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.     

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.     

Sexual Morph of <i>Furcasterigmium furcatum</i> (Plectosphaerellaceae) from <i>Magnolia liliifera</i> Collected in Northern Thailand

We isolated an interesting fungus from dead leaves of Magnolia liliifera collected from Chiang Mai, Thailand. The novel strain is related to Plectosphaerellaceae based on the morphology of its asexual morph and the analysis of sequence data. Phylogenetic analyses using a combined gene analysis of LSU and ITS sequence data showed that this strain is clustered in the same clade with Furcasterigmium furcatum with high statistical support. The new strains produced the asexual morph in culture which is morphologically similar to F. furcatum. Thus, we identified this strain as the sexual morph of F. furcatum. This is the first record of sexual morph for the monotypic genus Furcasterigmium and the first record of this genus on Magnolia.     

Phytochemical Analysis and Antioxidant Activity of <i>Crocus speciosus</i> Leaves

The numerous studies indicate leaves of plants are a rich source of bioactive compounds that can be a valuable source of compounds used in the pharmaceutical and cosmetic industries. Aim of this study was to investigate the chemical composition and the antioxidant property of Crocus speciosus leaves. Primary phytochemical screening of C. speciosus leaves revealed the presence of some following compound categories such as phenolic compounds, aminoacids, saponins, proteins, tannins, triterpenoids, glycosides, polysaccharides. The total flavonoids and phenolic compounds content were determined spectrophotometrically and by HPLC-DAD and HPLC-MS. Antiradical activity was determined by ABTS radical-cation scavenging method, spectrophotometrically. The total amount of flavonoids in C. speciosus leaves was 1.07 ± 0.02 mg RE/g (p < 0.05), the total amount of phenolic compounds was 0.41 ± 0.01 mg GAE/g (p < 0.05). By HPLC-DAD-MS analysis the presence of the mangiferin, chlorogenic acid, isoorientin, kaempferol, hyperoside, and isoquercitin was established for the first time in Crocus leaves. The antiradical activity of C. speciosus leaves extracts was 150.08 ± 4.5 μmol/g (p < 0.05) and its was mainly attributed to phenolic compounds content. The high amounts of flavonoids and antiradical activity in C. speciosus leaves suggests promising phytochemical and pharmacological study of this Crocus species.     

Effects of Barium Stress in <i>Brassica juncea</i> and <i>Cakile maritima</i>: The Indicator Role of Some Antioxidant Enzymes and Secondary Metabolites

Soil contamination by toxic trace metal elements, like barium (Ba), may stimulate various undesirable changes in the metabolic activity of plants. The plant responses are fast and with, direct or indirect, generation of reactive oxygen species (ROS). To cope with the stress imposed by the ROS production, plants developed a dual cellular system composed of enzymatic and non-enzymatic players that convert ROS, and their by-products, into stable nontoxic molecules. To assess the Ba stress response of two Brassicaceae species (Brassica juncea, a glycophyte, and Cakile maritime, a halophyte), plants were exposure to different Ba concentrations (0, 100, 200, 300 and 500 μM). The plants response was evaluated through their morphology and development, the determination of plant leaves antioxidant enzymatic activities and by the production of plants secondary metabolites. Results indicated that the two Brassicaceae species have the ability to survive in an environment containing Ba (even at 500 μM). The biomass production of C. maritima was slightly affected whereas an increase in biomass B. juncea was noticed. The stress imposed by Ba activated the antioxidant defense system in the two species, noticed by the changes in the leaves activity of catalase (CAT), ascorbate peroxidase (APX) and guaicol peroxidase (GPX), and of the secondary metabolites, through the production of total phenols and flavonoids. The enzymatic response was not similar within the two plant species: CAT and APX seem to have a more important role against the oxidative stress in C. maritima while in B. juncea is GPX. Overall, total phenols and flavonoids production was more significant in the plants aerial part than in the roots, of the both species. Although the two Brassicaceae species response was different, in both plants catalytic and non-catalytic transformation of ROS occurs, and both were able to overcome the Ba toxicity and prevent the cell damage.     

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.     

