Monitoring Corynespora cassiicola Resistance to Boscalid, Trifloxystrobin, and Carbendazim in China.

Cucumber leaf spot (CLS), caused by Corynespora cassiicola, is a serious disease of greenhouse cucumbers. With frequent use of existing fungicides, C. cassiicola has developed resistance to some of them, with serious implications for the control of CLS in the field. With a lack of new fungicides, it is necessary to use existing fungicides for effective control. Therefore, this study monitored the resistance of C. cassiicola to three commonly used and effective fungicides, boscalid, trifloxystrobin, and carbendazim, from 2017 to 2021. The frequency of resistance to boscalid showed an increasing trend, and the highest frequency was 85.85% in 2020. The frequency of resistance to trifloxystrobin was greater than 85%, and resistance to carbendazim was maintained at 100%. Among these fungicides, strains with multiple resistance to boscalid, trifloxystrobin, and carbendazim were found, accounting for 32.00, 25.25, 33.33, 43.06, and 37.24%, respectively. Of the strains that were resistant to boscalid, 87% had CcSdh mutations, including seven genotypes: B-H278L/Y, B-I280V, C-N75S, C-S73P, D-D95E, and D-G109V. Also, six mutation patterns of the Ccß-tubulin gene were detected: E198A, F167Y, E198A&M163I, E198A&F167Y, M163I&F167Y, and E198A&F200C. Detection of mutations of the CcCytb gene in resistant strains showed that 98.8% were found to have only the G143A mutation. A total of 27 mutation combinations were found and divided into 14 groups for analysis. The resistance levels differed according to genotype. The development of genotypes showed a complex trend, increasing from 4 in 2017 to 13 in 2021 and varying by region. Multiple fungicide resistance is gradually increasing. Therefore, it is necessary to understand the types of mutations and the trend of resistance to guide the use of fungicides to achieve disease control.

The mechanisms of target and non-target resistance to QoIs in Corynespora Cassiicola.

Corynespora leaf spot, caused by Corynespora cassiicola, is a foliar disease in cucumber. While the application of quinone outside inhibitors (QoIs) is an effective measure for disease control, it carries the risk of resistance development. In our monitoring of trifloxystrobin resistance from 2008 to 2020, C. cassiicola isolates were categorized into three populations: sensitive isolates (S, 0.01 < EC50 < 0.83 µg/mL), moderately resistant isolates (MR, 1.18 < EC50 < 55.67 µg/mL), and highly resistant isolates (HR, EC50 > 56.98 µg/mL). The resistance frequency reached up to 90% during this period, with an increasing trend observed in the annual average EC50 values of all the isolates. Analysis of the CcCytb gene revealed that both MR and HR populations carried the G143A mutation. Additionally, we identified mitochondrial heterogeneity, with three isolates carrying both G143 and A143 in MR and HR populations. Interestingly, isolates with the G143A mutation (G143A-MR and G143A-HR) displayed differential sensitivity to QoIs. Further experiments involving gene knockout and complementation demonstrated that the major facilitator superfamily (MFS) transporter (CcMfs1) may contribute to the disparity in sensitivity to QoIs between the G143A-MR and G143A-HR populations. However, the difference in sensitivity caused by the CcMfs1 transporter is significantly lower than the differences observed between the two populations. This suggests additional mechanisms contributing to the variation in resistance levels among C. cassiicola isolates. Our study highlights the alarming level of trifloxystrobin resistance in C. cassiicola in China, emphasizing the need for strict prohibition of QoIs use. Furthermore, our findings shed light on the occurrence of both target and non-target resistance mechanisms associated with QoIs in C. cassiicola.

First report of Pectobacterium brasiliense causing soft rot on leaf mustard in China.

