Growth and Physiological Responses of Magnoliaceae to NaCl Stress.

The growth and physiological characteristics of four Magnoliaceae plants (Yulania biondii, Yulania denudata, and two varieties of Magnolia wufengensis (Jiaohong 1 and Jiaohong 2)) were investigated. Four Magnoliaceae plants were subjected to various concentrations of NaCl for 60 days: 0 mM, 60 mM, 120 mM, 180 mM, and 240 mM. The leaf water content (LWC), relative growth rate of plant height and stem diameter, photosynthetic pigments, and photosynthetic rate (Pn) decreased during the NaCl treatments, indicating slowed growth and photosynthesis. Malondialdehyde (MDA), Na+, superoxide dismutase (SOD) activity, peroxidase (POD) activity, ascorbic acid (AsA) content, and soluble sugar content all increased while K+ decreased. Ascorbate peroxidase (APX) activity, glutathione (GSH), soluble protein, and proline first increased after decreasing with increasing NaCl concentration. Principal component 1 (PC1) had larger loading values for growth and photosynthesis indices, while principal component 2 (PC2) exhibited larger loading values for antioxidant substances and osmotic adjustment substances; the correlation analysis showed that PC1 and PC2 had negative correlations. The four Magnoliaceae plants exhibited largely variable growth and physiological activities in response to NaCl. Yulania denudata exhibited greater reductions in growth and photosynthesis and greater decreases in antioxidant enzyme activities and osmotic adjustment substances, which indicated poor tolerance to salt stress. Among the four Magnoliaceae plants, Jiaohong 1 exhibited the greatest salt tolerance, followed by Jiaohong 2, Yulania biondii, and Yulania denudata.

First report of Fusarium solani (FSSC 6) associated with the root rot of Magnolia denudata in China.

Magnolia denudata (Lilytree or Yulan magnolia) is an important ornamental species of the genus Magnolia. It has considerable economical value because of its beautiful fragrant flowers and excellent tree structure (Wang et al. 2010). In Beijing, nurseries cultivate M. denudata as an ornamental plant and traditional medicine. In May 2020, patches of root rotted plants were observed in a field in Beijing, China, with an estimated incidence of approximately 31%. Early symptoms comprised leaves melanocratic shrunken, and the vascular tissue of roots turned brown. Progressively, the roots rotted and the whole plant died (Fig. 1 a-d). Infected roots tissue was surface disinfested and plated on potato dextrose agar (PDA) medium at 25±2 °C and incubated in the dark for 7 days. Pure cultures were obtained by hyphal tip excision (strain MFR1215.4). Fungal colonies were entire margins, and the aerial mycelium was copious, early white, and gradually developed into cream white. Colonies developed to 45.1 mm in 4 days at 25±2 °C on PDA media. On Spezieller Nährstoffarmer Agar (SNA) medium at 25±2 °C for 10 days. The morphological characteristics including macroconidia, microconidia, and chlamydospores were shown in Fig.1 (i-p). These morphological characteristics of the isolate corresponded to the description given for Fusarium solani sensu lato (Nelson et al. 1983, Summerell, 2003). Molecular identification was confirmed via amplification of translation elongation factor 1α (EF-1α), RNA polymerase I beta subunit gene (RPB1), and RNA polymerase II beta subunit gene (RPB2) regions using EF1/EF2, RPB1-Fa/G2R, RPB2-5f2/7cR, and RPB2-7cF/11aR primers (O'Donnell, 2010). Sequences were registered in GenBank. In the Fusarium-ID database, the EF-1α, RPB1, and RPB2 sequences showed 100% (677/677 bp), 99.8% (1568/1571 bp), and 100% (1457/1457 bp) identity with the F. solani species complex (FSSC). The same species-level identification was also found using Fusarium MLST. A best maximum likelihood tree was constructed using PhyloSuite v1.2.2 (Zhang et al. 2020), and the sequences of the MFR1215.4 isolation showed the same homology with FSSC 6. Pathogenicity tests were conducted on healthy one-year-old M. denudata potted seedlings. 200 ml spore suspension (1×106 spores/ml) was poured over the roots of twenty seedlings, and sterile distilled water was irrigated into twenty seedlings as controls in a greenhouse with 25/15°C day/night temperature and 80% relative humidity. The experiment was repeated three times. All inoculated seedlings showed similar symptoms to those in the field after 65 days, whereas the controls remained symptomless. The reisolating pathogens from symptomatic tissues were identical to the original isolates by morphology and EF-1α sequence identification. Based on morphological, molecular, and pathogenic characterization, the isolated pathogen was identified as FSSC 6. Fusarium species have been recorded in various places of the world and are known to be harmful to numerous plants (Trabelsi et al. 2017). It has been reported that FSSC has infected soybeans (Coleman, 2016, Nelson et al. 1989), oil palm (Hafizi et al. 2013), tobacco (Yang et al. 2020), resulting in sudden death syndrome, crown disease, and root rot. To our knowledge, this is the first report of FSSC-induced root rot in M. denudata in China. This research may contribute to the development of epidemiology and management strategies for root rot caused by FSSC on M. denudata.