Physiological and Biochemical Mechanisms of Salinity Tolerance in <i>Carex morrowii</i> Boott

Carex species are widely used in many parts of the world and contain a large number of ecologically diverse species. Among the Carex species, some of them are known to be glycophytes, while others are halophytes. Carex morrowii Boott (Cyperaceae) is resistant to trample through their root structure and has an essential ornamental value in the landscape with their leaves. However, no information was found about the level of salinity tolerance/ sensitivity of the Carex morrowii among these species. In the present study, changes in trace element contents (Na, K, Ca, Cu, Mn, Mg, Ni, Fe, P, Zn, and N) and their transport from roots to leaves, osmotic regulation, alterations in chlorophyll and carotenoid contents, nitrogen assimilation (nitrate reductase activity; NRA) and total soluble protein content in both roots and leaves of Carex morrowii under different salinity concentrations (50 mM, 100 mM, 200 mM and 300 mM NaCl) were examined in detail. Our study provides the first detailed data concerning the responses of leaves and roots and the determination of the level of salinity tolerance/sensitivity of the Carex morrowii. The K+ /Na+ ratio was preserved up to 200 mM NaCl, and accordingly, the element uptake and transport ratios showed that they could control moderate NaCl levels. Ca homeostasis that is maintained even in 200 mM NaCl concentration can be effective in maintaining the structural integrity and selective permeability of the cell membranes, while 300 mM NaCl concentration caused decreased photosynthetic pigments, and deterioration in element content and compartmentation. Moreover, these data suggest that plant parts of Carex morrowii respond differently against varied levels of salinity stress. Although the decrease in NR activity at 200 mM and 300 mM NaCl concentrations in the leaves, NR activity was maintained in the roots. Consequently, Carex morrowii is moderately tolerant to salinity and the carotenoid content and osmotic regulation of Carex morrowii appears to be instrumental in its survival at different salinity levels. Especially the roots of Carex morrowii have a remarkable role in salinity tolerance.     

Identification and Evaluation of Insect and Disease Resistance in Transgenic <i>Cry1Ab13-1</i> and <i>NPR1</i> Maize

PCR detection, quantitative real-time PCR (q-RTPCR), outdoor insect resistance, and disease resistance identification were carried out for the detection of genetic stability and disease resistance through generations (T₂, T₃, and T₄) in transgenic maize germplasms (S3002 and 349) containing the bivalent genes (insect resistance gene Cry1Ab13-1 and disease resistance gene NPR1) and their corresponding wild type. Results indicated that the target genes Cry1Ab13-1 and NPR1 were successfully transferred into both germplasms through tested generations; q-PCR confirmed the expression of Cry1Ab13-1 and NPR1 genes in roots, stems, and leaves of tested maize plants. In addition, S3002 and 349 bivalent gene-transformed lines exhibited resistance to large leaf spots and corn borer in the field evaluation compared to the wild type. Our study confirmed that Cry1Ab13-1 and NPR1 bivalent genes enhanced the resistance against maize borer and large leaf spot disease and can stably inherit. These findings could be exploited for improving other cultivated maize varieties.     

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.     

Comparative Analysis of the Photosynthetic Characteristics and Active Compounds of <i>Semiliquidambar cathayensis</i> Chang Heteromorphic Leaves

In the present study, the variation patterns of leaf shape in different populations of individual Semiliquidambar cathayensis plants were analyzed to investigate the relationship among leaf shape variation, photosynthetic properties, and active compounds to understand the genetic characteristics of S. cathayensis and screen elite germplasms. The leaf shape of 18 offspring from three naturalS. cathayensis populations was analyzed to investigate the level of diversity and variation patterns of leaf shape. Furthermore, photosynthetic pigment content, physiological parameters of photosynthesis, and the active compounds in leaves of different shapes were determined. Statistical analysis showed that the leaf shape variation in  S. cathayensis indicated a high level of genetic diversity among and within the populations. Cluster analysis showed that the three natural populations formed two clusters, one whose offspring was dominated by entire leaves and another characterized by palmately trifoliate leaves. The differences in photosynthetic characteristics and active compounds of leaves of three different shapes were comprehensively evaluated using principal component analysis. Two principal components with a cumulative contribution rate of 92.768% were extracted, of which the highest comprehensive score was for asymmetrically lobed leaves. The leaf shape in different S. cathayensis germplasms exhibited distinct patterns, and there were some correlations between the photosynthetic properties and active compounds in leaves of different shapes. Thus, the leaf shape can be used to predict active compound content, and in turn, select varieties based on that purpose; it also provides a simple and effective method to classify S. cathayensis germplasms.     

