Selection and Verification of Reference Genes for qRT-PCR Analysis in <i>Iris domestica</i> under Drought

Iris domestica is a plant of the Iridaceae family and is drought-tolerant, but its drought-resistance mechanism is not yet clear. Analysing the gene expression changes of I. domestica by qRT-PCR is an important mean to understand its drought resistance characteristics. Nevertheless, a lack of reference genes greatly hinders investigation and research on the adaptation of I. domestica to drought at the molecular and genetic levels. In this study, we assessed the expression stability of 11 candidate gene in I. domestica under drought stress conditions and different tissues using geNorm, NormFinder, BestKeeper and RefFinder tools. The results showed that EF1β was the most stable reference genes under drought stress and in different tissues. To validate further the stability of the identified reference genes, the expression patterns of VP gene in I. domestica was analysed. These results will be conducive to more accurate quantification of gene expression levels in I. domestica.     

Colonization Characteristics and Diversity of Arbuscular Mycorrhizal Fungi in the Rhizosphere of <i>Iris lactea</i> in Songnen Saline-alkaline Grassland

To understand arbuscular mycorrhizal (AM) fungi resources and develop AM fungal species in ornamental plants with saline-alkaline tolerances, Iris lactea, which grows in the Songnen saline-alkaline grassland with a high ornamental value, was selected as the experimental material, and the colonization characteristics of its roots and the AM fungal diversity in its rhizosphere were explored. The results of the observations and calculations of mycorrhizae from ten different samples showed that AM fungi colonized the roots of I. lactea and formed Arum-type mycorrhizal structures. There was a significant correlation between soil spore density and pH value, while the colonization rate showed a fluctuating trend with increasing pH values. The observed colonization intensities were of Levels II (1%–10%) or III (11%–50%), and the vesicle abundances were of grades A₂ or A₃ among different sites. AM fungi produced a large number of mycelia and vesicles in the roots of I. lactea after colonization. Thirty-seven species belonging to 15 genera of AM fungi were isolated from the rhizosphere of I. lactea and identified by morphological identification. Funneliformis and Glomus were the dominant genera, accounting for 21.79% and 20.85% of the total number, respectively. F. mosseae and Rhizophagus intraradices were isolated in all samples with importance values of 58.62 and 51.19, respectively. These results are expected to provide a theoretical basis for the analysis of the salt tolerance mechanism of I. lactea and for the discovery, exploration and further screening of AM fungal resources with salinity tolerances in saline-alkaline soils.     

Differential Responses of <i>NHX1</i> and <i>SOS1</i> Gene Expressions to Salinity in two <i>Miscanthus sinensis</i> Anderss. Accessions with Different Salt Tolerance

The lignocellulosic crop Miscanthus spp. has been identified as a good candidate for biomass production. The responses of Miscanthus sinensis Anderss. to salinity were studied to satisfy the needs for high yields in marginal areas and to avoid competition with food production. The results indicated that the relative advantages of the tolerant accession over the sensitive one under saline conditions were associated with restricted Na⁺ accumulation in shoots. Seedlings of two accessions (salt-tolerant ‘JM0119’ and salt-sensitive ‘JM0099’) were subjected to 0 (control), 100, 200, and 300 mM NaCl stress to better understand the salt-induced biochemical responses of genes involved in Na⁺ accumulation in M. sinensis. The adaptation responses of genes encoding for Na⁺ /H⁺ antiporters, NHX1 and SOS1 to NaCl stress were examined in JM0119 and JM0099.The cDNA sequences of genes examined were highly conserved among the relatives of M. sinensis based on the sequencing on approximate 600 bp-long cDNA fragments obtained from degenerate PCR. These salt-induced variations of gene expression investigated by quantitative real-time PCR provided evidences for insights of the molecular mechanisms of salt tolerance in M. sinensis. The expression of NHX1 was up-regulated by salt stress in JM0119 shoot and root tissues. However, it was hardly affected in JM0099 shoot tissue except for a significant increase at the 100 mM salt treatment, and it was salt-suppressed in the JM0099 root tissue. In the root tissue, the expression of SOS1 was induced by the high salt treatment in JM0119 but repressed by all salt treatments in JM0099. Thus, the remarkably higher expression of NHX1 and SOS1 were associated with the resistance to Na⁺ toxicity by regulation of the Na⁺ influx, efflux, and sequestration under different salt conditions.     