First Report of Leaf Spot Caused by Colletotrichum fructicola on Magnolia wufengensis in Hubei, China.

Magnolia wufengensis belongs to the Magnoliaceae family. Its variation-rich flowers (tepal number from 9 to 46, tepal color from pink to bright red) and excellent wood characteristics (strong, straight, texture) have important ornamental and economic value (Duan et al. 2019; Luyi et al. 2006). M. wufengensis is popularly cultivated in parks, courtyards, mountains, and along roadsides. In May 2020, leaf spot symptoms were observed on over 85% of M. wufengensis in Yuyangguan Township, Wufeng County, Hubei Province (110.60°E, 30.21°N). The damaged area was over 18.7 hectares. Early symptoms began as small brown spots with a light-yellow halo. Gradual lesions expanded, and the center was withered, gray, and necrotic with a dark brown border. Eventually, several spots combined with larger irregular lesions, turning the leaves yellow and causing them to fall off. The border of lesions and healthy tissues were cut into small pieces (5×5 mm), and surface sterilized with 1% sodium hypochlorite solution for three minutes, rinsed three times with sterile water, and plated on potato dextrose agar (PDA) medium at 25±2 °C with a 12h photoperiod under fluorescent lighting. Pure isolates (MCS1228.1, MCS1228.4, MCS1228.9) were gray to pale grayish, and their average growth rate was 10.5±1.23 mm/day. Conidiophores were hyaline, aseptate, branched. Conidia were hyaline, aseptate, cylindrical, and 14.00 to 25.17 × 4.74 to 6.56 µm in size (average 17.48 × 5.58 µm) (n=50). Appressoria were brown and showed multivariate shape. The morphological characteristics of the isolates corresponded to the description given for Colletotrichum fructicola (Liu et al. 2015). Molecular identification was accomplished through amplification of the internal transcribed spacer (IST), actin (ACT), calmodulin (CAL), chitin synthase (CHS-1) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin (TUB2) genes (Fu et al. 2018). The ITS (OL800580.1, OL800581.1, OL800582.1), ACT (GenBank accession No. OL873155- OL873157), CAL (GenBank accession No. OL873158- OL873160), CHS-1 (GenBank accession No. OL873161- OL873163), GAPDH (GenBank accession No. OL873164- OL873166) and TUB2 (GenBank accession No. OL873167- OL873169) sequences were deposited in GenBank. A Bayesian inference phylogenetic tree based on multilocus sequences was constructed, and the sequences of the 3 isolations showed the same homology with C. fructicola (Fu et al. 2018). To fulfill Koch's postulates, 30 potted seedlings were inoculated with 1×10^6 conidia/ml suspension of each isolate by spraying the leaves, and 30 potted seedlings were sprayed with sterile distilled water as control. Inoculated and control plants were kept in a greenhouse with 25/15°C (day/night) temperature and 80% relative humidity. In addition, 30 healthy detached leaves free of pests and diseases were washed three times with sterile distilled water, air-dried, and artificially inoculated using a 6 mm (diameter) PDA medium (5 days incubation) with mycelium. 30 leaves were inoculated with sterile PDA medium as control. All leaves were sprayed with sterile water every 24 hours, covered with plastic wrap, and incubated at 25±2 °C, 100% humidity. The experiment was repeated three times. Similar symptoms to those found initially were both observed on all the inoculated potted seedlings and detached leaves after 14 days and 5 days post inoculation (dpi), respectively. Whereas the controls remained symptomless. The reisolated pathogens from symptomatic tissues were identical to the original isolates. In this study, isolated fungi associated with M. wufengensis leaf spot were identified as C. fructicola based on morphological and multiloci phylogenetic analyses, and Koch's postulates. Colletotrichum species are important plant pathogens and cause diseases in a wide variety of woody and herbaceous plants (Cannon et al. 2012). C. fructicola has been identified as a responsible pathogen for apple (Casanova et al. 2016), Fatsia japonica (Shi et al. 2017), and Rubus corchorifolius (Wu et al. 2021) leaf spot. To our knowledge, this is the first report of C. fructicola causing leaf spot in M. wufengensis in China. This research may contribute to the development of management strategies for this disease.