Leaf mustard (Brassica juncea [L.] Czern. et Coss.) belongs to Brassicaceae and is an important leaf vegetable widely cultivated in the Yangtze River basin and various southern provinces in China. In August 2023, the rhizome decay symptoms were observed at the stem base of leaf mustard plants (cv. Huarong) in the field of Changde City (29.05 °N; 111.59 °E), Hunan Province, China. The incidence of symptomatic leaf mustard was approximately 30% in several fields (2 ha in total). Brown and water-soaked symptoms appeared at the base of the outer leaves, and hollow rot at the base of the stem, accompanied by a fishy odor. To identify the causal agent, six infected stem samples were collected and surface sterilized by soaking in 75% ethanol for 60 seconds, rinsed three times with sterile distilled water, and finally cut into pieces (5 × 5 mm) in the sterile water. The extract was streaked on nutrient agar medium. After incubation at 28°C for 24 h, 17 strains were obtained and the colonies of all strains were creamy white, roughly circular, and convex elevation. Six single bacterial strains JC23121001-JC23121006, individually isolated from six different diseased stem samples, were selected as representative strains for further study. For preliminary identification, DNA from the six strains was extracted and identified by 16S rDNA sequencing using the universal primer pair 27F/1492R (Weisburg et al. 1991), and the sequences (accession nos. PP784484 to PP784489) showed 99% query coverage and 99.65% identity to Pectobacterium brasiliense type strain IBSBF1692T (Nabhan et al. 2012). In addition, five housekeeping genes acnA, mdh, mltD, pgi, and proA of the six strains were amplified with specially designed primers (Ma et al. 2007), and the resulting sequences from all six strains were 100% identical. The sequences of the representative strain JC23121001 were deposited into GenBank with accession numbers PP108247, PP066857, PP108248, PP066858, and PP066860, respectively. The maximum-likelihood phylogenetic tree clustered JC23121001 with P. brasiliense type strain IBSBF1692T (Nabhan et al. 2012). The pathogenicity test of six strains was carried out on the six-week-old leaf mustard (cv. Huarong) plants grown in the greenhouse by inoculating 10 µl of each bacterial suspension (108 CFU/ml) on needle-like wounds on the stem base of three healthy leaf mustard plants (Singh et al. 2013). Control plants were treated with sterile distilled water. After inoculation, the plants were incubated at 28°C and 90% relative humidity in a growth chamber. This trial was repeated three times. All inoculated mustard stems were slightly water-soaked after 24 hours and eventually developed into soft rot symptoms, consistent with the original symptoms observed. The control plants remained symptom-free. The strains were re-isolated from inoculated plants and re-identified as P. brasiliense by sequencing five housekeeping genes, thus fulfilling Koch's postulates. P. brasiliense has a broad host range and has been reported on other Brassica species, such as Bok choy (Brassica rapa var. chinensis) in China (Li et al. 2023). Soft rot of leaf mustard caused by Pectobacterium aroidearum has also been reported previously (Chu et al. 2023). To our knowledge, this is the first report of P. brasiliense causing soft rot on leaf mustard in China. The soft rot poses a significant threat to the local leaf mustard industry and requires further research into epidemiology and disease management options.

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.

Room-Temperature Preparation of Covalent Organic Framework Membrane for Nanofiltration.

The uniquely tunable nature of covalent organic frameworks (COFs), whose pore size and stability can be controlled by choosing diverse organic building blocks and linkage types, makes COFs potential candidates for the membrane separation. Therefore, the preparation of membranes with effective separation efficiency based on COFs has aroused great interest among researchers. Although solvothermal approach has been the most popular method for the preparation of COF membranes, fabricating COF membranes at room temperature (RT) will provide a simple and captivating strategy for separation membranes. Herein, a P-COF membrane on porous alumina substrate at RT, showing 99.7% rejection of rhodamine B and excellent water permeance up to 52 L m-2 h-1 bar-1 , which can effectively purify wastewater is successfully obtained. P-COF is directly grown on alumina to form the composite membrane, which enhances the mechanical strength of COF membrane and avoids the risk of damaging the membrane structure during the transfer process of self-standing membrane. Moreover, P-COF membrane is grown at RT, which is more energy efficient than the conventional solvothermal method. Thus, it is of great significance to obtain COF membranes with excellent nanofiltration performance in a simple and mild condition to alleviate environmental and energy concerns.

First Report of Fusarium oxysporum f. sp. apii race 4 Causing Fusarium Yellows on Celery in China.

