First Report of Fusarium annulatum Causing Blight on Bletilla striata (Baiji) in China.

Bletilla striata (Thunb. ex A. Murray) Rchb (known as baiji in Chinese), a herbal plant distributed mainly in China, has become a focus of scientific attention recently due to its medicinal value (He et al. 2017). In May 2023, blight symptoms on leaves and stems were observed approximately 60% of Bletilla striata in Hangzhou, Zhejiang, China (29.80° N, 119.67° E). Brown spots initially appear on the infected leaves, which gradually decay as the spots expand. The wilting is accompanied with fading and yellowing, and eventually leading to defoliation. The infected stem initially appears brown spots, which gradually decay as the spots expand, resulting in the death of the whole plant, affecting the yield and quality of the herbs ultimately. To isolate the pathogen, small symptomatic leaves and stems (5×5 mm) were surface-disinfected with 75% ethanol for 30 s and 1% NaClO for 2 min, then rinsed in distilled water 3 times. Subsequently, the disinfected tissues were placed on PDA and incubated at 27 ℃ for 3 days. A total of 8 fungal isolates with similar morphological characteristics were obtained. The colony by single-spore purification was light purple to dark purple with abundant aerial mycelium. Macroconidia were relatively slender with a curve, mainly three to five septate and measuring 24.34 to 54.64 µm (average 40.29 µm) × 3.59 to 5.45 µm (average 4.49 µm) (n=30). Microconidia appeared obovoid to pyriform, with sizes of 5.31 to 8.43 µm (average 7.12 µm) × 2.30 to 4.29 µm (average 3.22 µm) (n=30). The morphological characteristics were consistent with Fusarium annulatum (Yilmaz et al. 2021). To further confirm the isolate's identification, the genomic DNA of isolates were extracted and identified by phylogenetic analyses of multilocus sequences of the RNA polymerase largest subunit (rpb1, primers Fa and G2R), RNA polymerase second largest subunit (rpb2, primers 7cf and 11ar) and the translation elongation factor 1-alpha (tef1, primers EF1 and EF2) (O'Donnell et al. 2022). The sequences were deposited in GenBank (rpb1: OR493933, OR493934, OR753402; rpb2: OR753398, OR753399, OR753400; tef1: OR493935, OR493936, OR753401). BLAST searches of the rpb1, rpb2, and tef1 sequences revealed 99.83% (1775/1778 nt), 99.79% (957/959 nt), and 98.98% (678/685 nt) homology with those of Fusarium annulatum CBS:258.54 from New Caledonia (rpb1: MT010944; rpb2: MT010983; tef1: MT010994). To confirm pathogenicity, one-year-old B. striata leaves and stems were disinfected with 75% ethanol, wounded with a sterile syringe on 3 healthy leaves and stems, inoculated with 5 × 5 mm mycelial discs of strain BJ-L1 and BJ-S1, respectively. And the control were treated similarly except that they were inoculated with PDA discs. The experiment was replicated 3 times. After 5 days, all inoculated leaves and stems showed similar symptoms to those initially observed on infected plants. The same pathogen was re-isolated and identified by morphological characterization and molecular analysis, confirming Koch's postulates. Thus, the pathogen causing blight of B. striata was determined to be F. annulatum. To our knowledge, this is the first report of F. annulatum causing blight on B. striata in China. F. annulatum has a wide range of hosts and has been reported to infect a wide range of crops, fruits and vegetables (Bacon et al. 1991). This study provides the basis for further research on this disease and is important for the management of this disease and the improvement of the economic benefits of B. striata.

Time-Course Analysis and Transcriptomic Identification of a Group III ERF CmTINY2 Involved in Waterlogging Tolerance in Chrysanthemums × morifolium Ramat.

'Hangju' is a variety of Chrysanthemum × morifolium Ramat. with both edible and medicinal value, cultivated as a traditional Chinese medicine for four centuries. The cultivation of 'Hangju' is currently at risk due to waterlogging, yet there is a lack of comprehensive understanding regarding its response to waterlogging stress. This study compared the waterlogging-tolerant 'Hangju' variety Enhanced Waterlogging Tolerance (EWT) with the waterlogging-sensitive variety CK ('zaoxiaoyangju'). EWT exhibited a more developed aeration tissue structure and demonstrated rapid growth regarding the adventitious roots following waterlogging. The time-course transcriptome analysis indicated that EWT could swiftly adjust the expression of the genes involved in the energy metabolism signaling pathways to acclimate to the waterlogged environment. Through WGCNA analysis, we identified Integrase-Type DNA-Binding Protein (CmTINY2) as a key factor in regulating the waterlogging tolerance in EWT. CmTINY2, a transcription factor belonging to the ethylene-responsive factor (ERF) subfamily III, operated within the nucleus and activated downstream gene expression. Its role in enhancing the waterlogging tolerance might be linked to the control of the stomatal aperture via the Ethylene-Responsive Element (ERE) gene. In summary, our research elucidated that the waterlogging tolerance displayed by EWT is a result of a combination of the morphological structure and molecular regulatory mechanisms. Furthermore, the study of the functions of CmTINY2 from ERF subfamily III also broadened our knowledge of the role of the ERF genes in the waterlogging signaling pathways.

