First Report of Pectobacterium polaris Causing Soft Rot on Broccoli in China.

In April 2023, soft rot symptoms were observed in broccoli (Brassica oleracea L. var. italica) commercial fields in Songming County, Yunnan province, China (103°12'E, 25°31'N). The disease incidence in these fields (6 ha in size) was high, exceeding 50%, and it caused significant yield loss. The affected plants displayed characteristic symptoms, with the roots and stems of broccoli becoming soft, yellowish-brown, rotten, and emitting a foul odor. To identify the causal agent, soft rot symptomatic stems were surface sterilized by dipping them in 75% ethanol for 30 seconds, followed by three successive rinses with sterile distilled water. Tissue specimens were then plated onto nutrient agar (NA) plates and incubated at 28°C for 24 hours. (Wang et al. 2022). Three representative bacterial isolates HYC22041801-HYC22041803 from broccoli were selected for further analysis. The colonies on NA plates appeared as white, small, round, and translucent with smooth edges. Physiological and biochemical tests were performed, along with 96 phenotypic screenings using the BIOLOG GENIII microplate system (Biolog, Hayward, CA, USA). Three isolates were negative for D-arabitol, maltose, and sorbitol, but were positive for cellobiose, α-D-glucose, sucrose, glycerol and gentiobiose tests, which are consistent with the reported type strain P. polaris NIBIO1006T (Chen et al. 2021). Total genomic DNA was extracted from three bacterial isolates using the QIAamp DNA Mini Kit (QIAGEN, USA). The 16S rRNA region and nine housekeeping genes (gapA, icdA, mdh, mtlD, pel, pgi, pmrA, proA and rpoS) were amplified with universal primers 27F/1492R (Monciardini et al., 2006) and designed specific primers (Xie et al., 2018), respectively. All amplicons were sequenced and deposited in GenBank with accession numbers ON723841-ON723843 and ON723846-ON723872. The BLASTn analysis of the 16S rRNA amplicons confirmed that the isolates HYC22041801-HYC22041803 belonged to the genus Pectobacterium. Phylogenetic trees based on 16S rRNA gene sequences and multilocus sequence analysis of other nine housekeeping genes of the three isolates were constructed and the results revealed that three isolates clustered with P. polaris type strain NIBIO1006T, which was previously isolated from potato (Dees et al., 2017). To confirm the pathogenicity, nine broccoli seedlings were stab inoculated with a bacterial suspension (108 CFU·ml-1), while sterile distilled liquid LB medium was used as a negative control. The seedlings were kept at 80% relative humidity and 28°C in a growth chamber. Three trials were conducted per isolate (HYC22041801-HYC22041803). After 3 days, the inoculated petioles showed soft rot symptoms similar to those observed initially in the field, while control plants remained asymptomatic. All three isolates were re-isolated successfully from symptomatic tissues to complete Koch's postulates. P. polaris has been previously reported as the causative agent of blackleg in potato in several countries, including Norway, Poland, Russia, and China (Handique et al. 2022; Wang et al. 2022). Additionally, it was reported to cause soft rot in Chinese cabbage in China (Chen et al. 2021). However, this is the first report of P. polaris causing soft rot disease in broccoli in China. This discovery is of great importance for vegetable growers because this bacterium is well established on Cruciferous vegetables in the local area, and effective measures are needed to manage this disease.

First Report of Root Rot Caused by Fusarium incarnatum on Mongolian Snake gourd in China.

