Warm-season Turfgrass Research Articles

Assessment of Genetic Diversity Using Morphological and Molecular Characteristics of Indonesian Zoysiagrass Genotypes

Zoysiagrass is warm-season turfgrass thriving in tropical regions. Despite the adaptive nature, the existence of Indonesian zoysiagrass as well as morphological and genetic characteristics are not available. Therefore, this study aimed to explore Indonesian zoysiagrass from western and eastern parts of the country, as well as its morphological and molecular characteristics. Morphological characteristics was conducted to measure vegetative and reproductive characters while genotyping was performed using 15 simple sequence repeat markers. Morphological characteristics cluster three major groups, namely Group 1 corresponded to short, shorter, and fine leaves. Group 2 corresponded to tall, longer, and fine leaves, fewer seeds, and short spikelet, while Group 3 corresponded to tall, long, and wider leaves, more seeds, and longer spikelet. The results showed that the expected heterozygosity (He = 0.256) was lower than homozygosity (Ho = 0.341). The high level of discriminating capacity, polymorphism, and informativeness of SSR marker was observed (Effective Multiplex Ratio = 4.20, Marker Index = 2.394, and Resolving Power = 1.574). Additionally, population structure generated two subpopulations. Group 1 corresponded to Z. japonica from Sumatera Island and mixed province while Group 2 corresponded to Z. japonica from Central Java and Bali with Z. japonica and Z. matrella from mixed province. In conclusion, the exploration of morphological and genetic diversity from Indonesian zoysiagrass provided useful insight for conservation and future breeding improvement.

Horizontal and Vertical Distribution of Clarireedia spp. in Asymptomatic and Symptomatic Creeping Bentgrass Cultivars.

Dollar spot is an important disease of both cool- and warm-season turfgrasses caused by six fungal species in the genus Clarireedia, yet the ecology and epidemiology of these pathogens remains poorly understood. The goal of this study was to determine the distribution of Clarireedia in asymptomatic and symptomatic creeping bentgrass (Agrostis stolonifera) in the field using a previously developed quantitative PCR assay. To determine the horizontal distribution of the pathogen, the abundance of Clarireedia spp. was measured in leaf and crown tissue from 90, 1-cm-diameter cores spaced 10 cm apart in May 2019 and 2020 (asymptomatic tissue) and August 2019 and July 2020 (symptomatic tissue). Thirty-seven to 69% of cores sampled from asymptomatic turfgrass and 77 to 95% of cores taken from symptomatic turfgrass yielded positive detections for Clarireedia. Spatial analysis indicated that Clarireedia was randomly distributed in the field in both asymptomatic and symptomatic turfgrass. To assess the vertical distribution of the pathogen, the abundance of Clarireedia was measured in the foliar, crown, and thatch layers of 39, 1-cm-diameter × 2.5-cm-deep cores of creeping bentgrass maintained at fairway height (9.5 mm) during 2019 and 2020. Clarireedia was most abundant in foliar tissue, followed by crown tissue and thatch (lowest abundance) throughout the 2-year study. Both studies provide evidence that Clarireedia is widely distributed in turfgrass swards prior to symptom development and can persist within turfgrass as an endophyte. These findings will improve our understanding of Clarireedia epidemiology and may lead to more sustainable dollar spot management.

Evaluating the tradeoffs between water conservation, aesthetic value, evaporative cooling and CO2 emissions in St. augustinegrass and buffalograss

Urban lawns comprise a significant portion of urban greenery and provide several ecosystem services. Nevertheless, maintaining lawns comes with significant water costs in semi-arid inland southern California, as they require consistent irrigation to stay healthy and productive. The main objective of this study was to evaluate the effect of a wide range of irrigation rates and frequencies applied autonomously by a smart ET-based controller on the aesthetic value, cooling potential, and CO2 efflux of two warm-season turfgrass species. For three years, we studied the responses of Buffalograss and St. Augustinegrass to six irrigation rates and two irrigation frequencies in Riverside, California. Under historical average climate conditions, the minimum irrigation rate for Buffalograss was 93 % ETo, and for St. Augustinegrass, it was 74 % ETo. Under projected future climate conditions, the estimated minimum irrigation rate for Buffalograss did not change, but for St. Augustinegrass, it increased by 4 % and 7 % in 2100 under low emission (RCP 4.5) and high emission (RCP 8.5) scenarios, respectively. On average, canopy minus air temperature in Buffalograss was 6.2 ℃, and in St. Augustinegrass, it was 1.1 ℃. The average CO2 efflux in Buffalograss was 122.3 µg CO2-C m−2 s−1, and in St. Augustinegrass, it was 182.8 µg CO2-C m−2 s−1. Our results showed that turfgrass aesthetic values, cooling potential, and CO2 efflux diminished as the irrigation rate decreased, but at different rates for each turfgrass species.