First Report of Leaf Spot Caused by Botrytis cinerea on Strawberry in China.

In the winter of 2022, circular or irregular leaf spots were observed on strawberry (Fragaria × ananassa) planted in commercial fields (cultivar 'xuetu', 'mengzhifu') in Yinzhou, Ningbo, Zhejiang, China (N29°48'48″, E121°39'47″), with disease incidence ranging from 10 to 15% in a field approximately 0.67 ha in size. The estimated crop loss associated with this disease was ~10%. Symptoms included circular or irregular lesions with brown halos and wheel marks, which eventually developed into leaf blight and petiole decay, but spore masses were seldom found on the leaf surface. In severe cases, leaves withered and abscissed. To isolate the causal agent, ten diseased leaves from ten different plants were collected, surface-sterilized with 75% ethanol for 50 s, rinsed twice with sterile distilled water, cut into small pieces (0.5 cm × 0.5 cm), and plated on potato dextrose agar (PDA), then incubated at 25°C in darkness for 5 days. Isolates , which displayed one kind of colony morphology were consistently obtained from each of the ten samples, and 58 single-conidium isolates with the same colony morphology were obtained. The isolation frequency was 58 of 60 samples. The colonies that grew on PDA produced white mycelia, which sporulated after 1 week, producing typical Botrytis-like gray spores. Three isolates (NBCM-1, NBCM-2, NBCM-3) were selected for identification and pathogenicity assays. Conidia were round to ellipsoid, 9.2 to 14.3 µm long (n=50), and 6.4 to 9.2 µm wide (n=50). Sclerotia were not observed on PDA. Based on these characteristics, the pathogen was tentatively identified as Botrytis cinerea (Zhang 2001). PCR was conducted for each of the three isolates to amplify the G3PDH, HSP60, RPB2, NEP1, and NEP2 genes, which are typically used for molecular identification of Botrytis species (Staats et al. 2005; Liu et al. 2016). The resulting amplicons were sequenced, and the sequences were processed using BLAST in the National Center for Biotechnology Information. Sequences of the three isolates were deposited in GenBank (accession nos. OR052082 to OR052086, OR493405 to OR493414). BLASTn analyses showed that isolates were 99 to 100% identical to B.cinerea reported causing leaf spot on strawberry in California; accession numbers MK919496 (G3PDH, 883/883 bp), MK919494 (HSP60, 992/992 bp), and MK919495 (RPB2, 1081/1081 bp). The resulting concatenated data set of G3PDH-HSP60-RPB2-NEP1-NEP2 was used to conduct a multilocus phylogenetic analysis (MLSA) using the maximum likelihood method. The MLSA tree indicated that the three isolates belonged to Botrytis cinerea. To test for pathogenicity, three 1-month-old strawberry (cultivar 'xuetu') plants were inoculated with each isolate (NBCM-1, NBCM-2, NBCM-3). A noninoculated control (sterile water only) was also included. The strawberry plants were inoculated by spraying with conidia suspension (1.0 × 105/ml) until run-off. Inoculations with sterile water served as controls. All plants were kept at 28/25°C (day/night), under a 12:12-h light/dark photoperiod. All plants were covered with transparent plastic bags to maintain humidity for the first 48 h, after which the bags were removed. After 4 to 7 days, leaf spot symptoms similar to those observed in the field were observed in all inoculated plants, while the controls remained healthy. The experiment was repeated three times. The pathogen was reisolated from the inoculated leaves and again identified as B. cinerea, with the same methodology used for the initial identification. Leaf spot caused by B. cinerea on strawberry was recently reported in California (Mansouripour and Holmes 2020) and Florida (Marin and Peres 2022). To our knowledge, this is the first report of B. cinerea causing leaf spot on strawberry in China. The pathogen is also the causal agent of Botrytis fruit rot on strawberry. Given the high variability of this pathogen (Marin and Peres 2022), further studies on its occurrence, spread, management, and control are required. The identification of this pathogen provides a basis for further research on its management and control.

Identification and Characterization of Fusarium nirenbergiae Associated with Saffron Corm Rot Disease.