Mongolian snake gourd (Trichosanthes kirilowii Maxim) is a precious traditional Chinese herbal medicine and perennial liana plant in the family Cucurbitaceae, and the root, fruit, seed and peel all possess the medicinal value (Zhang et al. 2016). During 2021-2022, the root rot was observed in a 20-ha commercial farm and became a major disease limiting Mongolian snake gourd production in Zhenjiang City, Jiangsu Province, China (119°27'E, 32°12'N). Field investigations showed that disease incidence was estimated at approximately 70% and resulted in up to a 50% decrease in total production. Symptoms on snake gourd initially appeared as yellow mottling produced on the surface of the infected new leaves and systemic wilting on the upper leaves. With the development of the infection, the base of the stem began to brown and die, and has lots of filamentous hyphae attached to it. As the lesions coalesced, the whole plant gradually wilted and died. In order to explore the cause of the disease, six infected plants were randomly collected from the commercial farm. The roots of the plants were rinsed in sterile water to remove soil debris, and symptomatic roots were surface sterilized using 75% ethanol for 60s, rinsed three times in sterile water, then plated onto the potato dextrose agar (PDA), and incubated at 25°C for 3 days in the dark. White fungal colonies grew from the tissue pieces, then hyphal tips were transferred to PDA to obtain pure cultures. A total of six isolates with similar morphological characteristics were obtained from six of the infected plants. One representative isolate GL21091501 was chosen for further analysis. At 5 days after inoculation, the colonies on PDA began to grow as white, and with the incubated time was extended, the hyphae turned yellowish-brown with a yellowish-brown center on the reverse side. Observations under a light microscope showed conidia that were falculate, slender and slightly curved, and the cells at both ends were sharp. Macroconidia had four to five septa, measuring 22.4 ~ 33.5 µm. Microconidia without septa, elliptical, measuring 4.36 ~ 9.88 µm. On the tip of aerial hyphae can form conidiophore, and produce macroconidia (Wonglom et al. 2020; Lin et al 2018). The pathogen was typical Fusarium spp. by morphological characteristics. To identify the species level, the mycelia of the representative isolate GL21091501 was used for genomic DNA extraction (Tiangen, China). The internal transcribed spacer (ITS) region and partial translational elongation factor subunit 1-α (TEF-1α) of the cultures were amplified and sequenced using the primer pairs EF1/EF2 and ITS1/ITS4 respectively (White et al. 1990; O'Donnell et al. 1998). The obtained sequences were deposited in GenBank under the accesion numbers OP311409 and OP311410. BLAST searches of the deposited sequences showed 100% identity with the existing TEF sequences (MT563420.1) and ITS sequences (MN539094.1) of Fusarium incarnatum isolates in GenBank. In addition, BLASTn analysis of these in FUSARIUM-ID database showed 99.62% and 100% similarity with F. incarnatum-equiseti species complex (FIESC) NRRL13379 [ITS] and NRRL34004 [TEF-1α]), respectively. Phylogenetic analysis was conducted with the neighbor-joining (NJ) method using MEGA6.0 (Tamura et al. 2007). Combined phylogenetic analysis revealed that the isolate shared a common clade with the reference sequence of F. incarnatum in the F. incarnatum-equiseti species complex. Therefore, according to morphological and molecular characteristics confirming the identity of the isolated pathogen as F. incarnatum. In order to fulfill Koch's postulates, fresh isolate GL21091501 hyphae were cut into 3 × 3 mm agar plugs from a 7 cm PDA plate and inoculated in 200 mL the Potato Dextrose (PD) liquid medium on a shaker at 170 rpm, 25°C for 5 days. Spores were filtered through four layers of gauze, adjusted to 1 × 106 spores/ml with sterilized water. Then Mongolian snake gourd seedlings at the two true leaves stage were transplanted in (15-cm-diameter) pots (1 plants/pot) filled with mixture of sterilized soil: vermiculite: pearlite (2:1:1, v/v). The pathogenicity test was conducted on seedlings plants by root irrigation method (50 ml/plant, 1×106 conidia/mL), control plants were irrigation with sterilized water (50 ml/plant). Each treatment was repeated three times. After 15 days, all inoculated plants showed the same symptoms observed on the original diseased plants in the field, whereas, the control plants remained symptomless. The same pathogen was successfully re-isolated from the inoculated plants, and identical to those of the originals based on morphological and sequence data. To our knowledge, this is the first report of F. incarnatum causing root rot on Mongolian snake gourd in China. F. incarnatum has been reported to cause root and stem rot in many plants worldwide, including muskmelon (Wonglom et al. 2020), Cucurbita pepo (Thomas et al. 2019) and Bambusa multiplex (Lin et al. 2018). This discovery is of great importance for Mongolian snake gourd planters because the fungus is accurately identified in a certain geographic area and effective field management strategies are necessary to control this disease.

