First Report of Leaf Spot Disease Caused by Alternaria brassicae on Cercidiphyllum japonicum in China.

Cercidiphyllum japonicum is a deciduous tree belonging to the genus Cercidiphyllum of the family Cercidiphyllaceae (Li et al., 2008). Fossil records indicated that this tree was once distributed throughout the Northern hemisphere during the tertiary period, whereas it is now only found in Japan and China as a consequence of quaternary glaciation. In 1989, C. japonicum was listed as a Rare and Endangered plant in China (Song et al., 1989). It is also highly valued for use in ornamental, medicinal, and research contexts, leading to its widespread planting and cultivation throughout China. In September 2021, a severe leaf spot infection (FigS1.A) was first detected on C. japonicum trees in Meigu County, Sichuan Province, China (N 28°33', E 103°14'). In a survey of twenty 100-year-old C. japonicum trees in this region, the incidence of such leaf spot was found to be approximately 95%. During the early stages of disease, infected leaves exhibited small punctate spots along the leaf center or margins. These spots were brown in the center with black edges. As the disease progressed, these spots expanded until they coalesced to yield large circular or irregularly shaped regions of necrotic tissue, and finally produced mildew. Samples of leaf tissue between symptomatic and healthy regions (5 mm×5 mm) were excised from five symptomatic leaves, surface disinfected for 30 s with 75% ethanol, soaked for 2 min in 3% NaClO, rinsed then plated on potato dextrose agar (PDA) medium supplemented with ampicillin and carbenicillin (50 µg/ml each). After cultured for 3 days in the dark at 25°C, emergent hyphae were purified by subculturing them on fresh PDA medium. In total, single spore culturing was performed by collecting and purifying seven fungal isolates. These isolates exhibited largely comparable morphological characteristics. Aerial hyphae had a cotton-like appearance and were white to pale gray in color (FigS1.B), turning pale reddish-brown with profuse sporulation (FigS1.C). Conidia were present in long chains, with conidiophores being present in clusters or in isolation (FigS1.D), with 1-5 transverse septa, 0-3 oblique and longitudinal septa and an ellipsoidal to obpyriform structure, measuring 9.0-38.6 µm in length and 5.1-12.6 µm in width (n = 40) (FigS1.E). These seven isolates thus exhibited morphological characteristics consistent with those of members of the Alternaria genus (Simmons, 2008). Molecular identification of a representative isolate (LGB9) was performed by amplifying the internal transcribed spacer (ITS) rDNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), and partial RNA polymerase II largest subunit (RPB2) gene sequences with the ITS1/ITS4 (White et al.,1990), GDF/GDR (Templeton et al., 1992), TEF-728F/TEF-986R (Carbone & Kohn 1999) and RPB2-5F2/RPB2-7cR (Sung et al., 1990; Liu et al., 1999), and Bt-2a/Bt-2b (Glass and Donaldson 1995) primer pairs, respectively. The resultant sequences were deposited in GenBank (ITS, OL659190; GAPDH, OL685343; TEF, ON340848; RPB2, OL685344). Further phylogenetic analyses of isolate LGB9 revealed it to cluster in the A. brassicae clade with 97% bootstrap support. To confirm the pathogenicity of isolate LGB9, 15 healthy leaves from five one-year-old C. japonicum plants were spray-inoculated with a suspension containing 3×105 LGB9 conidia/mL, with control leaves instead being sprayed with distilled water. After 8 days, inoculated leaves exhibited symptoms similar to those observed on naturally infected leaves (FigS1.F-I), whereas the mock leaves were free of any symptoms. This is the first report to our knowledge of a case of leaf spot disease caused by A. brassicae affecting C. japonicum in China or anywhere else in the world. To ensure the protection of this living fossil species, appropriate interventional measures should be adopted to manage the development and spread of this disease.

De novo transcriptome analysis provides insights into the salt tolerance of Podocarpus macrophyllus under salinity stress.

BACKGROUND: Soil salinization is causing ecosystem degradation and crop yield reduction worldwide, and elucidation of the mechanism of salt-tolerant plants to improve crop yield is highly significant. Podocarpus macrophyllus is an ancient gymnosperm species with a unique environmental adaptation strategy that may be attributed to its lengthy evolutionary process. The present study investigated the physiological and molecular responses of P. macrophyllus plants to salt stress by analyzing its photosynthetic system and antioxidant enzyme activity. We also analyzed the differentially expressed genes (DEGs) in P. macrophyllus under salt stress using RNA sequencing and de novo transcriptome assembly. RESULTS: Salt treatment significantly affected the photosynthetic system in P. macrophyllus seedlings, which decreased chlorophyll content, altered chloroplast ultrastructure, and reduced photosynthesis. The activities of antioxidant enzymes increased significantly following salt stress treatment. Transcriptome analysis showed that salt stress induced a large number of genes involved in multiple metabolic and biological regulation processes. The transcription levels of genes that mediate phytohormone transport or signaling were altered. K+ and Ca2+ transporter-encoding genes and the MYB transcription factor were upregulated under salt stress. However, the genes involved in cell wall biosynthesis and secondary metabolism were downregulated. CONCLUSION: Our research identified some important pathways and putative genes involved in salt tolerance in P. macrophyllus and provided clues for elucidating the mechanism of salt tolerance and the utilization of the salt tolerance genes of P. macrophyllus for crop improvement.