Saffron (Crocus sativus L.) is the most expensive spice plant and is distributed widely around the world. However, its production is limited by corm rot, a disastrous disease, attributed to Fusarium oxysporum in many regions of the world. In 2020, extensive surveys were carried out in Zhejiang, Shanghai, Anhui, and Guizhou provinces as saffron growing areas of China. Fourteen single-spore isolates were obtained from rotted corms and identified as F. nirenbergiae according to morphological appearance and multilocus phylogenetic analysis with translation elongation factor 1-α (tef1), DNA-directed RNA polymerase II largest subunit (rpb2), and ß-tubulin (tub2). Results of the pathogenicity assay supported the conclusion that F. nirenbergiae is the pathogen responsible for corm rot. In this study, we obtained the whole genome sequence of two highly virulent F. nirenbergiae strains via the Illumina HiSeq platform. Genome sequence assemblies of approximately 52.7 and 52.2 Mb were generated for isolates WY5 and SH1, respectively. To the best of our knowledge, this is the first report of F. nirenbergiae causing C. sativus corm rot in China and indeed worldwide. Results from this research contribute to our understanding of genetic diversity, genomic information, and host determination, which will enable researchers to design appropriate management measures for this hazardous disease.

First Report of Pantoea ananatis Causing Crown Necrobiosis on Strawberry in China.

Strawberry (Fragaria × ananassa) is an economically important crop in Zhejiang, China. In the autumn of 2021, crown necrobiosis and angular leaf spot was observed in commercial strawberry fields (cultivar 'fenyu') in Cixi, Ningbo, Zhejiang, China (N30°9'55″, E121°21'13″). The disease incidence ranged from 5 to 8 % in the field, but could reach 50 to 60 % in some heavily affected plastic tunnels. In the affected field, this disease could reduce strawberry production by 50%. Early symptoms were water-soaked lesions around the vein of the abaxial leaves; subsequently, reddish-brown irregular spots and coalesced lesions developed. In humid conditions, a sticky bacterial ooze exuding from lesions was observed. Finally, the crown of the diseased plant was necrotized, and several pockets were observed inside the crown after dissection. To isolate the causal agent, the infected leaves and crown tissues from six different plants were surface-sterilized with 75% ethanol for 1 min, rinsed twice with sterile distilled water, cut into small pieces, and soaked in 5 ml of sterile distilled water for 20 min. The supernatant from the cut-up pieces was serially diluted and spread on nutrient agar medium. After 2 to 3 days at 28℃, several yellow colonies were grown on the medium. The colonies from five infected plants were gram-negative, anaerobic rods, yellow, viscous, and gloss, which are typical characteristics of Erwinia anana (Wells et al. 1986). To confirm the identity of the causal bacteria, PCR was conducted for six randomly selected colonies to amplify 16S rRNA (Monciardini et al. 2002), fusA, and gyrB (Stice et al. 2002). The amplicons were sequenced and blasted, and the results showed that the six colonies were identical. The 16S rRNA, fusA, gyrB sequences of the isolate CM3 were deposited in GenBank with accession number ON754076.1, OP587277, and OP587278; BLAST search showed 99.93% (1445 bp out of 1446 bp), 100% (746 bp out of 746 bp), 99.64% (1371 bp out of 1376 bp) similarity with strains of Pantoea ananatis (KT741001.1, MH015093.1 and CP066803.1 accessions, respectively). The resulting concatenated data set of 16S rRNA-fusA-gyrB was used to build a multilocus phylogenetic analysis (MLSA) by maximum likelihood criteria. The MLSA tree indicated that the isolate CM3 belonged to Pantoea ananatis. The isolate's identity was further confirmed by P. ananatis-specific primers pagyrB-F/R (Xiao et al. 2022). Thus, this isolate was designated as P. ananatis CM3. To fulfill Koch's postulates, two old leaves were broken off each of the ten 2-month-old strawberry (cultivar 'fenyu') plants to create wounds, each plants was sprayed with a cell suspension of P. ananatis (107CFU/ml, 0.5 ml) on the stem base. Ten plants were sprayed with water to serve as a control. All plants were kept at 28/25°C (day/night) under a 12-h/12-h photoperiod. All plants were covered with transparent plastic bags to maintain humidity. After 48 h, the bags were removed. After 2 weeks, water-soaked lesions on some leaves were observed similar to those in the field . Three to five weeks after inoculation, the crown of the inoculated plants was necrotized, which was similar to the symptoms in the field. No symptoms were observed in the control plants. The experiment was repeated three times. The bacteria were successfully reisolated from the inoculated crown tissues and leaves and confirmed as CM3 according to the same methodologies used for the initial identification. Bacterial leaf blight in strawberry caused by Pantoea ananatis has been reported in Nova Scotia, Canada, and Egypt (Bajpai et al. 2019; Abdel-Gaied et al. 2022). To our knowledge, this is the first report of Pantoea ananatis causing crown necrobiosis on strawberry in China. This report provides a basis for further research on this disease and its management and control.

Metabolic Profiling of Terpene Diversity and the Response of Prenylsynthase-Terpene Synthase Genes during Biotic and Abiotic Stresses in Dendrobium catenatum.