Reclassification of a likely pathogenic Dutch founder variant in KCNH2; implications of reduced penetrance.

BACKGROUND: Variants in KCNH2, encoding the human ether a-go-go (hERG) channel that is responsible for the rapid component of the cardiac delayed rectifier K+ current (IKr), are causal to long QT syndrome type 2 (LQTS2). We identified eight index patients with a new variant of unknown significance (VUS), KCNH2:c.2717C > T:p.(Ser906Leu). We aimed to elucidate the biophysiological effect of this variant, to enable reclassification and consequent clinical decision-making. METHODS: A genotype-phenotype overview of the patients and relatives was created. The biophysiological effects were assessed independently by manual-, and automated calibrated patch clamp. HEK293a cells expressing (i) wild-type (WT) KCNH2, (ii) KCNH2-p.S906L alone (homozygous, Hm) or (iii) KCNH2-p.S906L in combination with WT (1:1) (heterozygous, Hz) were used for manual patching. Automated patch clamp measured the variants function against known benign and pathogenic variants, using Flp-In T-rex HEK293 KCNH2-variant cell lines. RESULTS: Incomplete penetrance of LQTS2 in KCNH2:p.(Ser906Leu) carriers was observed. In addition, some patients were heterozygous for other VUSs in CACNA1C, PKP2, RYR2 or AKAP9. The phenotype of carriers of KCNH2:p.(Ser906Leu) ranged from asymptomatic to life-threatening arrhythmic events. Manual patch clamp showed a reduced current density by 69.8 and 60.4% in KCNH2-p.S906L-Hm and KCNH2-p.S906L-Hz, respectively. The time constant of activation was significantly increased with 80.1% in KCNH2-p.S906L-Hm compared with KCNH2-WT. Assessment of KCNH2-p.S906L-Hz by calibrated automatic patch clamp assay showed a reduction in current density by 35.6%. CONCLUSION: The reduced current density in the KCNH2-p.S906L-Hz indicates a moderate loss-of-function. Combined with the reduced penetrance and variable phenotype, we conclude that KCNH2:p.(Ser906Leu) is a low penetrant likely pathogenic variant for LQTS2.

First Report of Leaf Spot on water convolvulus Caused by Stemphylium solani in Yunnan Province, China.