Molecular Genetic Insights into the Stress Responses and Cultivation Management of Zoysiagrass: Illuminating the Pathways for Turf Improvement

Zoysiagrass (Zoysia spp.) and its hybrids are known for their low maintenance requirements and are widely utilized as warm-season turfgrass, which offers considerable ecological, environmental, and economic benefits in various environments. Molecular genetic approaches, including the identification and genetic engineering of valuable gene resources, present a promising opportunity to enhance the quality and performance of zoysiagrass. This review surveys the recent molecular genetic discoveries in zoysiagrass species, with a focus on elucidating plant responses to various abiotic and biotic stresses. Furthermore, this review explores the notable advancements in gene function exploration to reduce the maintenance demands of zoysiagrass cultivation. In addition, we discuss the achievements and potential of contemporary molecular and genetic tools, such as omics approaches and gene editing technologies, in developing zoysiagrass cultivars with desirable traits. Overall, this comprehensive review highlights future strategies that may leverage current molecular insights to accelerate zoysiagrass improvement and further promote sustainable turf management practices.

Genome-Wide Identification and Expression Analysis of the Cyclic Nucleotide-Gated Channel Gene Family in Zoysia japonica under Salt Stress.

Salt stress severely inhibits plant growth. Understanding the mechanism of plant salt tolerance is highly important to improving plant salt tolerance. Previous studies have shown that nonselective cyclic nucleotide-gated ion channels (CNGCs) play an important role in plant salt tolerance. However, current research on CNGCs mainly focuses on CNGCs in glycophytic plants, and research on CNGCs in halophytes that exhibit special salt tolerance strategies is still scarce. This study used the halophilic plant Zoysia japonica, an excellent warm-season turfgrass, as the experimental material. Through bioinformatics analysis, 18 members of the CNGC family were identified in Zoysia japonica; they were designated ZjCNGC1 through ZjCNGC18 according to their scaffold-level chromosomal positions. ZjCNGCs are divided into four groups (I-IV), with the same groups having differentiated protein-conserved domains and gene structures. ZjCNGCs are unevenly distributed on 16 scaffold-level chromosomes. Compared with other species, the ZjCNGCs in Group III exhibit obvious gene expansion, mainly due to duplication of gene segments. The collinearity between ZjCNGCs, OsCNGCs, and SjCNGCs suggests that CNGCs are evolutionarily conserved among gramineous plants. However, the Group III ZjCNGCs are only partially collinear with OsCNGCs and SjCNGCs, implying that the expansion of Group III ZjCNGC genes may have been an independent event occurring in Zoysia japonica. Protein interaction prediction revealed that ZjCNGCs, calcium-dependent protein kinase, H+-ATPase, outwardly rectifying potassium channel protein, and polyubiquitin 3 interact with ZjCNGCs. Multiple stress response regulatory elements, including those involved in salt stress, are present on the ZjCNGC promoter. The qPCR results revealed differences in the expression patterns of ZjCNGCs in different parts of the plant. Under salt stress conditions, the expression of ZjCNGCs was significantly upregulated in roots and leaves, with ZjCNGC8 and ZjCNGC13 showing the greatest increase in expression in the roots. These results collectively suggest that ZjCNGCs play an important role in salt tolerance and that their expansion into Group III may be a special mechanism underlying the salt tolerance of Zoysia japonica.

