reference recommendation for improving grass quality and grass management suitable for soccer stadiums in Indonesia. This study used a Split Plot design with two factors. The results showed that the interaction of shade treatment and auxin hormone had a very significant effect on all observation parameters. In an environment with 70% shade, Zoysia japonica can still survive, but its growth will be better if given 100% auxin hormone.

Anthocyanins play diverse roles in plants, including attracting pollinators and protecting cells from oxidative damage. In zoysiagrass, a warm season turfgrass, their accumulation in seed heads and stolons can decrease the aesthetic appeal. In this study, a high-density genetic map with ~8000 single nucleotide polymorphism (SNP) markers organized into 20 linkage groups was generated in a Zoysia japonica acc. Meyer × Zoysia matrella acc. PI 231146 F2 population. Using this genetic map, a large-effect quantitative trait locus (QTL) for anthocyanin variation in stolons and seed heads was mapped to chromosome 12 (PP locus). Variant analysis of a candidate gene for PP, Zjn_sc00004.1.g07010.1.sm.mk, which encodes an MYC-bHLH transcription factor that regulates anthocyanin biosynthesis, revealed a SNP at an exon–intron boundary in Meyer that led to intron retention. Interestingly, an F1 population derived from the same parents segregated for seed head color but uniformly displayed purple stolons. Seed head color in the F1 population comapped with the PP locus which, combined with genotypic and yeast two-hybrid analyses, revealed that a SNP in PI 231146 leading to an Ala163Ser substitution in the MYB-interacting N-terminal domain of the same MYC-bHLH transcription factor was likely causal. The Ala163Ser substitution affected interaction of MYC-bHLH with MYB in an MYB-dependent manner. The identified mutations can be exploited to develop cultivars with green seed heads and stolons. The high-marker-density interspecific Z. japonica × Z. matrella F2 genetic map also provides a robust tool for identifying genomic regions and genes of agronomic interest that differentiate the two species.

Evaluating herbicide resistance and cold tolerance in interspecific F1 hybrids of Zoysia Japonica and Zoysia Matrella using NDVI and percentage green cover

Gray leaf spot (GLS) is a damaging disease of perennial ryegrass, tall fescue, and St. Augustinegrass (Harmon, 2023). Zoysiagrass samples exhibiting GLS-like symptoms were collected from ‘Zenith’ zoysiagrass in Raleigh (KL027603) and from an experimental line of zoysiagrass in Rolesville, NC (KL027571) in late July 2022. Symptoms included oval or round lesions, tan color, and a dark brown border (Fig. 1A). Over time, the affected leaves exhibited dieback from the tip, and significant damage was observed. Temperatures in the Raleigh area averaged 26°C in July 2022 and local precipitation was greater than average. Zoysiagrass is an important turfgrass for golf course fairways and home lawns in the transition zone and Southeastern US. Thus, knowledge of a new host for a devastating disease such as GLS is critical. Following incubation of affected zoysiagrass in a humid environment at 23°C for 24 hours, portions of leaves with typical GLS symptoms were viewed microscopically, where numerous three-celled, pear-shaped conidia were observed (Fig. 1B). Symptomatic leaves were placed onto antibiotic-amended potato dextrose agar (PDA) containing 0.05 g L-1 of chloramphenicol, streptomycin, and tetracycline. After subculturing, 95% of all samples plated yielded colonies that matched colony descriptions for P. oryzae (Harmon, 2023, Ma and Uddin, 2009, and Nanayakkara, U. N. et al., 2008). Once pure cultures were obtained, actively growing isolates were transferred to oatmeal agar and grown at 22°C for 5 days. After 5 days, sterilized alfalfa stems were placed in the cultures to induce sporulation. Cultures were incubated at 26°C under consistent fluorescent light for 10 to 12 days. Spores were harvested by flooding the plates with 15 mL of distilled water, and conidia were scraped from the surface of the plates from ten isolates with a sterilized needle (Ma and Uddin, 2009, and Nanayakkara, U. N. et al., 2008). Five spores per isolate were measured using a micrometer embedded in a Zeiss AX10 Imager A1 compound microscope. Conidia were hyaline, pyriform in shape, two-septate, and averaged 8.5 ± 1.2 (wide) x 27.3 ± 6.3 (length) μm, consistent with the descriptions presented in Harmon (2023). The ITS1, 5.8S, and ITS2 region along with the actin gene from two zoysiagrass isolates were amplified by polymerase chain reaction using fungal ITS5/4 primers (KL027603 / KL027571 - 509 bp) and ACT-512F/ACT-783R (KL027603 - 240 bp / KL027571 - 249 bp)(Couch and Kohn 2002, and White et. al 1990). These sequences were uploaded to NCBI GenBank under accession numbers (KL027603 rRNA: PV477962, KL027603 ACT: PV495209; KL027571 rRNA: PV477982, KL027571 ACT: PV495208). Nucleotide blasting resulted in ≥99% sequence identity to Pyricularia oryzae Cavara accession CBS:365.52. (ITS1,5.8S,ITS2: Genbank accession MH857082.1)(ACT: Genbank accession KM485191.1). The rRNA region and actin gene were concatenated, and a clustal omega alignment was performed. A Kamura-Nei neighbor joining tree with 1,000 bootstraps was generated and visualized in Geneious Prime (Kamura et al 2004) (Fig. 2). KL027603 and KL027571 clustered with P. oryzae with 100% bootstrap support, providing more evidence that these isolates are P. oryzae. Koch’s postulates were performed on ‘Zenith’ zoysiagrass plants established from seed. Pots containing sterilized potting mix were seeded with ‘Zenith’ zoysiagrass and allowed to grow and develop for 7 weeks prior to inoculation. All ten isolates of P. oryzae were plated onto PDA and allowed to grow at 26°C for three days. After 3 days of growth, agar plugs from the edge of the colonies were transferred into sterilized rye grain to produce inoculum. Five infested rye grains were placed in the canopy of ‘Zenith’ zoysiagrass and kept moist by putting them in a clear plastic bin in the greenhouse, where temperatures ranged from 23-28°C. Symptoms developed 5 to 7 days after inoculation and were consistent with those described above and those attributed to GLS on other turfgrasses. We successfully isolated P. oryzae from the affected tissue in the pathogenicity tests. To our knowledge, this is the first report of GLS disease on zoysiagrass in the United States.