Development of a New Cold-Tolerant Maize (<i>Zea mays</i> L.) Germplasm Using the <i>ICE1</i> Gene from <i>Arabidopsis thaliana</i>

To develop cold-tolerant maize germplasms and identify the activation of INDUCER OF CRT/DRE-BINDING FACTOR EXPRESSION (ICE1) expression in response to cold stress, RT-PCR was used to amplify the complete open reading frame sequence of the ICE1 gene and construct the plant expression vector pCAMBIA3301-ICE1-Bar. Immature maize embryos and calli were transformed with the recombinant vector using Agrobacterium tumefaciens-mediated transformations. From the regenerated plantlets, three T1 lines were screened and identified by PCR. A Southern blot analysis showed that a single copy of the ICE1 gene was integrated into the maize (Zea mays L.) genomes of the three T1 generations. Under low temperature-stress conditions (4°C), the relative conductivity levels decreased by 27.51%–31.44%, the proline concentrations increased by 12.50%–17.50%, the malondialdehyde concentrations decreased by 16.78%–18.37%, and the peroxidase activities increased by 19.60%–22.89% in the T1 lines compared with those of the control. A real-time quantitative PCR analysis showed that the ICE1 gene was ectopically expressed in the roots, stems, and leaves of the T1 lines. ICE1 positively regulates the expression of the CBF genes in response to cold stress. Thus, this study showed the successful transformation of maize with the ICE1 gene, resulting in the generation of a new maize germplasm that had increased tolerance to cold stress.     

Genome-Wide Identification,Evolution and Expression Analyses of <i>GA2ox</i> Gene Family in <i>Brassica napus</i> L.

Gibberellin 2-oxidases (GA2ox) are important enzymes that maintain the balance of bioactive GAs in plants. GA2ox genes have been identified and characterized in many plants, but these genes were not investigated in Brassica napus. Here, we identified 31 GA2ox genes in B. napus and 15 of these BnaGA2ox genes were distributed in the A and C subgenomes. Subcellular localization predictions suggested that all BnaGA2ox proteins were localized in the cytoplasm, and gene structure analysis showed that the BnaGA2ox genes contained 2–4 exons. Phylogenetic analysis indicated that BnGA2ox family proteins in monocotyledons and dicotyledons can be divided into four groups, including two C₁₉-GA2ox and two C₂₀-GA2ox clades. Group 4 is a C₂₀-GA2ox Class discovered recently. Most BnaGA2ox genes had a syntenic relationship with AtGA2ox genes. BnaGA2ox genes in the C subgenome had experienced stronger selection pressure than genes in the A subgenome. BnaGA2ox genes were highly expressed in specific tissues such as those involved in growth and development, and most of them were mainly involved in abiotic responses, regulation of phytohormones and growth and development. Our study provided a valuable evolutionary analysis of GA2ox genes in monocotyledons and dicotyledons, as well as an insight into the biological functions of GA2ox family genes in B. napus.     

Effects of Different Geographical Aspects and Ontogenetic Variability on Total Hypericin Content of <i>Hypericum triquetrifolium</i> Turra. and <i>Hypericum scabrum</i> L.

The present study was conducted to determine the total hypericin contents of Hypericum triquetrifolium Turra. and Hypericum scabrum L. species which are naturally distributed in the flora of Siirt province, Turkey. Hypericin contents of Hypericum species grown in different geographical aspects (North, South, East, and West), and it was measured at different harvest times (full blooming and post blooming period). In the current study, it has been determined that total hypericin content varies considerably according to aspects, plant developmental stages (ontogenetic variance), and species. According to species x aspect interaction, the highest total hypericin content was recorded from the west aspect (3.13 mg/g) in Hypericum triquetrifolium, while, the lowest hypericin content was also obtained from the west aspect (1.22 mg/g) in Hypericum scabrum. When the highest total hypericin content was analyzed according to aspect x species x harvest time interaction, the highest total hypericin content was produced from Hypericum triquetrifolium at the harvest of west aspect with 5.28 mg/g, while the minimum amount of hypericin was obtained from the same aspect in Hypericum scabrum with 0.50 mg/g. In species x harvest time interaction, the highest total hypericin content was obtained from the full bloom (3.10 mg/g) harvest in Hypericum triquetrifolium, while the lowest hypericin was obtained from the full bloom (1.26 mg/g) harvest in Hypericum scabrum. The data suggest that the average total hypericin content was 2.26 mg/g in Hypericum triquetrifolium and 1.28 mg/g in Hypericum scabrum.     