Identification of Shaker Potassium Channel Family Members and Functional Characterization of SsKAT1.1 in Stenotaphrum secundatum Suggest That SsKAT1.1 Contributes to Cold Resistance.

Stenotaphrum secundatum is an excellent shade-tolerant warm-season turfgrass. Its poor cold resistance severely limits its promotion and application in temperate regions. Mining cold resistance genes is highly important for the cultivation of cold-resistant Stenotaphrum secundatum. Although there have been many reports on the role of the Shaker potassium channel family under abiotic stress, such as drought and salt stress, there is still a lack of research on their role in cold resistance. In this study, the transcriptome database of Stenotaphrum secundatum was aligned with the whole genome of Setaria italica, and eight members of the Shaker potassium channel family in Stenotaphrum secundatum were identified and named SsKAT1.1, SsKAT1.2, SsKAT2.1, SsKAT2.2, SsAKT1.1, SsAKT2.1, SsAKT2.2, and SsKOR1. The KAT3-like gene, KOR2 homologous gene, and part of the AKT-type weakly inwardly rectifying channel have not been identified in the Stenotaphrum secundatum transcriptome database. A bioinformatics analysis revealed that the potassium channels of Stenotaphrum secundatum are highly conserved in terms of protein structure but have more homologous members in the same group than those of other species. Among the three species of Oryza sativa, Arabidopsis thaliana, and Setaria italica, the potassium channel of Stenotaphrum secundatum is more closely related to the potassium channel of Setaria italica, which is consistent with the taxonomic results of these species belonging to Paniceae. Subcellular location experiments demonstrate that SsKAT1.1 is a plasma membrane protein. The expression of SsKAT1.1 reversed the growth defect of the potassium absorption-deficient yeast strain R5421 under a low potassium supply, indicating that SsKAT1.1 is a functional potassium channel. The transformation of SsKAT1.1 into the cold-sensitive yeast strain INVSC1 increased the cold resistance of the yeast, indicating that SsKAT1.1 confers cold resistance. The transformation of SsKAT1.1 into the salt-sensitive yeast strain G19 increased the resistance of yeast to salt, indicating that SsKAT1.1 is involved in salt tolerance. These results suggest that the manipulation of SsKAT1.1 will improve the cold and salt stress resistance of Stenotaphrum secundatum.

Single-cell RNA-sequencing provides new insights into the cell-specific expression patterns and transcriptional regulation of photosynthetic genes in bermudagrass leaf blades

As an important warm-season turfgrass species, bermudagrass (Cynodon dactylon L.) flourishes in warm areas around the world due to the existence of the C4 photosynthetic pathway. However, how C4 photosynthesis operates in bermudagrass leaves is still poorly understood. In this study, we performed single-cell RNA-sequencing on 5296 cells from bermudagrass leaf blades. Eight cell clusters corresponding to mesophyll, bundle sheath, epidermis and vascular bundle cells were successfully identified using known cell marker genes. Expression profiling indicated that genes encoding NADP-dependent malic enzymes (NADP-MEs) were highly expressed in bundle sheath cells, whereas NAD-ME genes were weakly expressed in all cell types, suggesting C4 photosynthesis of bermudagrass leaf blades might be NADP-ME type rather than NAD-ME type. The results also indicated that starch synthesis-related genes showed preferential expression in bundle sheath cells, whereas starch degradation-related genes were highly expressed in mesophyll cells, which agrees with the observed accumulation of starch-filled chloroplasts in bundle sheath cells. Gene co-expression analysis further revealed that different families of transcription factors were co-expressed with multiple C4 photosynthesis-related genes, suggesting a complex transcription regulatory network of C4 photosynthesis might exist in bermudagrass leaf blades. These findings collectively provided new insights into the cell-specific expression patterns and transcriptional regulation of photosynthetic genes in bermudagrass.

Plant-Parasitic Nematode Genera Associated with Turfgrass in Maryland Golf Courses and Athletic Fields.