Understanding how flowering phenology varies across spatial scales is essential for assessing plant responses to environmental heterogeneity under climate change. In this study, we investigated the flowering phenology of the plant species Taraxacum officinale across five sites in an agricultural region of Wanju, Republic of Korea. Each site contained five 1 m × 1 m quadrats, where the number of flowering heads was recorded at 1- to 2-day intervals during the spring flowering period (February to May). We applied the nlstimedist package in R to model flowering distributions and to estimate key phenological metrics including flowering onset (5%), peak (50%), and end (95%). The results revealed substantial variation in flowering timing and duration at both the intra-site (quadrat-level) and inter-site (site-level) scales. Across all sites, the mean onset, peak, end, and duration of flowering were day of year (DOY) 89.6, 101.5, 117.6, and 28.0, respectively. Although flowering onset showed relatively small variation across sites (DOY 88 to 92), flowering peak (DOY 97 to 108) and end dates (DOY 105 to 128) exhibited larger differences at the site level. Sites with dry soils and regularly mowed Zoysia japonica vegetation with minimal understory exhibited shorter flowering durations, while those with moist soils, complex microtopography, and diverse slope orientations showed delayed and prolonged flowering. These findings suggest that microhabitat variability—including landform type, slope direction, soil water content, and soil temperature—plays a key role in shaping local flowering dynamics. Recognizing this fine-scale heterogeneity is essential for improving phenological models and informing site-specific climate adaptation strategies.

Zoysia sinica is a perennial grass that thrives in intertidal zones, even under extreme waterlogging stress. In this study, we present a high-quality chromosome-level genome assembly of Z. sinica, with a total size of 312.67 Mb. Through genome annotation, we identified 29,551 protein-coding genes in the Z. sinica genome and re-annotated 32,925, 53,226, and 53,656 genes in the previously reported Zoysia genomes: Zoysia japonica, Zoysia matrella, and Zoysia pacifica, respectively. Genome divergence analysis indicated that the Zoysia species diverged relatively recently, approximately 3.63 million years ago. Comparative genomic analysis revealed an expansion of ethylene response factors and identified Z. sinica-specific genes related to the response to auxin and ethylene. Transcriptome data from intertidal environments with different levels of waterlogging showed significant upregulation of nitrate transporters (NRT2.2 and NRT2.4) in roots and genes involved in suberin biosynthesis in shoots. Additionally, various transporters were responsive to the intertidal environment. Our study provides insights into the intertidal adaptation of Zoysia species and offers a foundation for the development of stress-tolerant cultivars.

