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.

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.

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.

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.

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.

Plasma membrane localization and potential endocytosis of constitutively expressed XA21 proteins in transgenic rice.

The rice pattern recognition receptor (PRR) XA21 confers race-specific resistance in leaf infection by bacterial blight Xathomonas oryzae pv. oryzae (Xoo), and was shown to be primarily localized to the endoplasmic reticulum (ER) when expressed with its native promoter or overexpressed in the protoplast. However, whether the protein is still ER-localization in the intact cell when overexpressed remains to be identified. Here, we showed that XA21, its kinase-dead mutant XA21P(K736EP), and the triple autophosphorylation mutant XA21P(S686A/T688A/S699A) GFP fusions were primarily localized to the plasma membrane (PM) when overexpressed in the intact transgenic rice cell, and also localized to the ER in the transgenic protoplast. The transgenic plants constitutively expressing the wild-type XA21 or its GFP fusion displayed race-specific resistance to Xoo at the adult and seedling stages. XA21 and XA21P(K736EP) could be internalized probably via the SCAMP-positive early endosomal compartment in the protoplast, suggesting that XA21 might be endocytosed to initiate resistance responses during pathogen infection. We also established a root infection system and demonstrated that XA21 also mediated race-specific resistance responses to Xoo in the root. Our current study provides an insight into the nature of the XA21-mediated resistance and a practical approach using the root cell system to further dissect the cellular signaling of the PRR during the rice-Xoo interaction.

Effects of osmotic stress on antioxidant enzymes activities in leaf discs of PSAG12-IPT modified Gerbera.

Leaf senescence is often caused by water deficit and the chimeric gene P(SAG12)-IPT is an auto-regulated gene delaying leaf senescence. Using in vitro leaf discs culture system, the changes of contents of chlorophylls, carotenoids, soluble protein and thiobarbituric acid reactive substance (TBARS) and antioxidant enzymes activities were investigated during leaf senescence of P(SAGl2)-IPT modified gerbera induced by osmotic stress compared with the control plant (wild type). Leaf discs were incubated in 20%, 40% (w/v) polyethylene glycol (PEG) 6000 nutrient solution for 20 h under continuous light [130 micromol/(m(2) x s)]. The results showed that the contents of chlorophylls, carotenoids and soluble protein were decreased by osmotic stress with the decrease being more pronounced at 40% PEG, but that, at the same PEG concentration the decrease in the transgenic plants was significantly lower than that in the control plant. The activities of superoxide dismutase (SOD), catalases (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and dehydroascorbate reductase (DHAR) were stimulated by PEG treatment. However, the increases were higher in P(SAG12)-IPT transgenic plants than in the control plants, particularly at 40% PEG treatment. Lipid peroxidation (TBARS content) was increased by PEG treatment with the increase being much lower in transgenic plant than in the control plant. It could be concluded that the increases in the activities of antioxidant enzymes including SOD, CAT, APX, GPX and DHAR were responsible for the delay of leaf senescence induced by osmotic stress.

[Induction of embryogenic calli from caryopses of common bermudagrass and ultrastructure of embryogenic cells as well as plant regeneration].

A tissue culture system for embryogenic callus (EC) induction and plant regeneration of common bermudagrass using mature caryopsis (embryos) as explants was developed. The results showed that embryogenic calli could be induced from caryopsis with high frequency, in MS medium with 2,4-D 2.0-6.0 mg/L, and the best concentration of 2,4-D was 4.0 mg/L. The best method for maintaining EC and tissue differentiation was to subculture EC in MS+2,4-D 4.0 mg/L 1-2 times, follwed by subculturing in 1/2 MS+2,4-D 2.0 mg/L for 1-2 times, then to transfer EC to 1/2 MS without hormone for a 10-d-preregeneration in light, followed by transferring to MS+6-BA 3.0 mg/L for regeneration, with regeneration frequency 31.7%. Morphological and micro-structural differences between EC and non-embryogenic callus (NEC) were observed by electron microscope. Ultrastructrual characteristics of the EC cells are described in this paper.

Alpha-picolinic acid, a fungal toxin and mammal apoptosis-inducing agent, elicits hypersensitive-like response and enhances disease resistance in rice.

Alpha-picolinic acid (PA), a metabolite of tryptophan and an inducer of apoptosis in the animal cell, has been reported to be a toxin produced by some of plant fungal pathogens and used in screening for disease resistant mutants. Here, we report that PA is an efficient apoptosis agent triggering cell death of hypersensitive-like response in planta. Confirmed by Fluorescence Activated Cell Sorter (FACS), rice suspension cells and leaves exhibited programmed cell death induced by PA. The PA-induced cell death was associated with the accumulation of reactive oxygen species that could be blocked by diphenylene iodonium chloride, indicating that the generation of reactive oxygen species was NADPH-oxidase dependent. We also demonstrated the induction of rice defense-related genes and subsequent resistant enhancement by PA against the rice blast fungus Magnaporthe grisea. Hence, it was concluded that the PA-stimulated defense response likely involves the onset of the hypersensitive response in rice, which also provides a simple eliciting tool for studying apoptosis in the plant cell.