Field surveys were conducted to assess the occurrence and diversity of plant-parasitic nematodes (PPNs) in golf courses and athletic fields across Maryland, USA, during 2022 and 2023. A total of 28 golf courses and ten athletic fields were surveyed, revealing the prevalence and abundance of 13 PPNs taxa in the region. Criconemoides was identified as the most prevalent (94.9%) and Tylenchorhynchus as the most abundant (2.3) across all samples. Central golf courses (west side of the Chesapeake Bay) exhibited a high prevalence of Criconemoides and Tylenchorhynchus, while Eastern Shore golf courses and athletic fields displayed a higher prevalence of Helicotylenchus and Criconemoides. Further, Belonolaimus longicaudatus was reported for the first time from turfgrass in Maryland, raising concerns due to its potential to cause severe damage on both cool- and warm-season turfgrass. Biodiversity analysis indicated that richness (R2) was higher in athletic fields, while diversity (H') and evenness (J') were significantly greater in golf courses. This study provides baseline information for monitoring PPNs distribution in Maryland and also for the development of effective nematode management approaches in turfgrass ecosystems.

AcEXPA1, an α-expansin gene, participates in the aluminum tolerance of carpetgrass (Axonopus compressus) through root growth regulation.

AcEXPA1, an aluminum (Al)-inducible expansin gene, is demonstrated to be involved in carpetgrass (Axonopus compressus) root elongation under Al toxicity through analyzing composite carpetgrass plants overexpressing AcEXPA1. Aluminum (Al) toxicity is a major mineral toxicity that limits plant productivity in acidic soils by inhibiting root growth. Carpetgrass (Axonopus compressus), a dominant warm-season turfgrass widely grown in acidic tropical soils, exhibits superior adaptability to Al toxicity. However, the mechanisms underlying its Al tolerance are largely unclear, and knowledge of the functional genes involved in Al detoxification in this turfgrass is limited. In this study, phenotypic variation in Al tolerance, as indicated by relative root elongation, was observed among seventeen carpetgrass genotypes. Al-responsive genes related to cell wall modification were identified in the roots of the Al-tolerant genotype 'A58' via transcriptome analysis. Among them, a gene encoding α-expansin was cloned and designated AcEXPA1 for functional characterization. Observed Al dose effects and temporal responses revealed that Al induced AcEXPA1 expression in carpetgrass roots. Subsequently, an efficient and convenient Agrobacterium rhizogenes-mediated transformation method was established to generate composite carpetgrass plants with transgenic hairy roots for investigating AcEXPA1 involvement in carpetgrass root growth under Al toxicity. AcEXPA1 was successfully overexpressed in the transgenic hairy roots, and AcEXPA1 overexpression enhanced Al tolerance in composite carpetgrass plants through a decrease in Al-induced root growth inhibition. Taken together, these findings suggest that AcEXPA1 contributes to Al tolerance in carpetgrass via root growth regulation.

Quality and Establishment of Some Water-Conserving Turfgrass Species for Sustainable Development and Some Ecosystem Services in Arid Urban Environments

Turfgrasses are essential landscape plants with social, environmental, and aesthetic services for urban ecosystems. However, more is needed to know how to establish them so that they can benefit from their ecosystem services in urban environments. This research examined some quality and morphological and physiological factors for the establishment and social and environmental service assessment of three warm-season turfgrasses, including Kikuyu grass (Pennisetum clandestinum), bermuda grass (Cynodon dactylon), and buffalo grass (Buchloe dactyloides), compared to the cool-season grass of tall fescue (Festuca arundinacea Schreb.). The experiment was split-plot in time, based on a randomized complete block design with eight replications. The main plot was the season with four levels, and the subplot was the four turfgrass species types. The results indicated that seasons and turfgrass types and their interaction significantly impacted most measured variables (p ≤ 0.01). Some quality measurements like turf density, color, texture, coverage, and quality after clipping and establishment confirmed the superiority of Buchloe dactyloides over the other species. Also, kikuyu grass showed higher turfgrass density, more potential for weed control, and higher coverage and growth rate but also showed invasiveness features. Tall fescue had the lowest visual aesthetic compared with the other turfgrass species. Warm-season turfgrasses adaptable to the ecology of the region should be used compared to tall fescue to achieve better turfgrass quality and social and ecosystem services for the sustainable development of arid urban environments.