Transcription factor WRKY30 mediates resistance to Cucumber mosaic virus in Arabidopsis.

WRKY transcription factors are key regulators in regulating abiotic or biotic stress response in Arabidopsis. Previous studies showed that WRKY30 expression was induced by oxidative stress treatment, fungal elicitor, SA and ABA. However, functions of WRKY30 on viral defense are not well studied. Here, we found that Arabidopsis WRKY DNA binding protein 30 (WRKY30) plays essential roles in regulating Cucumber mosaic virus (CMV) resistance. The expression of WRKY30 was induced by CMV infection and wrky30 mutant displayed more susceptibility (including higher oxidative damages, induced reactive oxygen species synthesis and more PSII photochemistry compromise), while WRKY30 overexpression plants (WRKY30OX) exhibited more resistance to CMV infection. Moreover BRs-induced CMV tolerance is partly dependent on WRKY30. And WRKY30 expression increased after BL treatment. All these demonstrated that WRKY30 works as a positive regulator in plant CMV resistance process.

Simple additive simulation overestimates real influence: altered nitrogen and rainfall modulate the effect of warming on soil carbon fluxes.

Experiments and models have led to a consensus that there is positive feedback between carbon (C) fluxes and climate warming. However, the effect of warming may be altered by regional and global changes in nitrogen (N) and rainfall levels, but the current understanding is limited. Through synthesizing global data on soil C pool, input and loss from experiments simulating N deposition, drought and increased precipitation, we quantified the responses of soil C fluxes and equilibrium to the three single factors and their interactions with warming. We found that warming slightly increased the soil C input and loss by 5% and 9%, respectively, but had no significant effect on the soil C pool. Nitrogen deposition alone increased the soil C input (+20%), but the interaction of warming and N deposition greatly increased the soil C input by 49%. Drought alone decreased the soil C input by 17%, while the interaction of warming and drought decreased the soil C input to a greater extent (-22%). Increased precipitation stimulated the soil C input by 15%, but the interaction of warming and increased precipitation had no significant effect on the soil C input. However, the soil C loss was not significantly affected by any of the interactions, although it was constrained by drought (-18%). These results implied that the positive C fluxes-climate warming feedback was modulated by the changing N and rainfall regimes. Further, we found that the additive effects of [warming × N deposition] and [warming × drought] on the soil C input and of [warming × increased precipitation] on the soil C loss were greater than their interactions, suggesting that simple additive simulation using single-factor manipulations may overestimate the effects on soil C fluxes in the real world. Therefore, we propose that more multifactorial experiments should be considered in studying Earth systems.

[Induction on callus culture and regeneration of Orostachyis fimbriatae].

OBJECTIVE: To explore the effects of different hormonal combinations on induction, proliferation and differentiation of Orostachyis fimbriatae callus culture. METHODS: Aseptic seedling leaves were used as explants,the different concentrations of IAA,NAA, 6-BA and KT on induction proliferation of callus were optimized by orthogonal test to explore the optimum medium for differentiation of callus by tissue culture techniques. RESULTS: The best medium for induction was MS + IAA 1.0 mg/L + NAA 0.5 mg/L + KT 1.0 mg/L, and the best hormonal combination for proliferation was MS + IAA 0.5 mg/L + 6-BA 0.5 mg/I. + KT 1.0 mg/L. The best medium for differentiation was MS + IAA 0.1 mg/L + KT 2.0 mg/L, and 1/2MS + IAA 0.2 mg/L was the optimum medium for rooting culture. CONCLUSION: The system of regeneration of Orostachyis fimbriatae is establishd by tissue culture techniques in this study.

[Callus induction and plant regeneration of Sambucus chinensis].

OBJECTIVE: To optimize the medium for callus and multiple shoot induction and cultural plantlets rooting of Sambucus chinensis, and to provide a basis for the development of the seedling of mass production and germplasm conservation. METHODS: The plantlet leaves, petioles, stems and shoot tips were used as explants for the study of influences of different explants and different hormones on callus and multiple shoot induction, and cultural plantlets rooting. RESULTS: The optimal medium for callus induction of leaves was MS +2, 4-D 1.5 mg/L; and optimum mediums for petiole were MS + NAA 0.2 mg/L + KT 1.0 mg/L and MS +2, 4-D 0. 2 mg/L + KT 1.0 mg/L, MS + NAA 0.2 mg/L + KT 3.0 mg/L + GA, 2.0 mg/L medium was best for multiple shoot induction. And the best medium for tissue culture rooting was 1/2 MS + NAA 0. 3 mg/L. On the optimized medium, the roots were well-developed and the plants were healthier. CONCLUSION: Both the leaves and petioles can be well induced to callus on suitable medium, the stems and shoot tips are the best materials for multiple shoot induction and plant regeneration.

A Novel and Efficient In Vitro Organogenesis Approach for Ajuga lupulina Maxim.