Dendrobium catenatum is a widely cultivated Chinese orchid herb rich in abundant secondary metabolites, such as terpenes. However, terpene distribution and characterization of terpene biosynthesis-related genes remain unknown in D. catenatum. In this study, metabolic profiling was performed to analyze terpene distribution in the root, stem, leaf, and flower of D. catenatum. A total of 74 terpene compounds were identified and classified. Clustering analysis revealed that terpene compounds exhibited a tissue-specific accumulation, including monoterpenes in the flowers, sesquiterpenes in the stems, and triterpenes in the roots. Transcriptome analysis revealed that the 'terpenoid backbone biosynthesis' pathway was only significantly enriched in root vs. flower. The expression of terpene biosynthesis-related genes was spatiotemporal in the flowers. Prenylsynthase-terpene synthases (PS-TPSs) are the largest and core enzymes for generating terpene diversity. By systematic sequence analysis of six species, 318 PS-TPSs were classified into 10 groups and 51 DcaPS-TPSs were found in eight of them. Eighteen DcaPS-TPSs were regulated by circadian rhythm under drought stress. Most of the DcaPS-TPSs were influenced by cold stress and fungi infection. The cis-element of the majority of the DcaPS-TPS promoters was related to abiotic stress and plant development. Methyl jasmonate levels were significantly associated with DcaTPSs expression and terpene biosynthesis. These results provide insight into further functional investigation of DcaPS-TPSs and the regulation of terpene biosynthesis in Dendrobium.

Overexpression of PvCO1, a bamboo CONSTANS-LIKE gene, delays flowering by reducing expression of the FT gene in transgenic Arabidopsis.

BACKGROUND: In Arabidopsis, a long day flowering plant, CONSTANS (CO) acts as a transcriptional activator of flowering under long day (LD) condition. In rice, a short day flowering plant, Hd1, the ortholog of CO, plays dual functions in respond to day-length, activates flowering in short days and represses flowering in long days. In addition, alleles of Hd1 account for ~ 44% of the variation in flowering time observed in cultivated rice and sorghum. How does it work in bamboo? The function of CO in bamboo is similar to that in Arabidopsis? RESULTS: Two CO homologous genes, PvCO1 and PvCO2, in Phyllostachys violascens were identified. Alignment analysis showed that the two PvCOLs had the highest sequence similarity to rice Hd1. Both PvCO1 and PvCO2 expressed in specific tissues, mainly in leaf. The PvCO1 gene had low expression before flowering, high expression during the flowering stage, and then declined to low expression again after flowering. In contrast, expression of PvCO2 was low during the flowering stage, but rapidly increased to a high level after flowering. The mRNA levels of both PvCOs exhibited a diurnal rhythm. Both PvCO1 and PvCO2 proteins were localized in nucleus of cells. PvCO1 could interact with PvGF14c protein which belonged to 14-3-3 gene family through B-box domain. Overexpression of PvCO1 in Arabidopsis significantly caused late flowering by reducing the expression of AtFT, whereas, transgenic plants overexpressing PvCO2 showed a similar flowering time with WT under LD conditions. Taken together, these results suggested that PvCO1 was involved in the flowering regulation, and PvCO2 may either not have a role in regulating flowering or act redundantly with other flowering regulators in Arabidopsis. Our data also indicated regulatory divergence between PvCOLs in Ph. violascens and CO in Arabidopsis as well as Hd1 in Oryza sativa. Our results will provide useful information for elucidating the regulatory mechanism of COLs involved in the flowering. CONCLUSIONS: Unlike to the CO gene in Arabidopsis, PvCO1 was a negative regulator of flowering in transgenic Arabidopsis under LD condition. It was likely that long period of vegetative growth of this bamboo species was related with the regulation of PvCO1.

The Systemic Acquired Resistance Regulator OsNPR1 Attenuates Growth by Repressing Auxin Signaling through Promoting IAA-Amido Synthase Expression.

Systemic acquired resistance is a long-lasting and broad-spectrum disease resistance to pathogens. Our previous study demonstrated that overexpression of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (OsNPR1), a master gene for systemic acquired resistance in rice (Oryza sativa), greatly enhanced resistance to bacterial blight caused by Xanthomonas oryzae pv oryzae However, the growth and development of the OsNPR1 overexpression (OsNPR1-OX) plants were restrained, and the mechanism remained elusive. In this study, we dissected the OsNPR1-induced growth inhibition. We found that the OsNPR1-OX lines displayed phenotypes mimicking auxin-defective mutants, with decreases in root system, seed number and weight, internode elongation, and tiller number. Whole-genome expression analysis revealed that genes related to the auxin metabolism and signaling pathway were differentially expressed between the OsNPR1-OX and wild-type plants. Consistently, the indole-3-acetic acid (IAA) content was decreased and the auxin distribution pattern was altered in OsNPR1-OX plants. Importantly, we found that some GH3 family members, in particular OsGH3.8 coding IAA-amido synthetase, were constitutively up-regulated in OsNPR1-OX plants. Decreased OsGH3.8 expression by RNA interference could partially restore IAA level and largely rescue the restrained growth and development phenotypes but did not affect the disease resistance of OsNPR1-OX plants. Taken together, we revealed that OsNPR1 affects rice growth and development by disrupting the auxin pathway at least partially through indirectly up-regulating OsGH3.8 expression.