Directional Breeding Generates Distinct Genetic Diversity in Hybrid Turf Bermudagrass as Probed with Simple Sequence Repeat Markers

Bermudagrass (Cynodon spp.) is a drought-resistant warm-season turfgrass adapted to the southern and transitional zones in the United States. Multiple hybrid cultivars have been developed and released for use as turfgrass, and others are still undergoing development. Increasing genetic diversity of commercial cultivars is vital to stress tolerance. A DNA profiling study of 21 experimental selections from the Oklahoma State University turfgrass breeding program and 11 cultivars was conducted using 51 simple sequence repeat primer pairs across the bermudagrass genome. A pairwise genetic relationship analysis of the genotypes using 352 polymorphic bands showed genetic similarity coefficients ranging from 0.59 to 0.89. The average pairwise population differentiation values were 0.012 for the 11 cultivars and 0.169 for the 21 selections. A cluster analysis using the unweighted paired group with the arithmetic average method grouped the entries into six clusters. A correlation analysis identified different levels of pairwise genetic relationships among the entries that largely reflected parental relationship. Directional breeding and selection for cold hardiness or drought resistance created progeny that had distinct genetic diversity in the tested bermudagrasses. It is evident that an increase in genetic diversity of the existing cultivar pool with the release of one or more experimental selections for commercial use will strengthen and improve bermudagrass systems.

Whole-genome sequencing of allotetraploid bermudagrass reveals the origin of Cynodon and candidate genes for salt tolerance.

Bermudagrass (Cynodon dactylon) is a globally distributed, extensively used warm-season turf and forage grass with high tolerance to salinity and drought stress in alkaline environments. However, the origin of the species and genetic mechanisms for salinity tolerance in the species are basically unknown. Accordingly, we set out to study evolution divergence events in the Cynodon genome and to identify genes for salinity tolerance. We developed a 604.0 Mb chromosome-level polyploid genome sequence for bermudagrass 'A12359' (n = 18). The C. dactylon genome comprises 2 complete sets of homoeologous chromosomes, each with approximately 30 000 genes, and most genes are conserved as syntenic pairs. Phylogenetic study showed that the initial Cynodon species diverged from Oropetium thomaeum approximately 19.7-25.4 million years ago (Mya), the A and B subgenomes of C. dactylon diverged approximately 6.3-9.1 Mya, and the bermudagrass polyploidization event occurred 1.5 Mya on the African continent. Moreover, we identified 82 candidate genes associated with seven agronomic traits using a genome-wide association study, and three single-nucleotide polymorphisms were strongly associated with three salt resistance genes: RAP2-2, CNG channels, and F14D7.1. These genes may be associated with enhanced bermudagrass salt tolerance. These bermudagrass genomic resources, when integrated, may provide fundamental insights into evolution of diploid and tetraploid genomes and enhance the efficacy of comparative genomics in studying salt tolerance in Cynodon.

Genome-Wide Identification and Expression Analysis of the PUB Gene Family in Zoysia japonica under Salt Stress.

The U-box protein family of ubiquitin ligases is important in the biological processes of plant growth, development, and biotic and abiotic stress responses. Plants in the genus Zoysia are recognized as excellent warm-season turfgrass species with drought, wear and salt tolerance. In this study, we conducted the genome-wide identification of plant U-box (PUB) genes in Zoysia japonica based on U-box domain searching. In total, 71 ZjPUB genes were identified, and a protein tree was constructed of AtPUBs, OsPUBs, and ZjPUBs, clustered into five groups. The gene structures, characteristics, cis-elements and protein interaction prediction network were analyzed. There were mainly ABRE, ERE, MYB and MYC cis-elements distributed in the promoter regions of ZjPUBs. ZjPUBs were predicted to interact with PDR1 and EXO70B1, related to the abscisic acid signaling pathway. To better understand the roles of ZjPUBs under salt stress, the expression levels of 18 ZjPUBs under salt stress were detected using transcriptome data and qRT-PCR analysis, revealing that 16 ZjPUBs were upregulated in the roots under salt treatment. This indicates that ZjPUBs might participate in the Z. japonica salt stress response. This research provides insight into the Z. japonica PUB gene family and may support the genetic improvement in the molecular breeding of salt-tolerant zoysiagrass varieties.