Antibacterial Activity and Mechanisms of Ethanol Extracts from <i>Scutellaria baicalensis</i> Georgi and <i>Magnolia officinalis</i> against <i>Phytophthora nicotianae</i>

Phytophthora nicotianae causes substantial economic losses in most countries where tobacco is produced. At present, the control of P. nicotianae mainly depends on chemical methods, with considerable environmental and health issues. We investigated the effects of ethanol extracts from Scutellaria baicalensis Georgi (SBG) and Magnolia officinalis (MO). On mycelial growth, sporangium formation, and zoospore release of P. nicotianae. Both extracts inhibited the growth of P. nicotianae, with mycelial growth inhibition rates of 88.92% and 93.92%, respectively, at 40 mg/mL, and EC50 values of 5.39 and 5.74 mg/mL, respectively. The underlying mechanisms were the inhibition of sporangium formation, the reduction of zoospore number, and the destruction of the mycelium structure. At an SBG extract concentration of 16.17 mg/mL, the inhibition rates for sporangia and zoospores were 98.66% and 99.39%, respectively. At an MO extract concentration of 2.87 mg/mL, the production of sporangia and zoospores was completely inhibited. The hyphae treated with the two plant extracts showed different degrees of deformation and damage. Hyphae treated with SBG extract showed adhesion and local swelling, whereas treatment with MO extract resulted in broken hyphae. Mixture of the extracts resulted in a good synergistic effect.     

Genome-Wide Analysis of the <i>KANADI</i> Gene Family and Its Expression Patterns under Different Nitrogen Concentrations Treatments in <i>Populus trichocarpa</i>

KANADI (KAN) is a plant-specific gene that controlled the polarity development of lateral organs. It mainly acted on the abaxial characteristics of plants to make the lateral organs asymmetrical. However, it had been less identified in woody plants. In this study, the members of the KAN gene family in Populus trichocarpa were identified and analyzed using the bioinformatics method. The results showed that a total of 8 KAN family members were screened out, and each member contained the unique GARP domain and conserved region of the family proteins. Phylogenetic analysis and their gene structures revealed that all KAN genes from P. trichocarpa, Arabidopsis thaliana, and Nicotiana benthamiana could be divided into four subgroups, while the eight genes in P. trichocarpa were classified into three subgroups, respectively. The analysis of tissue-specific expression indicated that PtKAN1 was highly expressed in young leaves, PtKAN6 was highly expressed in young leaves and mature leaves, PtKAN2, PtKAN5, and PtKAN7 were highly expressed in nodes and internodes, PtKAN8 was highly expressed in roots, and PtKAN3 and PtKAN4 showed low expression levels in all tissues. Among them, PtKAN2 and PtKAN6, and PtKAN4 and PtKAN5 might have functional redundancy. Under high nitrogen concentrations, PtKAN2 and PtKAN8 were highly expressed in mature stems and leaves, respectively, while PtKAN4, PtKAN5, and PtKAN7 were highly expressed in roots. This study laid a theoretical foundation for further study of the KAN gene-mediated nitrogen effect on root development.     

Screening of <i>Bacillus subtilis</i> HAAS01 and Its Biocontrol Effect on <i>Fusarium</i> wilt in Sweet Potato

Fusarium wilt, a disease caused by Fusarium oxysporum f.sp batatas (Fob) is an important disease in sweet potato production. Using endophytic bacteria for biological control of sweet potato diseases is one of the important ways. A Bacillus subtilis with antagonistic effect on Fusarium wilt of sweet potato was isolated from soil by confrontation culture. According to the biological characteristics, 16S rDNA sequence analysis, and physiological and biochemical analysis, the Bacillus subtilis HAAS01 was named. A pot experiment was conducted for the biological control experiment of strain HAAS01, and the endogenous hormone content, antioxidant enzyme activity, soluble protein content, and related gene expressions of sweet potato plants were detected. The results showed that the HAAS01 strain could promote the production of endogenous hormones and resist the infection of plant diseases together with defensive enzymes and upregulation of related gene expressions. In summary, Bacillus subtilis HAAS01 was effective in controlling Fusarium wilt of sweet potato and has potential for application and development.     

Cloning and Characterization of <i>EuGID1</i> in <i>Eucommia ulmoides</i> Oliver

Gibberellic acid controlled the key developmental processes of the life cycle of landing plants, and regulated the growth and development of plants. In this study, a novel gibberellin receptor gene EuGID1 was obtained from Eucommia ulmoides Oliver. The cDNA of EuGID1 was 1556 bp, and the open reading frame was 1029 bp, which encoded 343 amino acids. EuGID1 had the homology sequence with the hormone-sensitive lipase family. Amino acid sequence alignment confirmed EuGID1 protein had the highest homology with the GID1 protein of Manihot esculenta. EuGID1 was located in the nucleus and cell membrane and had expression in four plant organs. Overexpression of EuGID1 in transgenic Arabidopsis plants promoted plant elongation and increased siliques yield.     