In a hypothetical climate change scenario, zoysiagrass species could be a good choice for turfgrass areas due to their adaptation to heat conditions and the great variability in species and cultivars. Knowledge of the root system’s characteristics is paramount for predicting cultivar adaptation to different heat–drought scenarios and therefore for designing proper turfgrass management programs, especially irrigation. A field experiment was conducted in the Mediterranean environment of Valencia (Spain) to study the root weight density (RWD), root length density (RLD), and average root diameter (RDI) at three different soil depths (0–5, 5–15, and 15–30 cm) of five new zoysiagrass genotypes (Zoysia matrella (L.) Merr., Zoysia japonica Steud., and their hybrid), relating these parameters to the performance of these experimental lines during their establishment. All the tested experimental lines had a higher RWD and RLD in the upper soil layer (0–5 cm), while the RDI was higher in the lowest layer of the sampled soil profile (0.269 mm compared with 0.249 mm and 0.241 mm in the upper layers). All the tested genotypes showed the same RWD and RLD, while the Zoysia matrella experimental line A showed a higher RDI value (0.2683 mm) than those for the Z. japonica (0.2369 mm) and the hybrid (0.2394 mm) genotypes. This last finding could have influenced its more rapid establishment, although it was not linked to its NDVI values during autumn. In conclusion, different morphological root characteristics were detected among new zoysiagrass genotypes and soil depths, which could have affected their canopy performance, and they are expected to affect irrigation management in a possible future drought scenario.

Abstract Bermudagrasses (Cynodon Rich. species) easily contaminate fields of zoysiagrasses (Zoysia Willd. species) through vegetative propagules and seeds, and once established, can be hard to eradicate. Zoysiagrasses and bermudagrasses vary in color, texture, and canopy architecture; infestations can be visually identified when dew is present. Field research conducted in 2024 quantified the volume of dew retained in Tifway hybrid bermudagrass (Cynodon dactylon (L.) Pers. × C. transvaalensis Burtt Davy) and Meyer Japanese lawngrass (Zoysia japonica Steud.) canopies. The hybrid bermudagrass canopy held a greater volume of dew than Japanese lawngrass at all collection times between sunrise and 150 min after sunrise. Aerial imagery was collected from three sites comprising varying degrees of contamination of bermudagrasses in Japanese lawngrass to test the effectiveness of an artificial intelligence‐based, supervised classification tool (Magic selection tool in PIX4Dfields) in delineating bermudagrasses from Japanese lawngrass. The Magic selection tool's ability to distinguish bermudagrasses from Japanese lawngrass depended on the density of the turfgrass stand, background soil color, presence of other grasses, presence of clippings, and dew volume. Based on dew volume, the ideal time to scout fields for contamination of bermudagrasses is within 90 min of sunrise and when dense stands of both grasses are present.

Abstract This study was conducted to identify genetic diversity and spatial genetic structure in natural populations of zoysiagrasses using inter‐simple sequence repeat markers for the purpose of establishing highly efficient measures for preserving zoysiagrass. The genetic diversity indices based on the Nei's gene diversity (H) and Shannon's information index (I) were 0.048 and 0.072 in site I and 0.024 and 0.034 in site II. Compared to site II, site I contained more diverse genotypes per unit area. The analysis of molecular variance showed that 56.2% of the genetic variations were attributed to variations among populations, while 43.8% were attributed to the variation within populations. It was deemed that the high rate of inter‐plot genetic variations reflected the species‐specific characteristics in reproduction mode. The spatial genetic structures of the zoysiagrasses based on the correlogram analysis indicated that genetically similar individuals were found within 2 m of each other in the Zoysia japonica site I, but genetically different when two genotypes were grown more than 3.5 m apart. On the other hand, there was a random distribution of individuals at all distances, and no distinct genetic colonies were formed in the Zoysia sinica site II. Results from this study demonstrated that for ex situ genetic conservation of zoysiagrasses, the sampling strategy should be considered in intervals of at least 2 m.

Abstract A total of 277 zoysiagrass growing naturally in the local regions was collected per region, and a morphological classification was conducted through statistical analysis using morphological modification. For a diversified analysis through morphological characteristics, the main trait related to morphological characteristics was analyzed, and as a result of analyzing the classificational relationships of zoysiagrass, zoysiagrass that were collected according to leaf width, stolon internode thickness, number of stolon internode, number of seed per spikelet, seed length, and seed length/seed width ratio were classified into four groups. The major morphological traits observed in each individual group were compared to the morphological characteristics of native zoysiagrasses described in the reference, and three of the groups were assumed to be Zoysia japonica (group A), Zoysia sinica (group B), and Zoysia matrella (group D). One of the groups was assumed to be a hybrid and/or mutant with intermediate characteristics.

Abstract Sod installation rooting vigor is an important characteristic for establishment of turfgrass sod. This study evaluated rooting traits of two zoysiagrass cultivars, ‘SS‐500’ (Empire®, Sod Solutions, Inc., Zoysia japonica Steud) and ‘Zeon’ (Zoysia matrella L. Merr), under controlled settings to simulate sod installation. The experiment, conducted twice, followed a randomized complete block design with a split‐plot arrangement, where cultivars were the main plots and weeks of harvest were the split plots. Significant differences were observed between genotypes and harvest weeks for all root traits. Empire generally had higher values for the evaluated characteristics, and values increased over time for subsequent harvest dates. Results indicate that the methodology has merit for evaluation of sod installation rooting vigor of zoysiagrass.