Transcriptomic Analysis Reveals the Positive Role of Abscisic Acid in Endodormancy Maintenance of Leaf Buds of Magnolia wufengensis.

Magnolia wufengensis (Magnoliaceae) is a deciduous landscape species, known for its ornamental value with uniquely shaped and coloured tepals. The species has been introduced to many cities in south China, but low temperatures limit the expansion of this species in cold regions. Bud dormancy is critical for plants to survive in cold environments during the winter. In this study, we performed transcriptomic analysis of leaf buds using RNA sequencing and compared their gene expression during endodormancy, endodormancy release, and ecodormancy. A total of 187,406 unigenes were generated with an average length of 621.82 bp (N50 = 895 bp). In the transcriptomic analysis, differentially expressed genes involved in metabolism and signal transduction of hormones especially abscisic acid (ABA) were substantially annotated during dormancy transition. Our results showed that ABA at a concentration of 100 µM promoted dormancy maintenance in buds of M. wufengensis. Furthermore, the expression of genes related to ABA biosynthesis, catabolism, and signalling pathway was analysed by qPCR. We found that the expression of MwCYP707A-1-2 was consistent with ABA content and the dormancy transition phase, indicating that MwCYP707A-1-2 played a role in endodormancy release. In addition, the upregulation of MwCBF1 during dormancy release highlighted the enhancement of cold resistance. This study provides new insights into the cold tolerance of M. wufengensis in the winter from bud dormancy based on RNA-sequencing and offers fundamental data for further research on breeding improvement of M. wufengensis.

Physiological and transcriptome analysis of Magnolia denudata leaf buds during long-term cold acclimation.

BACKGROUND: Magonlia denudata is an important perennial tree species of the Magnoliaceae family, known for its ornamental value, resistance to smoke pollution and wind, role in air purification, and robust cold tolerance. In this study, a high-throughput transcriptome analysis of leaf buds was performed, and gene expression following artificial acclimation 22 °C, 4 °C and 0 °C, was compared by RNA sequencing. RESULTS: Over 426 million clean reads were produced from three libraries (22 °C, 4 °C and 0 °C). A total of 74,503 non-redundant unigenes were generated, with an average length of 1173.7 bp (N50 = 1548). Based on transcriptional results, 357 and 235 unigenes were identified as being upregulated and downregulated under cold stress conditions, respectively. Differentially expressed genes were annotated using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analyses. The transcriptomic analysis focused on carbon metabolism and plant hormone signal transduction associated with cold acclimation. Transcription factors such as those in the basic helix-loop-helix and AP2/ERF families were found to play an important role in M. denudata cold acclimation. CONCLUSION: M. denudata exhibits responses to non-freezing cold temperature (4 °C) to increase its cold tolerance. Cold resistance was further strengthened with cold acclimation under freezing conditions (0 °C). Cold tolerance genes, and cold signaling transcriptional pathways, and potential functional key components for the regulation of the cold response were identified in M. denudata. These results provide a basis for further studies, and the verification of key genes involved in cold acclimation responses in M. denudata lays a foundation for developing breeding programs for Magnoliaceae species.

Conservation and divergence of ancestral AGAMOUS/SEEDSTICK subfamily genes from the basal angiosperm Magnolia wufengensis.