Complementary RNA-Sequencing Based Transcriptomics and iTRAQ Proteomics Reveal the Mechanism of the Alleviation of Quinclorac Stress by Salicylic Acid in Oryza sativa ssp. japonica.

To uncover the alleviation mechanism of quinclorac stress by salicylic acid (SA), leaf samples of Oryza sativa ssp. Japonica under quinclorac stress with and without SA pre-treatment were analyzed for transcriptional and proteomic profiling to determine the differentially expressed genes (DEGs) and proteins (DEPs), respectively. Results showed that quinclorac stress altered the expression of 2207 DEGs (1427 up-regulated, 780 down-regulated) and 147 DEPs (98 down-regulated, 49 up-regulated). These genes and proteins were enriched in glutathione (GSH) metabolism, porphyrin and chlorophyll metabolism, the biosynthesis of secondary metabolites, glyoxylate and dicarboxylate metabolism, and so on. It also influenced apetala2- ethylene-responsive element binding protein (AP2-EREBP) family, myeloblastosis (MYB) family and WRKY family transcription factors. After SA pre-treatment, 697 genes and 124 proteins were differentially expressed. Pathway analysis showed similar enrichments in GSH, glyoxylate and dicarboxylate metabolism. Transcription factors were distributed in basic helix-loop-helix (bHLH), MYB, Tify and WRKY families. Quantitative real-time PCR results revealed that quinclorac stress induced the expression of glutathion reductase (GR) genes (OsGR2, OsGR3), which was further pronounced by SA pre-treatment. Quinclorac stress further mediated the accumulation of acetaldehyde in rice, while SA enhanced the expression of OsALDH2B5 and OsALDH7 to accelerate the metabolism of herbicide quinclorac for the protection of rice. Correlation analysis between transcriptome and proteomics demonstrated that, under quinclorac stress, correlated proteins/genes were mainly involved in the inhibition of intermediate steps in the biosynthesis of chlorophyll. Other interesting proteins/genes and pathways regulated by herbicide quinclorac and modulated by SA pre-treatment were also discussed, based on the transcriptome and proteomics results.

Comparative transcriptome profiling of two Brassica napus cultivars under chromium toxicity and its alleviation by reduced glutathione.

BACKGROUND: Chromium (Cr) being multifarious industrial used element, is considered a potential environmental threat. Cr found to be a prospective water and soil pollutant, and thus it is a current area of concern. Oilseed rape (Brassica napus L.) is well known as a major source of edible oil around the globe. Due to its higher growth, larger biomass and capability to uptake toxic materials B. napus is considered a potential candidate plant against unfavorable conditions. To date, no study has been done that described the Cr and GSH mechanism at RNA-Seq level. RESULTS: Both digital gene expression (DGE) and transcriptome profile analysis (TPA) approaches had opened new insights to uncover the several number of genes related to Cr stress and GSH alleviating mechanism in two leading cultivars (ZS 758 and Zheda 622) of B. napus plants. Data showed that Cr inhibited KEGG pathways i.e. stilbenoid, diarlyheptanoid and gingerol biosynthesis; limonene and pentose degradation and glutathione metabolism in ZS 758; and ribosome and glucosinolate biosynthesis in Zheda-622. On the other hand, vitamin B6, tryptophan, sulfur, nitrogen and fructose and manose metabolisms were induced in ZS 758, and zeatin biosynthesis, linoleic acid metabolism, arginine and proline metabolism, and alanine, asparate and glutamate metabolism pathways in Zheda 622. Cr increased the TFs that were related to hydralase activity, antioxidant activity, catalytic activity phosphatase and pyrophosphatase activity in ZS 758, and vitamin binding and oxidoreductase activity in Zheda 622. Cr also up-regulated the promising proteins related to intracellular membrane bounded organelles, nitrile hyrdatase activity, cytoskeleton protein binding and stress response. It also uncovered, a novel Cr-responsive protein (CL2535.Contig1_All) that was statistically increased as compared to control and GSH treated plants. Exogenously applied GSH successfully not only recovered the changes in metabolic pathways but also induced cysteine and methionine metabolism in ZS 758 and ubiquinone and other terpenoid-quinone biosynthesis pathways in Zheda 622. Furthermore, GSH increased the level of TFs i.e. the gene expression of antioxidant and catalytic activities, iron ion binding and hydrolase activity as compared with Cr. Moreover, results pointed out a novel GSH responsive protein (CL827.Contig3_All) whose expression was found to be significantly increased when compared than Cr stress. Results further delineated that GSH induced TFs such as glutathione disulphide oxidoreducatse and aminoacyl-tRNA ligase activity, and beta glucosidase activity in ZS 758. Similarly in Zheda 622, GSH induced the TFs for instance DNA binding and protein dimerization activity. GSH also highlighted the proteins that were involved in transportation, photosynthesis process, RNA polymerase activity, and against the metal toxicity. These results indicated that cultivar ZS 758 had better metabolism and showed higher tolerance against Cr toxicity. CONCLUSION: The responses of ZS 758 and Zheda 622 differed considerably at both physiological and transcriptional level. Moreover, RNA-Seq method explored the hazardous behavior of Cr as well as GSH up-regulating mechanism by activating plant metabolism, stress responsive genes, TFs and protein encyclopedia.