Comparative Analysis of the Complete Chloroplast Genome Sequences of Four Origin Plants of Lonicerae Flos (<i>Lonicera</i>; Caprifoliaceae)

Lonicerae Flos (LF) derived from the dried flower buds or opening flowers of four Lonicera plants (Lonicera macranthoides, L. hypoglauca, L. confusa, and L. fulvotnetosa), is a popular traditional Chinese medicine. Because the four origin plants are very similar in morphology, it is difficult to control the quality of LF in actual production. Over the past decade, many reports have pointed out the differences among them, including the botanical characteristics and active ingredients. However, there is still a lack of rapid methods that can be applied to the identification of the four origins. In this study, comparative analysis of the four chloroplast genomes was performed, and they showed low diversity (Pi = 0.00267), three variation hotspots regions (rbcL-accD, rps12-ndhF and rps12-trnN-trnG) were identified as potentially molecular marker of highly informative. Meanwhile, the most obvious difference in SSR comparative analysis is reverse and complement repeats were only identified in L. confusa and L. hypoglauca, respectively. Lastly, the phylogenetic tree showed that L. confusa is more closely related to L. fulvotnetosa, while L. macranthoides is closer to L. hypoglauca. This study systematically revealed the differences among the four chloroplast genomes, and it provides valuable genetic information for identifying the origin of LF.     

<i>Aspergillus tubingensis</i> Causes Leaf Spot of Cotton (<i>Gossypium hirsutum</i> L.) in Pakistan

Cotton (Gossypium hirsutum L.) is a key fiber crop of great commercial importance. Numerous phytopathogens decimate crop production by causing various diseases. During July-August 2018, leaf spot symptoms were recurrently observed on cotton leaves in Rahim Yar Khan, Pakistan and adjacent areas. Infected leaf samples were collected and plated on potato dextrose agar (PDA) media. Causal agent of cotton leaf spot was isolated, characterized and identified as Aspergillus tubingensis based on morphological and microscopic observations. Conclusive identification of pathogen was done on the comparative molecular analysis of CaM and β-tubulin gene sequences. BLAST analysis of both sequenced genes showed 99% similarity with A. tubingensis. Koch’s postulates were followed to confirm the pathogenicity of the isolated fungus. Healthy plants were inoculated with fungus and similar disease symptoms were observed. Fungus was re-isolated and identified to be identical to the inoculated fungus. To our knowledge, this is the first report describing the involvement of A. tubingensis in causing leaf spot disease of cotton in Pakistan and around the world.     

Identification and Characterization of a Novel Yellow Leaf Mutant <i>yl1</i> in Rice

Leaf-color mutants play an important role in the study of chlorophyll metabolism, chloroplast development, and photosynthesis system. In this study, the yellow leaf 1 (yl1) rice mutant was identified from the ethyl methane sulfonate-treated mutant progeny of Lailong, a glutinous japonica rice landrace cultivated in Guizhou Province, China. Results showed that yl1 exhibited yellow leaves with decreased chlorophyll content throughout the growth period. Chloroplast development in the yl1 mutant was disrupted, and the grana lamellae was loosely packed and disordered. RNA sequencing and real-time quantitative polymerase chain reaction (qRT-PCR) analysis revealed that the chlorophyll synthesis-related genes OsCHLH, OsCHLM, OsCHLG, PORB, and YGL8, as well as the chloroplast development-related genes FtsZ, OsRpoTp, and RbcL, were down-regulated in the yl1 mutant. Genetic analysis revealed that the yellow leaf phenotype of yl1 was controlled by recessive nuclear gene. By employing the MutMap method, the mutation responsible for the phenotype was mapped to a 6.17 Mb region between 17.34 and 23.51 Mb on chromosome 3. Two non-synonymous single-nucleotide polymorphisms (SNPs) located in the gene locus LOC_Os03g31210 and LOC_Os03g36760 were detected in this region. The two SNPs were further confirmed by PCR and Sanger sequencing. The expression patterns of the two candidate genes indicated that LOC_Os03g36760 showed greater potential for functional verification. Subcellular protein localization revealed that the encoded product of LOC_Os03g36760 was localized in the nucleus, cytoplasm, and plasma membrane. These results will be useful for further characterization and cloning of the yl1 gene, and for research on the molecular mechanisms controlling biogenesis and chloroplast biochemical processes.