AGAMOUS/SEEDSTICK (AG/STK) subfamily genes play crucial roles in the reproductive development of plants. However, most of our current knowledge of AG/STK subfamily genes is restricted to core eudicots and grasses, and the knowledge of ancestral exon-intron structures, expression patterns, protein-protein interaction patterns and functions of AG/STK subfamily genes remains unclear. To determine these, we isolated AG/STK subfamily genes (MawuAG1, MawuAG2 and MawuSTK) from a woody basal angiosperm Magnolia wufengensis (Magnoliaceae). MawuSTK arose from the gene duplication event occurring before the diversification of extant angiosperms, and MawuAG1 and MawuAG2 may result from a gene duplication event occurring before the divergence of Magnoliaceae and Lauraceae. Gene duplication led to apparent diversification in their expression and interaction patterns. It revealed that expression in both stamens and carpels likely represents the ancestral expression profiles of AG lineage genes, and expression of STK-like genes in stamens may have been lost soon after the appearance of the STK lineage. Moreover, AG/STK subfamily proteins may have immediately established interactions with the SEPALLATA (SEP) subfamily proteins following the emergence of the SEP subfamily; however, their interactions with the APETALA1/FRUITFULL subfamily proteins or themselves differ from those found in monocots and basal and core eudicots. MawuAG1 plays highly conserved roles in the determinacy of stamen, carpel and ovule identity, while gene duplication contributed to the functional diversification of MawuAG2 and MawuSTK. In addition, we investigated the evolutionary history of exon-intron structural changes of the AG/STK subfamily, and a novel splice-acceptor mode (GUU-AU) and the convergent evolution of N-terminal extension in the euAG and PLE subclades were revealed for the first time. These results further advance our understanding of ancestral AG/STK subfamily genes in terms of phylogeny, exon-intron structures, expression and interaction patterns, and functions, and provide strong evidence for the significance of gene duplication in the expansion and evolution of the AG/STK subfamily.

De novo transcriptome sequencing and gene expression profiling of Magnolia wufengensis in response to cold stress.

BACKGROUND: Magnolia wufengensis is a new species of Magnolia L. and has considerable ornamental and economic value due to its unique characteristics. However, because of its characteristic of poor low temperature resistance, M. wufengensis is hardly popularization and application in the north of China. Furthermore, the mechanisms of gene regulation and signaling pathways involved in the cold-stress response remained unclear in this species. In order to solve the above-mentioned problems, we performed de novo transcriptome assembly and compared the gene expression under the natural (25 °C) and cold (4 °C) conditions for M. wufengensis seedlings. RESULTS: More than 46 million high-quality clean reads were produced from six samples (RNA was extracted from the leaves) and were used for performing de novo transcriptome assembly. A total of 59,764 non-redundant unigenes with an average length of 899 bp (N50 = 1,110) were generated. Among these unigenes, 31,038 unigenes exhibited significant sequence similarity to known genes, as determined by BLASTx searches (E-value ≤1.0E-05) against the Nr, SwissProt, String, GO, KEGG, and Cluster of COG databases. Based on a comparative transcriptome analysis, 3,910 unigenes were significantly differentially expressed (false discovery rate [FDR] < 0.05 and |log2FC (CT/CK)| ≥ 1) in the cold-treated samples, and 2,616 and 1,294 unigenes were up- and down-regulated by cold stress, respectively. Analysis of the expression patterns of 16 differentially expressed genes (DEGs) by quantitative real-time RT-PCR (qRT-PCR) confirmed the accuracy of the RNA-Seq results. Gene Ontology and KEGG pathway functional enrichment analyses allowed us to better understand these differentially expressed unigenes. The most significant transcriptomic changes observed under cold stress were related to plant hormone and signal transduction pathways, primary and secondary metabolism, and photosynthesis. In addition, 113 transcription factors, including members of the AP2-EREBP, bHLH, WRKY, MYB, NAC, HSF, and bZIP families, were identified as cold responsive. CONCLUSION: We generated a genome-wide transcript profile of M. wufengensis and a de novo-assembled transcriptome that can be used to analyze genes involved in biological processes. In this study, we provide the first report of transcriptome sequencing of cold-stressed M. wufengensis. Our findings provide important clues not only for understanding the molecular mechanisms of cold stress in plants but also for introducing cold hardiness into M. wufengensis.