Water convolvulus (Ipomoea aquatica Forsk.), a member of the Convolvulaceae family, is an important tropical vegetable cultivated in China (Liu et al. 2017). From 2016 to 2020, dark-brown leaf spots were observed in major water convolvulus (cv. Large leaf) growing areas (2 ha) in Honghe City (24°12' N, 103°6' E), Yunnan Province, China. Field investigations showed that a leaf spot disease occurred on water convolvulus in four fields with 15% incidence (50 plants in each field were investigated) and resulted in up to a 10% decrease in its total production. Symptoms on water convolvulus plants appeared as small lesions, yellowish-green and circular on the leaves. Ten plants were selected randomly from the growing area, with three diseased leaves collected from each plant. Symptomatic tissues were excised, surface sterilized with 75% ethanol for 30 s, washed in sterile-distilled water three times, and placed on the Potato Dextrose Agar (PDA) followed by incubation at 25°C in the dark for 7 days. Colonies on PDA were gray to green in color and fuzzy in the middle, with irregular borders. Conidiophore morphology showed single, yellowish-brown or brown structures with 1~6 septa, and long 22~125 µm, wide 3.5~5.5 µm. Conidia were elliptical, black-brow, solitary, with a smooth surface, 1~6 longitudinal septa and 1~3 transverse septa, 20~30 µm in length, and 15~22 µm in width. The morphological characteristics of the fungus were consistent with the description of Stemphylium solani (Chai et al. 2014; Weber, 1930). To further confirm the identity of the 30 isolates, the partial gapdh (glyceraldehyde-3-phosphate dehydrogenase), tef1 (translation elongation factor 1-alpha), cmdA (Calmodulin) and ITS (intemal transcribed spacers) sequences were amplified by PCR with the primer pairs of gpd1/gpd2, EF1-728F/EF1-986R, CALDF1/CALDR2 and ITS1/ITS4, respectively (Berbee et al. 1999; Carbone & Kohn. 1999; Lawrence et al. 2013; White et al. 1990). Multiple sequence alignments were generated using MEGA7, and phylogenetic analysis was conducted with the neighbor-joining (NJ) method (Tamura et al. 2007), the results indicated that all sequences from the 30 isolates were identical. Thus, one representative isolate, KXC11033003 was chosen for further analysis. The ITS, gapdh, cmdA and tef1 sequences of this isolate were submitted to the NCBI GenBank database (accession nos. OL444947~OL444950). The strain KXC11033003 and S. solani (CBS-408.54) formed a clade with 82% bootstrap value (Figure S2). To fulfill Koch's postulates, 30 plants were inoculated for each of the thirty isolates. Conidia were sprayed on leaves of water convolvulus (8-true-leaf stage) in a suspension of 107 conidia/mL or water as a healthy control in a greenhouse at 15~18℃ (night) / 25~28℃ (day) with 95% humidity. Symptoms of dark brown spots appeared on the leaves after 7 days, whereas controls remained healthy The pathogens were reisolated from the lesions and confirmed identical to the original isolate by gene sequences. No pathogens were isolated from the control plants. To our knowledge, this is the first report of leaf spot caused by S. solani on water convolvulus in Yunnan Province, China. Further, Stemphylium leaf spot caused by S. solani has been reported previously on tomato, garlic, pepper (Zheng et al.2008; Nasehi et al.2018). This study stresses the need to identify appropriate management strategies for S. solani that help prevent quality and yield losses in water convolvulus in China.

Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing.

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by the lack of dystrophin, which maintains muscle membrane integrity. We used an adenine base editor (ABE) to modify splice donor sites of the dystrophin gene, causing skipping of a common DMD deletion mutation of exon 51 (∆Ex51) in cardiomyocytes derived from human induced pluripotent stem cells, restoring dystrophin expression. Prime editing was also capable of reframing the dystrophin open reading frame in these cardiomyocytes. Intramuscular injection of ∆Ex51 mice with adeno-associated virus serotype-9 encoding ABE components as a split-intein trans-splicing system allowed gene editing and disease correction in vivo. Our findings demonstrate the effectiveness of nucleotide editing for the correction of diverse DMD mutations with minimal modification of the genome, although improved delivery methods will be required before these strategies can be used to sufficiently edit the genome in patients with DMD.

First Report of Bacterial Leaf Spot of Cucurbita pepo Caused by Erwinia persicina in China.