Fine mapping and candidate gene analysis of the novel thermo-sensitive genic male sterility tms9-1 gene in rice.

KEY MESSAGE: Fine mapping of the novel thermo-sensitive genic male sterility locus tms9 - 1 in the traditional TGMS line HengnongS-1 revealed that the MALE STERILITY1 homolog OsMS1 is the candidate gene. Photoperiod-thermo-sensitive genic male sterility (P/TGMS) has been widely used in the two-line hybrid rice breeding system. HengnongS-1 is one of the oldest TGMS lines and is often used in indica two-line breeding programs in China. In this study, our genetic analysis showed that the TGMS gene in HengnongS-1 was controlled by a single recessive gene that was non-allelic with the other TGMS loci identified, including C815S, Zhu1S and Y58S. Using SSR markers and bulked segregant analysis, we located the TGMS locus on chromosome 9 and named the gene tms9-1. Fine mapping further narrowed the tms9-1 loci to a 162 kb interval between two dCAPS markers. Sequence analysis revealed that a T to C substitution results in an amino acid change in the tms9-1 candidate gene (Os09g27620) in HengnongS-1 as compared to Minghui63. Sequencing of other rice accessions, including six P/TGMS lines, seven indica varieties and nine japonica varieties, showed that this SNP was exclusive to HengnongS-1. With multiple sequence alignment and expression pattern analyses, the rice MALE STERILITY1 homolog OsMS1 gene was identified as the candidate gene for tms9-1. Therefore, our study identified a novel TGMS locus and will facilitate the functional identification of the tms9-1 gene. Moreover, the markers linked to the tms9-1 gene will provide useful tools for the development of new TGMS lines by marker-assisted selection in two-line hybrid rice breeding programs.

Co-expression of RCH10 and AGLU1 confers rice resistance to fungal sheath blight Rhizoctonia solani and blast Magnorpathe oryzae and reveals impact on seed germination.

Rice sheath blight and blast caused by Rhizoctonia solani Kühn and Magnorpathe oryzae respectively, are the two most destructive fungal diseases in rice. With no genetic natural traits conferring resistance to sheath blight, transgenic manipulation provides an obvious approach. In this study, the rice basic chitinase gene (RCH10) and the alfalfa ß-1,3-glucanase gene (AGLU1) were tandemly inserted into transformation vector pBI101 under the control of 35S promoter with its enhancer sequence to generate a double-defense gene expression cassette pZ100. The pZ100 cassette was transformed into rice (cv. Taipei 309) by Agrobacterium-mediated transformation. More than 160 independent transformants were obtained and confirmed by PCR. Northern analysis of inheritable progenies revealed similar levels of both RCH10 and AGLU1 transcripts in the same individuals. Disease resistance to both sheath blight and blast was challenged in open field inoculation. Immunogold detection revealed that RCH10 and AGLU1 proteins were initially located mainly in the chloroplasts and were delivered to the vacuole and cell wall upon infection, suggesting that these subcellular compartments act as the gathering and execution site for these anti-fungal proteins. We also observed that transgenic seeds display lower germination rate and seedling vigor, indicating that defense enhancement might be achieved at the expense of development.

Evaluation of Virus-Free Chrysanthemum 'Hangju' Productivity and Response to Virus Reinfection in the Field: Molecular Insights into Virus-Host Interactions.

The shoot apical meristem culture has been used widely to produce virus-free plantlets which have the advantages of strong disease resistance, high yield, and prosperous growth potential. However, this virus-free plant will be naturally reinfected in the field. The physiological and metabolic responses in the reinfected plant are still unknown. The flower of chrysanthemum 'Hangju' is a traditional medicine which is unique to China. In this study, we found that the virus-free 'Hangju' (VFH) was reinfected with chrysanthemum virus B/R in the field. However, the reinfected VFH (RVFH) exhibited an increased yield and medicinal components compared with virus-infected 'Hangju' (VIH). Comparative analysis of transcriptomes was performed to explore the molecular response mechanisms of the RVFH to CVB infection. A total of 6223 differentially expressed genes (DEGs) were identified in the RVFH vs. the VIH. KEGG enrichment and physiological analyses indicated that treatment with the virus-free technology significantly mitigated the plants' lipid and galactose metabolic stress responses in the RVFH. Furthermore, GO enrichment showed that plant viral diseases affected salicylic acid (SA)-related processes in the RVFH. Specifically, we found that phenylalanine ammonia-lyase (PAL) genes played a major role in defense-related SA biosynthesis in 'Hangju'. These findings provided new insights into the molecular mechanisms underlying plant virus-host interactions and have implications for developing strategies to improve plant resistance against viruses.