Microclimate simulation and model optimization of the effect of roadway green space on atmospheric particulate matter.

Urban green spaces have the potential to mitigate and regulate atmospheric pollution. However, existing studies have primarily focused on the adsorption effect of different plants on atmospheric particulate matter (PM), whereas the effect of green space on PM has not been adequately addressed. In this study, the effect of different urban green space structures and configurations on PM was investigated through the 3D computational fluid dynamics (CFD) model ENVI-met by treating the green space as a whole based on field monitoring, and at the same time, the regulatory effect of green space on PM was examined by integrating information about the forest stand, PM concentration, and meteorological factors. The results show that the green space primarily affected wind speed but had no significant effect on relative humidity, temperature, or wind direction (P > 0.05). The PM concentration was significantly positively correlated with the relative humidity (P < 0.01), significantly negatively correlated with temperature (P < 0.05), but not significantly correlated with wind speed and direction (P > 0.05). Comparison with the measured values reveals that the ENVI-met model well reflected the differences in PM concentrations between different green spaces and the effect of green space on PM. In different green space structures, the uniform-type structure performed rather poorly at purifying PM, the concave-shaped structure performed the best, and the purifying effectiveness of the incremental-type and convex-shaped structure of green space was higher in the rear region than in the front region; in contrast, the degressional-type green space structure was prone to cause aggregation of the PM in the middle region. Broadleaf and broadleaf mixed forests had a better purifying effectiveness on PM than did coniferous forests, mixed coniferous forests, and coniferous broadleaf mixed forests. The above results are of great significance for urban planning and maximizing the use of urban green space resources.

Gene duplication led to divergence of expression patterns, protein-protein interaction patterns and floral development functions of AGL6-like genes in the basal angiosperm Magnolia wufengensis (Magnoliaceae).

The MADS-box family genes play critical roles in the regulation of growth and development of flowering plants. AGAMOUS-LIKE 6 (AGL6)-like genes are one of the most enigmatic subfamilies of the MADS-box family because of highly variable expression patterns and ambiguous functions, which have long puzzled researchers. A lot of AGL6 homologs have been identified from gymnosperms and angiosperms. However, only a few have been characterized, especially for basal angiosperm taxa. Magnolia wufengensis is a woody basal angiosperm from the family Magnoliaceae. In the current study, the phylogenesis, expression and protein-protein interaction (PPI) patterns, and functions of two AGL6 homologs from M. wufengensis, MawuAGL6-1 and MawuAGL6-2, were analyzed. Phylogenetic analysis indicated that the two AGL6 duplicates may have arisen by gene duplication before the divergence of Magnoliaceae and Lauraceae, with the diversification of their expression and PPI patterns after gene duplication. Functional analysis revealed that, in addition to common functions in accelerating flowering, MawuAGL6-1 might be responsible for flower meristem determinacy, while MawuAGL6-2 is preferentially recruited to regulate tepal morphogenesis. These findings further advance our understanding of the evolution of phylogenesis, expression, interaction and functions of AGL6 lineage genes from basal angiosperms, as well as the entire AGL6 lineage genes, and the significance of AGL6 lineage genes in the evolution and biological diversity.

An investigation of the leaf retention capacity, efficiency and mechanism for atmospheric particulate matter of five greening tree species in Beijing, China.