In February 2020, the common symptoms of water-soaked spots on Cucurbita pepo L. cotyledon were observed in Guangrao county in Shandong province, China. Field investigation showed that 40% of the Cucurbita pepo cotyledons in an area of approximately 0.8 ha were infected. The disease resulted in a severe loss in seedling production. Samples of C. pepo with water-soaked leaf spots were collected and prepared for pathogen analysis. Symptomatic cotyledon tissue was surface disinfested in 75% ethanol for 30 sec, then rinsed three times in sterilized water. Bacteria were released in sterile water in Petri dish for 2 min by cutting symptomatic tissue into small sections and stirring the plant tissue mixture fully. The diffusate was streaked onto plates containing nutrient agar (NA) and plates were incubated at 28℃ for 2 days. Three representative isolates were purified eventually from each of the plates. Colonies on NA were small, round and with smooth margins. All bacterial isolates characterized as gram-negative, white to cream color, and pink pigment was formed on the plates over long-term culture. The isolates were positive for catalase, Voges-Proskauer, potato rot, methyl red, acetoin production, nitrate utilization and citrate utilization, and acid production from maltose, glucose, melezitose, sucrose, D-arabinose, D-trehalose, cellobiose, lactose, raffinose, mannitol, D-sorbitol, melibiose and xylitol. KOH production was demonstrated according to strand formation within the potassium hydroxide test (Suslow et al. 1982). Isolates were negative for oxidase, arginine dihydrolase, phenylalanine deaminase, gelatinase, esculine, indole production and H2S production. Total genomic DNA was extracted from isolate XHL2002230201 with TIANamp Bacteria DNA Kit (TIANGEN). Universal primers 27F and 1492R (Monciardini et al. 2002) were used in PCR to amplify a 1,307-bp DNA fragment of the 16S rRNA region for molecular identification. Furthermore, four additional housekeeping genes (gyrB, atpD, rho, and rpoS) were selected and amplified using specially designed primers. The amplification products of 16S rRNA were sequenced and submitted to GenBank under accession number (MT568607.1). Sequence analysis showed 99% similarity to Erwinia persicina strains B57 (LM651373.1) and B64 (CI789_17875) by BLAST search in GenBank database (Gálvez et al. 2015; Cho et al. 2019). A phylogenetic tree was constructed, and the taxonomic position of strain XHL2002230201 was determined from the multilocus sequence analysis (MLSA) on 16S rRNA and other four housekeeping genes with E. persicina and not with other closely related Erwinia species. Pathogenicity tests and re-isolation and re-identification of the bacteria were performed to confirm the isolate and fulfill the Koch' postulates. The strain XHL2002230201 suspensions (108 CFU ml-1) were spray inoculated onto fifteen Cucurbita pepo seedlings with two true leaves, and the same number of control plants were inoculated with water. Experiments were repeated three times. All inoculated plants were kept in a moist chamber placed in a greenhouse at 28℃. Initial symptoms were observed on leaves of inoculated plants at 5 days post-inoculation, whereas no symptoms appeared on the plants inoculated with sterile distilled water. Based on morphological and biochemical characteristics, phylogenetic analysis, and Koch's postulates, the bacterial isolates were identified as E. persicina. To our knowledge, this is the first report of E. persicina causing leaf spot disease on Cucurbita pepo in China.

First Report of Cercospora apiicola Causing Leaf Spots and Stem Lesions on Celery in China.

Celery (Apium graveolens L.) is a vegetable crop cultivated widely in the Mediterranean, Europe and parts of Asia. From March to May in 2014, leaf spots and stem lesions were observed on celery plants in Yanqing (116°03'E, 40°32'N), Beijing and Chengdu (104°06'E, 30°67'N), Sichuan Province. Plants developed 0.3-1.8 cm diameter subcircular leaf spots with brown centers surrounded by pale yellow halos. Spots on leaves were amphigenous. Necrotic areas on stems were subcircular to elongated, pale brown to brown. Plants in five greenhouses were surveyed with 30 to 60% disease incidence. Necrotic tissue from 8 stems and 12 leaves were cut from the margins of lesions and divided into two parts. One part was treated with lactophenol and used for microscopic examination. The other part was surface sterilized with 4% sodium hypochlorite for 2 min, rinsed three times in sterile water, placed onto 2% malt extract agar (MEA), and incubated at 26°C for seven days with natural daylight. Stromata on leaves and stems were not well developed. Four-to-ten conidiophores (15.3-56.5 × 2.8-5.5 µm) formed in fascicles, emerged through stomata or erupted through the cuticle. Conidia (n=50) were 60-135 × 2.5-4.5 µm, solitary, septate, cylindrical to obclavate-cylindrical, hila thickened and darkened. Colonies were white to smoke-gray, and aerial mycelia were sparse to moderate. Morphological characteristics of the pathogen were similar to Cercospora apiicola (Groenewald et al. 2006; Groenewald et al. 2013). The gDNA of 20 isolates was extracted from mycelium using the Plant Genomic DNA Kit (Tiangen, China). The internal transcribed spacers (ITS), actin (ACT), translation elongation factor 1-α (TEF1) and histone H3 (HIS3) regions were amplified with primer pairs ITS1/ITS4 (Groenewald et al. 2013), ACT-512F/ACT-783R (Carbone and Kohn 1999), EF1-728F/EF1-986R (Carbone and Kohn 1999), CYLH3F/CYLH3R (Crous et al. 2006). Phylogenetic analysis of multiple genes (Bakhshi et al. 2018) was conducted with the neighbor-joining method using MEGA7. The sequences of our isolate (QC14030702) and five published sequences of C. apiicola were clustered into one clade with a 99% confidence level. The sequences of QC14030702 have been deposited in GenBank with accessions KU870468 for ITS, KU870469 for ACT, KU870470 for TEF1, and KU870471 for HIS3. Pathogenicity of the isolates was tested on plants (cv. Jia Yuan Xi Yang Qin). Because the pathogen sporulated poorly on various media, mycelial fragments were sprayed on leaves in a suspension of 1×106 mL-1 in a greenhouse (temperature 26±0.5°C; RH 98%; photoperiod 12 h). Healthy plants were sprayed with sterilized water as controls. Three replicates of every isolate were conducted, and each replicate included 5 celery plants. After 7 days, leaf spots appeared on all inoculated plants, which were similar to those on celery in the field. All control plants remained asymptomatic. Re-isolation of the fungus from infected tissues showed same morphological and cultural characteristics of C. apiicola as the original isolates. C. apiicola has been reported in Greece, Korea, South Korea and Venezuela on celery, but never been reported in China (Farr and Rossman 2020). C. apiicola potential threatens celery production, and this the first report of the disease in China.