First Report of <i>Fusarium striatum</i> Causing Root Rot Disease of <i>Panax notoginseng</i> in Yunnan,China

Panax notoginseng is a traditional Chinese medicinal plant. Root rot of P. notoginseng is one of the most serious diseases affecting P. notoginseng growth and causes wilted leaves, fewer lateral roots and rotten roots. Root rot is a soil-borne disease, and mainly occurs from June to August in Yunnan Province when the temperatures are high and the air is humid. In this study, the endophytic fungal genus Fusarium isolate E-2018.1.22-#3.2 was obtained from a P. notoginseng embryo. Fusarium isolate E-2018.1.22-#3.2 was identified as Fusarium striatum based on morphological characteristics and molecular analysis. The fungus was found to have conidiophores and macroconidia, and its ITS, LSU and TEF-1α genes shared 100%, 99.2% and 99% identities with the homologous genes of Fusarium striatum, respectively. Isolate F. striatum E-2018.1.22-#3.2 can cause root rot symptoms, including black, soft roots, fewer lateral roots and leaf wilt, in 93% of the experimental P. notoginseng plants, and could be re-isolated, fulfilling Koch’s postulates. When the P. notoginseng plants were treated with the fungicide pyraclostrobin, isolate F. striatum E-2018.1.22-#3.2 was unable to cause root rot. We have therefore demonstrated that F. striatum E-2018.1.22-#3.2 is able to cause root rot disease in P. notoginseng. This is the first report of root rot disease caused by F. striatum on P. notoginseng in China.     

AM Fungi and <i>Piriformospora indica</i> Improve Plant Growth of <i>Pinus elliottii</i> Seedlings

Pinus elliottii is an exotic afforestation pine extensively distributed in southern parts of China. In order to understand whether endophytic fungi can affect seedling growth of P. elliottii, Piriformospora indica (Pi), Funnelifcrmis mosseae (Fm), and Diversispora tortuosa (Dt) were inoculated respectively, and the non-inoculated group was set as control. The growth indexes, the contents of soluble sugar and soluble protein, and plant endogenous hormone levels in the leaves of P. elliottii, were analyzed. The results showed that Fm, Dt and Pi colonized the P. elliottii roots to form mycorrhizal structure and chlamydospores arranged in beads respectively. Three fungal inoculants exhibited the stimulated growth responses, whilst Dt illustrated the most positive effect on plant height, single fresh weight, trunk diameter and root system structure, compared with the control. On the other hand, the soluble sugar and soluble protein contents were increased distinctively in mycorrhizal plants. The endogenous IAA, GA3, ZR contents were increased, while the ABA contents were reduced in mycorrhizal plants versus non-mycorrhizal plants. The fungi-induced endogenous hormone changes triggered plant growth improvement of P. elliottii seedlings. This research unraveled the positive effect of AM fungi and P. indica on growth of pine seedlings, while, more application of endophytic fungi to fields needs to be explored.     

Efficient Evergreen Plant Regeneration of <i>Cinnamomum japonicum</i> Sieb. through <i>in vitro</i> Organogenesis

Cinnamomum japonicum Sieb. is an excellent roadside tree and medicinal tree species with considerable ornamental and economic value. In this study, we successfully developed a large-scale micropropagation protocol for C. japonicum for the first time. Sterilized shoots were excised and used as explants for shoot induction on several basal media, supplemented with different concentrations of plant growth regulators (PGRs), such as Thidiazuron (TDZ), N⁶ -Benzyladenine (6-benzylaminopurine) (BA), α-naphthaleneacetic acid (NAA) and Gibberellic acid (GA₃). After comparison, the most efficient medium for shoot regeneration was 1/2 Murashige and Skoog (MS) medium containing 0.5 mg L^–1 BA, 0.05 mg L^–1 NAA and 0.2 mg L^–1 GA₃, which resulted in an average number of induced shoots per explant and shoot length of 5.2 and 1.62 cm at 28 d, respectively. Then, elongated adventitious shoots were transferred to induce roots. 86.7% of shoots was able to root on 1/2 MS medium supplemented with 0.5 mg L^–1 NAA and 0.1 mg L^–1 BA. The earliest rooting time observed was after 21 d and the average root length was up to 3.3 cm after 28 d. Our study shows that C. japonicum can be successfully regenerated through de novo organogenesis, which lays a foundation for future transformation research on this tree.