First Report and Molecular Variability of Belonolaimus longicaudatus Associated with Turfgrass in Maryland.

Turfgrass is a crop used extensively in athletic fields and golf courses in Maryland. A soil sample collected in July 2023 from an athletic field in Baltimore County, Maryland, part of a turfgrass nematode survey, contained Belonolaimus longicaudatus. In the southeastern United States, B. longicaudatus is an economically important pathogen of warm season turfgrass. The density was four individuals/100 cm3 of soil, and no visual symptoms were observed in the bermudagrass field. Morphological features and morphometrics of males and females were consistent with B. longicaudatus and placed the Maryland population in a subclade that was geographically represented by populations from north and west Florida, Texas, and South Carolina. Sequencing of the internal transcribed spacer region ITS1 and ITS2 and 28S large ribosomal subunit D2-23 expansion region confirmed the species' identity. Phylogenetic trees and parsimony network analysis placed the Maryland isolate in a large grouping of B. longicaudatus populations including those from Alabama, Delaware, Florida, Indiana, Mississippi, South Carolina, and Texas. To our knowledge, this is the first report of B. longicaudatus in Maryland.

Confirmation and differential metabolism associated with quinclorac resistance in smooth crabgrass (Digitaria ischaemum)

AbstractQuinclorac controls crabgrass (Digitaria spp.) in cool- and warm-season turfgrass species. Herbicide-resistant smooth crabgrass [Digitaria ischaemum (Schreb.) Schreb. ex Muhl.] biotypes have evolved due to recurrent usage of quinclorac. Two Mississippi populations (MSU1 and MSU2) of D. ischaemum were characterized using standard greenhouse dose–response screens to assess their resistance relative to known susceptible populations. Subsequent investigations explored mechanisms of resistance, including examining cyanide accumulation, glutathione S-transferase (GST) activity, and the potential involvement of cytochrome P450s in MSU1, MSU2, and a susceptible (SMT2). Resistant populations MSU1 and MSU2 required 80 and 5 times more quinclorac, respectively, to reach 50% biomass reduction than susceptible populations. The SMT2 biotype accumulated three times more cyanide than the resistant MSU1 and MSU2 populations. GST activity was elevated in resistant MSU1 and MSU2 populations. Furthermore, quinclorac concentrations in treated resistant populations were elevated when plants were pretreated with the P450 inhibitor malathion. These findings suggest a non–target site based mechanism of resistance involving the accumulation of cyanide. This may provide a scientific basis for understanding the occurrence of quinclorac-resistant D. ischaemum, although further research is needed to investigate potential target-site mechanisms of resistance.

CRISPR/Cas9-mediated knockout of NYC1 gene enhances chlorophyll retention and reduces tillering in Zoysia matrella (L.) Merrill

Key MessageGenome editing by CRISPR/Cas9 can be applied to Z. matrella ‘Wakaba’, and knockout mutants of ZmNYC1 gene exhibited stay-green phenotype and reduced tillering.Zoysia matrella is a widely used C4 warm-season turfgrass for landscaping, golf courses, and sports fields. Here, we used the CRISPR/Cas9 system to target the Non-Yellow Coloring1 (ZmNYC1) gene in the highly heterozygous allotetraploid Z. matrella ‘Wakaba’, aiming to generate a novel stay-green variety. Of 441 Agrobacterium-infected calli, 22 (5.0%) were transformed, and 14 of these (63.6%) showed targeted mutations through cleaved amplified polymorphic sequences analysis. Sequencing analysis revealed mutations mostly consisting of 1 or 2 bp indels, occurring 2 to 4 bp upstream of the PAM sequence. Regenerated plants exhibited five ZmNYC1 target locus genotypes, including homozygous mutants with a complete knockout of all four alleles in the T0 generation. Under dark treatment, ZmNYC1-mutated plants displayed suppressed chlorophyll b (Chl b) degradation, leading to higher chlorophyll content and Chl b, with a lower chlorophyll a/chlorophyll b ratio compared to the wild type (WT). However, the ZmNYC1 mutation also inhibited plant growth in homozygous mutant genotypes, exhibiting reduced tillering compared to WT. Additionally, during winter simulation, mutant with a complete knockout retained greenness longer than the WT. This is the first successful use of CRISPR/Cas9 genome editing in zoysiagrass. The mutants of the ZmNYC1 gene would serve as valuable breeding material for developing improved zoysiagrass varieties that can maintain their green color for longer periods, even during winter dormancy.