The rice hydroperoxide lyase OsHPL3 functions in defense responses by modulating the oxylipin pathway.

As important signal molecules, jasmonates (JAs) and green leaf volatiles (GLVs) play diverse roles in plant defense responses against insect pests and pathogens. However, how plants employ their specific defense responses by modulating the levels of JA and GLVs remains unclear. Here, we describe identification of a role for the rice HPL3 gene, which encodes a hydroperoxide lyase (HPL), OsHPL3/CYP74B2, in mediating plant-specific defense responses. The loss-of-function mutant hpl3-1 produced disease-resembling lesions spreading through the whole leaves. A biochemical assay revealed that OsHPL3 possesses intrinsic HPL activity, hydrolyzing hydroperoxylinolenic acid to produce GLVs. The hpl3-1 plants exhibited enhanced induction of JA, trypsin proteinase inhibitors and other volatiles, but decreased levels of GLVs including (Z)-3-hexen-1-ol. OsHPL3 positively modulates resistance to the rice brown planthopper [BPH, Nilaparvata lugens (Stål)] but negatively modulates resistance to the rice striped stem borer [SSB, Chilo suppressalis (Walker)]. Moreover, hpl3-1 plants were more attractive to a BPH egg parasitoid, Anagrus nilaparvatae, than the wild-type, most likely as a result of increased release of BPH-induced volatiles. Interestingly, hpl3-1 plants also showed increased resistance to bacterial blight (Xanthomonas oryzae pv. oryzae). Collectively, these results indicate that OsHPL3, by affecting the levels of JA, GLVs and other volatiles, modulates rice-specific defense responses against different invaders.

Regenerating Saffron (Crocus sativus L.) from Corm Lateral Buds via Indirect Somatic Embryogenesis.

Saffron (Crocus sativus L.), being one of the distinguished commercial spice crops in the world, is in demand for its culinary, colorant, and pharmaceutical benefits. In this study, a novel indirect somatic embryogenesis (SE) system was, thus, established for the study of this plant. To this end, firstly, the lateral buds were cultured. Then, the cultures were transformed using Murashige and Skoog (MS) medium supplemented with either 6-benzyladenine (BA: 5 and 10 mg/L), naphthalene acetic acid (NAA: 0, 1, and 2 mg/L), or trans-zeatin (tZ: 0, 0.5, and 1.0 mg/L), before being classified into four structures: white globular (WG), yellow compact nodular (YCN), yellow-brown fragile (YBF), and dark-brown porous (DBP). As soon as BA (10 mg/L) and NAA (2 mg/L) were added, elevated percentages of white globular calli (56.8%) and white globular calli (31.5%) structures were induced. Additionally, 6-benzyladenine (5 mg/L) and naphthalene acetic acid (1 mg/L) allowed the formation of yellow-brown fragile structures, and the combination of 6-benzyladenine (10 mg/L) with trans-zeatin (1 mg/L) formed the DBP structures. After three months, the white globular calli were incubated using the MS basal medium, before being augmented with thidiazuron (TDZ: 1 mg/L) and picloram (PIC: 2 mg/L), from which 60% of the cases matured into shoots and, ultimately, cormlets. Morphoanatomical analyses also showed that the white globular calli cells were closely arranged, as they had a dense cytoplasm, a significant vascular differentiation, and embryoids. Furthermore, the yellow compact nodular structures were characterized by a strong differentiation capacity and contained many meristematic cells with high caryomitosis centers. We observed that the yellow-brown fragile calli had looser cell arrangements, with a vascular structure located on the protoderm edge, while there was no obvious cellular arrangement in the dark-brown porous structures. The induction of the adventitious buds in vivo on the MS medium that was supplemented with thidiazuron and picloram accordingly demonstrated the highest rates (60%) of white globular calli.

Characterization and Control of Dendrobium officinale Bud Blight Disease.

Dendrobium officinale is an important traditional Chinese medicine (TCM). A disease causing bud blight in D. officinale appeared in 2021 in Yueqing city, Zhejiang Province, China. In this paper, 127 isolates were obtained from 61 plants. The isolates were grouped into 13 groups based on collected areas and morphological observations. Four loci (ITS, LSU, tub2 and rpb2) of 13 representative isolates were sequenced and the isolates were identified by constructing phylogenetic trees with the multi-locus sequence analysis (MLSA) method. We found the disease to be associated with three strains: Ectophoma multirostrata, Alternaria arborescens and Stagonosporopsis pogostemonis, with isolates frequencies of 71.6%, 21.3% and 7.1%, respectively. All three strains are pathogenic to D. officinale. A. arborescens and S. pogostemonis isolated from D. officinale were reported for the first time. Iprodione (50%), 33.5% oxine-copper and Meitian (containing 75 g/L pydiflumetofen and 125 g/L difenoconazole) were chosen to control the dominant pathogen E. multirostrata, with an EC50 value of 2.10, 1.78 and 0.09 mg/L, respectively. All three fungicides exhibited an effective inhibition of activities to the growth of the dominant pathogen E. multirostrata on potato dextrose agar (PDA) plates, with Meitian showing the strongest inhibitory effect. We further found that Meitian can effectively control D. officinale bud blight disease in pot trial.