Quantification of Viable Cells of Pseudomonas syringae pv. tomato in Tomato Seed Using Propidium Monoazide and a Real-Time PCR Assay.

Pseudomonas syringae pv. tomato is a seedborne pathogen that causes bacterial speck disease in tomato. P. syringae pv. tomato is typically detected in tomato seed using quantitative real-time PCR (qPCR) but the inability of qPCR to distinguish between viable and nonviable cells might lead to an overestimation of viable P. syringae pv. tomato cells. In the present study, a strategy involving a propidium monoazide (PMA) pretreatment followed by a qPCR (PMA-qPCR) assay was developed for quantifying viable P. syringae pv. tomato cells in contaminated tomato seed. PMA could selectively bind to the chromosomal DNA of dead bacterial cells and, therefore, block DNA amplification of qPCR. The primer pair Pst3F/Pst3R was designed based on gene hrpZ to specifically amplify and quantify P. syringae pv. tomato by qPCR. The PMA pretreatment protocol was optimized for selectively detecting viable P. syringae pv. tomato cells, and the optimal PMA concentration and light exposure time were 10 µmol liter-1 and 10 min, respectively. In the sensitivity test, the detection limit of PMA-qPCR for detecting viable cells in bacterial suspension and artificially contaminated tomato seed was 102 CFU ml-1 and 11.86 CFU g-1, respectively. For naturally contaminated tomato seed, viable P. syringae pv. tomato cells were quantified in 6 of the 19 samples, with infestation levels of approximately 102 to 104 CFU g-1. The results indicated that the PMA-qPCR assay is a suitable tool for quantifying viable P. syringae pv. tomato cells in tomato seed, which could be useful for avoiding the potential risks of primary inoculum sources from contaminated seed.

Verticillium Wilt of Okra Caused by Verticillium dahliae Kleb. in China.

Okra (Abelmoschus esculentus (L.) Moench) has gained more popularity as an economically significant plant for its nutritional and medicinal value, especially in China. During 2014-2016, the root disease of okra was discovered in four okra commercial fields surveyed in China. A fungul was isolated from the infected tissues, and was identified by Verticillium dahliae based on morphological characteristics. Pathogenicity test demonstrated that the fungus was pathogenic on okra, and fulfilled Koch's postulates. The analysis of three sequences revealed 99-100% identity with the reported V. dahliae strain in GenBank. Neighbor-joining analysis of the gene sequences revealed that the representative isolates were clustered with V. dahliae. To the best of our knowledge, this is the first report of Verticillium wilt of okra in China.