Overexpression of PvWAK3 from seashore paspalum increases salt tolerance in transgenic Arabidopsis via maintenance of ion and ROS homeostasis

Seashore paspalum (Paspalum vaginatum O. Swartz) is an important warm-season turfgrass species with extreme salt tolerance, but investigations on its salt tolerance mechanism are limited. A salt induced PvWAK3 from halophyte seashore paspalum was identified in this study. Overexpression of PvWAK3 in Arabidopsis led to increased salt tolerance. Transgenic plants had higher levels of seed germination rate, root length, number of lateral roots, shoot weight, survival rate, Fv/Fm, ETR, and NPQ compared with the wild type (WT) under salt stress. Na+ content was increased and K+ content was decreased after salinity treatment, with lower levels of Na+ and Na+/K+ ratio but higher level of K+ in transgenic plants than in WT under salt stress. The improved maintenance of Na+ and K+ homeostasis was associated with the higher transcript levels of K + -Uptake Permease 4 (KUP4), Potassium Transport 2/3 (AKT2), Salt Overly Sensitive 1 (SOS1) and High-Affinity K + Transporter 5 (HAK5) in transgenic plants compared with WT. Superoxide dismutase (SOD), catalase (CAT) and ascorbate-peroxidase (APX) activities, proline concentration, and P5CS1 transcript were increased after salinity treatment, with higher levels in transgenic lines compared with WT, which led to reduced accumulation of O2·− and H2O2 under salt stress. It is suggested that PvWAK3 regulates salt tolerance positively, which is associated with promoted Na+ and K+ homeostasis, activated antioxidant enzymes, and proline biosynthesis under salt stress.

A New Method for Hybrid Bermuda Grass (Cynodon dactylon × C. transvaalensis Burtt.-Davy) Vegetative Propagation

Hybrid Bermuda grasses (Cynodon dactylon × C. transvaalensis Burtt.-Davy) represent one of the greatest contributions to the growing quality of turfgrass in the warm season and transition zone areas of the world. Hybrid Bermuda grass production relies on vegetative propagation from sod or sprigs. In the past, efforts have focused on improving the technique of stolonizing (or sprigging) for establishment in new areas. Such propagation requires bulk harvesting and planting of all rhizomes and stolons. We have developed a novel method of propagation and establishment from a single node harvested from greenhouse grown stolons. Despite a stolon fraction bearing a single node being suitable for effectively propagating a warm-season turfgrass, the technique has been held as economically impractical until now. Our method has been developed to obtain the multiplication of plant material in soilless conditions by harvesting single-node sprigs, propagation of plants from the single nodes, and transplant of single plants in the field. The investigation aimed to identify values for method set-up. Indeed, node and internode size variability with differential between maximum diameters is crucial for discrimination. For Patriot Bermuda grass stolons, nodes exhibited a maximum diameter of 2.43 ± 0.46 mm, while internodes had a maximum diameter of 1.54 ± 0.16 mm. Based on these findings, a 2 mm sieve was selected, achieving an optimal ratio between the node fraction and internode residues. The sieve yielded 87% of node fractions and only 1% of internodes from the initial mix, demonstrating its efficacy. Further results for the transplanting phase indicated that a double release resulted in an average success rate of 98.8%, with only 6.9% blank cells when using a single release. The average was 149 plants per tray over 160 cells, representing a 93.1% success rate. These results underscore the efficiency and acceptability of the overall propagation process in alignment with market references.

Transcriptome Analysis Reveals the Stress Tolerance Mechanisms of Cadmium in Zoysia japonica.