Colletotrichum siamense Strain LVY 9 Causing Spot Anthracnose on Winterberry Holly in China.

Winterberry holly (Ilex verticillata) is an economically valuable landscaping ornamental plant. Serious outbreaks have been reported, in its leaf tips curl upward, irregular black brown spots appear on leaves, and extensive defoliation is commonly observed. The incidence in Hangzhou was estimated at 50% and resulted in large economic losses for growers in 2018. Samples were collected from the main cultivation area in Zhejiang Province. In total, 11 fungal isolates were obtained from diseased leaves through a single-spore purification method, and isolate LVY 9 exhibited strong pathogenicity. Based on morphology and molecular phylogenetic analyses based on multilocus sequence typing of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), and chitin synthase (CHS-1) genes, we identified the pathogen as Colletotrichum siamense, causative agent of anthracnose of winterberry holly.

Nigrospora oryzae Causing Leaf Spot Disease on Chrysanthemum × morifolium Ramat and Screening of Its Potential Antagonistic Bacteria.

Chrysanthemum × morifolium Ramat. is a famous perennial herb with medicinal, edible, and ornamental purposes, but the occurrence of plant diseases can reduce its value. A serious disease that caused leaf spots in C. morifolium appeared in 2022 in Tongxiang City, Zhejiang Province, China. The C. morifolium leaves with brown spots were collected and used for pathogen isolation. By completing Koch's postulates, it was proven that the isolate had pathogenicity to infect C. morifolium. It was determined that the pathogen isolated from chrysanthemum leaves was Nigrospora oryzae, through morphology and a multilocus sequence analysis method using a combination of the internal transcribed spacer gene (ITS), beta-tubulin gene (TUB2), and translation elongation factor 1-alpha gene (TEF1-α). This is the first report of C. morifolium disease caused by N. oryzae in the world. Through dual culture assay on PDA plates, 12 strains of bacteria with antagonistic effects were selected from 231 strains from the C. morifolium phyllosphere, among which Bacillus siamensis D65 had the best inhibitory effect on N. oryzae growth. In addition, the components of a strain D65 fermentation broth were profiled by SPME-GC-Q-TOF analysis, providing a foundation for further application and research of biological control.

A Gaijin-like miniature inverted repeat transposable element is mobilized in rice during cell differentiation.

BACKGROUND: Miniature inverted repeat transposable element (MITE) is one type of transposable element (TE), which is largely found in eukaryotic genomes and involved in a wide variety of biological events. However, only few MITEs were proved to be currently active and their physiological function remains largely unknown. RESULTS: We found that the amplicon discrepancy of a gene locus LOC_Os01g0420 in different rice cultivar genomes was resulted from the existence of a member of Gaijin-like MITEs (mGing). This result indicated that mGing transposition was occurred at this gene locus. By using a modified transposon display (TD) analysis, the active transpositions of mGing were detected in rice Jiahua No. 1 genome under three conditions: in seedlings germinated from the seeds received a high dose γ-ray irradiation, in plantlets regenerated from anther-derived calli and from scutellum-derived calli, and were confirmed by PCR validation and sequencing. Sequence analysis revealed that single nucleotide polymorphisms (SNPs) or short additional DNA sequences at transposition sites post mGing transposition. It suggested that sequence modification was possibly taken place during mGing transposition. Furthermore, cell re-differentiation experiment showed that active transpositions of both mGing and mPing (another well studied MITE) were identified only in regenerated plantlets. CONCLUSIONS: It is for the first time that mGing active transposition was demonstrated under γ-ray irradiation or in cell re-differentiation process in rice. This newly identified active MITE will provide a foundation for further analysis of the roles of MITEs in biological process.

4ß-Hy-droxy-12,13-ep-oxy-trichothec-9-ene.

The asymmetric unit in the crystal of the title compound, C(15)H(22)O(3), contains two independent mol-ecules with similar structures. Each mol-ecule contains two six-membered rings and one five-membered ring. The five-membered ring displays an envelope conformation with the C atom linking the epoxy group as the flap, while the two six-membered rings show half-chair conformations. The two independent mol-ecules are linked by an O-H⋯O hydrogen bond. These dimers are further linked by O-H⋯O hydrogen bonds, forming supra-molecular chains running along the a axis.