Urban trees have the potential to reduce air pollution, but the retention capacity and efficiency of different tree species for atmospheric particulate matter (PM) accumulation and the underlying mechanism hasn't been well understood. To select tree species with high air purification abilities, the supplementing ultrasonic cleaning (UC) procedure was first introduced into the conventional leaf cleaning methods [single water cleaning (WC) or plus brush cleaning (BC)] for eluting the leaf-retained PM. Further updates to the methodology were applied to investigate the retention capacity, efficiency, and mechanism for PM of five typical greening tree species in Beijing, China. Meanwhile, the particle size distribution of PM on the leaves, the PM retention efficiencies of easily removable (ERP), difficult-to-remove (DRP) and totally removable (TRP) particles on the leaf (AEleaf), and the individual tree scales were estimated. The experimental leaf samples were collected from trees with similar sizes 4 (SDR) and 14days (LDR) after rainfall. When the leaves were cleaned by WC+BC, there was, on average, 29%-46% of the PM remaining on the leaves of different species, which could be removed almost completely if UC was supplemented. From SDR to LDR, the mass of the leaf-retained PM increased greatly, and the particle size distribution changed markedly for all species except for Sophorajaponica. Pinus tabuliformis retains particles with the largest average diameter (34.2µm), followed by Ginkgo biloba (20.5µm), Sabina chinensis (16.4µm), Salix babylonica (16.0µm), and S. japonica (13.1µm). S. japonica and S. chinensis had the highest AEleaf to retain the TRP and ERP of both PM1 and PM1-2.5, respectively. Conversely, S. babylonica and P. tabuliformis could retain both TRP and ERP of PM2.5-5 and PM5-10, and PM>10 and TSP with the highest AEleaf, respectively. In conclusion, our results could be useful in selecting greening tree species with high air purification abilities.

Various response of Pinus tabulaeformis Carr. regeneration in artifical gaps.

Understanding the influence of gaps in promoting canopy recruitment will help to maintain structural stability and achieve continuous forest cover. We established three control plots and experimental plots with three replications each (gap sizes L-I, L-II, L-III, and L-IV) in a Chinese pine (Pinus tabulaeformis Carr.) plantation to test the short-term effects of gap size on the age distribution, density and growth, and annual height and ground diameter growth for regeneration established before (REBG) and after (REAG) gap creation. Age distribution exhibited an approximately normal distribution, with the numbers of REBG and REAG decreasing and increasing, respectively, as the age increased. Although there was no difference in density among gap size classes, regeneration growth positively responded to gap size, with maximum values observed in class L-III. Annual average height growth after (AAH-A) gap creation was significantly greater than that before (AAH-B) gap creation for REBG among gap sizes, suggesting that gaps promote the rapid growth of regeneration. However, the responses of height and ground diameter growth in REBG to gap size were not immediate and exhibited a response delay of 2-4 years. Similarly, for the height and ground diameter growth of REAG, significant differences were first observed within years 2-4 after germination in the same growing season for all gap size classes.

[Dynamic change of Yulania sap flow before dormancy in response to environmental factors].

From September 26 to November 5, 2011, the sap flow of Yulania wufengensis trees including cold-resistance type (HK) and non cold-resistance type (HF), Y. 'Sunspire' (HY), and Yulania x soulangeana (EQ) which were introduced into Beijing four years before was monitored by Flow-32 stem heat balance sensor, and, in combining with the environmental factors monitored synchronically, the changes of the sap flow before dormancy and the environmental factors were analyzed, with the responses of the sap flow to the environmental factors investigated at the scales of 0.5 h and 1 day. The sap flow of the Yulanias trees before dormancy displayed an obvious trend of declining day by day. The environmental factors affecting the sap flow could be divided into two categories, i. e., meteorological index (MI) and soil index (SI). The sap flow of the Yulanias trees had a synchronous variation rhythm with MI, and declined in parallel to SI. The combined effect of MI and SI on the diurnal changes of the sap flow was 69% - 73%. At both 0.5 h and 1 day scales, the sap flow showed significantly correlations with total radiation (Rs), air vapor pressure deficit (D), air relative humidity (RH), air temperature (Ta), and wind speed (w). The sap flow showed no significant correlations with soil temperature (Ts) and soil water content (SWC) at 0. 5 h scale, but had significant correlations with Ts, SWC, and day length (Z) at 1 day scale (the correlation efficient was about 0.8). Only Rs, Z, and D were included into the model at 1 day scale, but almost all environmental factors (except SWC and Ts) were included in the model at 0.5 h scale. Except for HF type, the regression coefficients of the model for the Yulanias trees at 1 day scale (0.92-0.96) were larger than those at 0.5 h scale (0.77-0.87), and the correlations between the dynamic changes of sap flow and the environmental factor were consistent, which was in accord with the fact that the HF could not overwinter in Beijing but the others could.