Cadmium (Cd) is a severe heavy metal pollutant globally. Zoysia japonica is an important perennial warm-season turf grass that potentially plays a role in phytoremediation in Cd-polluted soil areas; however, the molecular mechanisms underlying its Cd stress response are unknown. To further investigate the early gene response pattern in Z. japonica under Cd stress, plant leaves were harvested 0, 6, 12, and 24 h after Cd stress (400 μM CdCl2) treatment and used for a time-course RNA-sequencing analysis. Twelve cDNA libraries were constructed and sequenced, and high-quality data were obtained, whose mapped rates were all higher than 94%, and more than 601 million bp of sequence were generated. A total of 5321, 6526, and 4016 differentially expressed genes were identified 6, 12, and 24 h after Cd stress treatment, respectively. A total of 1660 genes were differentially expressed at the three time points, and their gene expression profiles over time were elucidated. Based on the analysis of these genes, the important mechanisms for the Cd stress response in Z. japonica were identified. Specific genes participating in glutathione metabolism, plant hormone signal and transduction, members of protein processing in the endoplasmic reticulum, transporter proteins, transcription factors, and carbohydrate metabolism pathways were further analyzed in detail. These genes may contribute to the improvement of Cd tolerance in Z. japonica. In addition, some candidate genes were highlighted for future studies on Cd stress resistance in Z. japonica and other plants. Our results illustrate the early gene expression response of Z. japonica leaves to Cd and provide some new understanding of the molecular mechanisms of Cd stress in Zosia and Gramineae species.

Identification of Accurate Reference Genes for qRT-PCR Analysis of Gene Expression in Eremochloa ophiuroides under Multiple Stresses of Phosphorus Deficiency and/or Aluminum Toxicity.

Centipedegrass (Eremochloa ophiuroides (Munro.) Hack.) is a species originating in China and is an excellent warm-season turfgrass. As a native species in southern China, it is naturally distributed in the phosphorus-deficient and aluminum-toxic acid soil areas. It is important to research the molecular mechanism of centipedegrass responses to phosphorus-deficiency and/or aluminum-toxicity stress. Quantitative Real-Time PCR (qRT-PCR) is a common method for gene expression analysis, and the accuracy of qRT-PCR results depends heavily on the stability of internal reference genes. However, there are still no reported stable and effective reference genes for qRT-PCR analysis of target genes under the acid-soil-related stresses in different organs of centipedegrass. For scientific rigor, the gene used as a reference for any plant species and/or any stress conditions should be first systematically screened and evaluated. This study is the first to provide a group of reliable reference genes to quantify the expression levels of functional genes of Eremochloa ophiuroides under multiple stresses of P deficiency and/or aluminum toxicity. In this study, centipedegrass seedlings of the acid-soil-resistant strain 'E041' and acid-soil-sensitive strain 'E089' were used for qRT-PCR analysis. A total of 11 candidate reference genes (ACT, TUB, GAPDH, TIP41, CACS, HNR, EP, EF1α, EIF4α, PP2A and actin) were detected by qRT-PCR technology, and the stability of candidate genes was evaluated with the combination of four internal stability analysis software programs. The candidate reference genes exhibited differential stability of expression in roots, stems and leaves under phosphorus-deficiency and/or aluminum-toxicity stress. On the whole, the results showed that GAPDH, TIP41 and HNR were the most stable in the total of samples. In addition, for different tissues under various stresses, the selected reference genes were also different. CACS and PP2A were identified as two stable reference genes in roots through all three stress treatments (phosphate deficiency, aluminum toxicity, and the multiple stress treatment of aluminum toxicity and phosphate deficiency). Moreover, CACS was also stable as a reference gene in roots under each treatment (phosphate deficiency, aluminum toxicity, or multiple stresses of aluminum toxicity and phosphate deficiency). In stems under all three stress treatments, GAPDH and EIF4α were the most stable reference genes; for leaves, PP2A and TIP41 showed the two highest rankings in all three stress treatments. Finally, qRT-PCR analysis of the expression patterns of the target gene ALMT1 was performed to verify the selected reference genes. The application of the reference genes identified as internal controls for qRT-PCR analysis will enable accurate analysis of the target gene expression levels and expression patterns in centipedegrass under acid-soil-related stresses.