This work was aimed at establishing an effective approach for in vitro propagation of Ajuga lupulina Maxim, a medicinal and ornamental plant mainly found in eastern Xizang, in the western Sichuan region of China. We report an optimum response in the proliferation of axillary shoots from nodal segment explants (10.2 shoots/explant) on MS medium containing 3.0 mg L-1 of 6-benzyladenine (BA). When BA and TDZ individually or in combination with NAA were employed for adventitious shoot regeneration, shoots and embryo-like structures (ELSs) were noted in the callus from leaf explants. The maximum response of 26.4 shoots /explant (81.6%) and 12.0 ELSs/explant were ascertained on MS medium with 4.0 mg L-1 TDZ and 0.1 mg L-1 NAA. The leaf despite browning still demonstrated a high regeneration capacity. TDZ (2.0 mg L-1) and BA (2.0 mg L-1) along with NAA (0.01 mg L-1) were found to perform well for shoot regeneration via callus from shoot tip explants. The best for rooting was MS medium (half-strength) containing indole-3-butyric acid (IBA: 1.5 mg L-1) and (NAA: 0.5 mg L-1) with the maximum number of roots (25.8 per shoot) and the highest rooting frequency (81.71%). The survival of the plantlets in the greenhouse was 78.2% indicative of successful acclimatization. This work is the first report of a consistent, definitive, and unique protocol for A. lupulina regeneration, paving the way for the in vitro preservation of such significant genetic resources and also further allied systems based on such callus-based or embryo-based approaches.

The complete chloroplast genome of Ottelia acuminate var. crispa, an endangered aquatic herb with extremely narrow distribution.

Ottelia acuminate var. crispa is an endangered aquatic herb with extremely narrow distribution. In this study, we assembled the chloroplast genome of this species based on the second-generation high-throughput sequencing. The genome is 157,783 bp in length with a typical quadripartite structure including a large single-copy region (LSC) of 88,294 bp, a small single-copy region (SSC) of 49,379 bp, and a pair of inverted repeats (IRs) of 10,055 bp each. A total of 128 genes were annotated, including 83 protein-coding genes (PCG), 37 tRNA genes, and 8 rRNA genes. The phylogenetic tree shows that O. acuminate var. crispa has a close relationship with the genus Elodea. The chloroplast genome presented here provides a valuable resource to conserve this endangered species.

Soil respiration in a subtropical forest of southwestern China: Components, patterns and controls.

Partitioning the components of soil respiration is crucial to understand and model carbon cycling in forest ecosystems. In this study, total soil respiration (RS), autotrophic respiration (RA), heterotrophic respiration (RH), litter respiration (RL), litterfall input and environmental factors were synchronously monitored for 2 years in a subtropical Michelia wilsonii forest of southwestern China. RH rates were often higher than RA rates during the two years except for the middle growing season (from July to September). The mean rate of Rs, RA, RH and RL was 1.94 µmol m-1 s-1, 0.85 µmol m-1 s-1, 1.09 µmol m-1 s-1 and 0.65 µmol m-1 s-1, respectively, during the 2-year experiment. Annual CO2 emission derived from RA, RH and RL was 3.26 Mg C ha-1 a-1, 4.67 Mg C ha-1 a-1 and 2.61 Mg C ha-1 a-1, respectively, which accounted for 41.4%, 58.6% and 32.9% of RS. Over the experimental period, the ratio of RA/RS increased with soil temperature but the opposite was true for RH/RS and RL/RS. The Q10 value was 2.01, 4.01, 1.34 and 1.30, respectively, for RS, RA, RH and RL. Path analysis indicated that environmental variables and litterfall production together explained 82.0%, 86.8%, 42.9% and 34.7% variations of monthly fluxes of RS, RA, RH and RL, respectively. Taken together, our results highlight the differential responses of the components of RS to environmental variables.

[Effects of drought stress on leaf gas exchange and chlorophyll fluorescence of Salvia miltiorrhiza].

Taking the seedlings of Salvia miltiorrhiza cv. Sativa (SA) and S. miltiorrhiza cv. Silcestris (SI) as test materials, this paper studied the effects of drought stress on their leaf gas exchange and chlorophyll fluorescence parameters. After 15 days of drought stress, the net photosynthetic rate (P(n)) and the maximal photochemical efficiency of PS II (F(v)/F(m)) of SA were decreased by 66.42% and 10.98%, whereas those of SI were decreased by 29.32% and 5.47%, respectively, compared with the control, suggesting that drought stress had more obvious effects on the P(n) and F(v)/F(m) of SA than of SI. For SI, the reduction of P, under drought stress was mainly due to stomatal limitation; while for SA, it was mainly due to non-stomatal limitation. Drought led to a decrease of leaf stomatal conductance (G(s)), but induced the increase of water use efficiency (WUE), non-photochemical quenching coefficient (q(N)), and the ratio of photorespiration rate to net photosynthetic rate (P(r)/P(n)), resulting in the enhancement of drought resistance. The increment of WUE, q(N), and P(r)/P(n) was larger for SI than for SA, indicating that SI had a higher drought resistance capacity than SA.