Celery (Apium graveolens var. dulce), which belongs to the family Apiaceae, is one of the most widely cultivated vegetable crops in the world. During 2020 and 2021, celery plants with Fusarium yellows and root rot were observed in four approximately 0.3 ha sized fields located in Zhaili village (118°74'E, 36°67'N) of Shouguang city, Shandong province, China. Almost 50% of the plants were infected. Disease symptoms were comprised of wilting of outer-older leaves, overall stunted growth, rotted roots and stems, with eventual death of plants. A total of 7 diseased plants were collected from 4 fields and used for isolation and identification of the causal agent. Diseased root tissues were cut into 3 × 3 mm pieces from the edge of the rotting region, surface sterilized by soaking in 75% ethanol for 1 min, followed by three washes with sterile distilled water, and then placed on potato dextrose agar (PDA), and incubated at 28°C for 6 days in the dark. A total of 19 morphologically similar fungal isolates were obtained by single-spore subcultures. The colonies produced abundant, loosely floccose, white aerial mycelia and pale purple pigmentation on PDA. Microconidia were hyaline, zero to one septate, and ranged from 1.7 - 3.6 × 5.3 - 13.7 µm (n = 70). Macroconidia were falciform, hyaline, mostly four to five septate, and ranged from 2.2 - 4.2 × 12.4 - 45.4 µm in size (n = 70). These morphological characteristics were consistent with Fusarium oxysporum (Leslie and Summerell 2006). The genomic DNA of 19 isolates was extracted using the Plant Genomic DNA Kit (Tiangen, China). The translation elongation factor-1α (TEF-1α) and IGS rDNA regions were amplified with primers EF1/EF2 (O' Donnell et al. 1998) and iNL11/FoIGS-R (Epstein et al. 2017). BLAST analysis showed that 19 isolates were highly similar to Fusarium oxysporum, with 100% for TEF-1α (MN507109) and 99% for IGS rDNA (MT671188), respectively. The resulting 683-bp TEF-1α and 930-bp IGS rDNA sequences of isolate QC20091622 were deposited in GenBank with accession nos. ON260806 for TEF-1α and ON260805 for IGS rDNA, respectively. In a maximum-likelihood phylogenetic analysis based on TEF-1α and IGS rDNA sequences of F. oxysporum, using MEGAX software, isolate QC20091622 was grouped in the same clade with F. oxysporum f. sp. apii race 4, with a low bootstrap value of 54 between race 3 and race 4, indicating that the races are not distinguishable using only these two loci, as reported by Epstein et al (2022). Additional loci and other diagnostic methods are required to identify the race. Furthermore, the total DNA of 19 isolates was amplified by race-specific primers N4851-F/R (F. oxysporum f. sp. apii race 2) and N3875-2F/R (race 4), respectively (Epstein et al. 2017), and 187 bp product was amplified with primer pair N3875-2F/R, but none with primer pair N4851-F/R, so the isolates were identified as F. oxysporum f. sp. apii race 4. Pathogenicity of the 19 isolates was tested on potted celery plants (cv. 'Baimiao'). Ten healthy 6-week-old celery plants were inoculated by dipping the roots in a conidial suspension (107 conidia/mL) for 30 min. Control plants were dipped in sterile distilled water. The plants were then grown in a greenhouse maintained at 15°C (night)/26°C (day) and 90% relative humidity with natural daylight. The pathogenicity test was repeated twice. All inoculated plants started to wilt and developed root rot symptoms 14 days later, which were similar to those observed in the fields. The control plants remained healthy. F. oxysporum f. sp. apii race 4 was reisolated from the symptomatic roots, and their identity was confirmed by PCR, fulfilling Koch's postulates. To our knowledge, this is the first report of F. oxysporum f. sp. apii race 4 causing root rot on celery in China. F. oxysporum f. sp. apii race 4 has been a destructive pathogen in celery, prevention and control measures should be considered.

A Rapid Molecular Detection System for Sdh Mutations Conferring Differential Succinate Dehydrogenase Inhibitor Resistance in Corynespora cassiicola.

Cucumber leaf spot, caused by Corynespora cassiicola, is a serious disease of cucumbers in greenhouses. Due to the frequent application of succinate dehydrogenase inhibitors (SDHIs), resistance caused by point mutations in the SDHB/C/D gene has been reported. Different mutations lead to different resistance levels, and mutations vary over time and regions. This means that it is necessary to know the type of mutation in the field to select the appropriate SDHIs. Here, the sensitivity of mutations to SDHIs was determined, and eight resistance patterns were obtained: pattern I (BosVHR, FluoMR, PenHR, CarR); pattern II (BosMR, FluoSS, PenS, CarS); pattern III (BosVHR, FluoSS, PenLR, CarS); pattern IV (BosLR, FluoLR, PenS, CarR); pattern V (BosMR, FluoLR, PenS, CarS); pattern VI (BosMR, FluoLR, PenLR, CarS); pattern VII (BosVHR, FluoHR, PenHR, CarS); and pattern VIII (BosLR, FluoLR, PenLR, CarS). We successfully established nine allele-specific PCR (AS-PCR) assays that can detect mutation types. The sensitivity and specificity of AS-PCR were also determined. The sensitivity results showed that most of the detection thresholds of the AS-PCR assays were 100 pg/µl, while the AS-PCR assay of the B-H278R and D-G109V mutations exhibited high sensitivity, with 10 pg/µl. To validate the use of the developed AS-PCR assay, DNA from leaves inoculated with known mutations was extracted, detected by AS-PCR, and sequenced. The results showed good similarity between the two methods. Additionally, to rapidly detect mutations in the CcSdhD gene, we developed a single-tube multiplex allele-specific PCR (MAS-PCR) assay. In conclusion, AS-PCR and MAS-PCR were established for mutation detection and targeted control of CLS.

Selection and evaluation of suitable reference genes for quantitative gene expression analysis during infection of Cucumis sativus with Pectobacterium brasiliense.

AIMS: Bacterial soft rot caused by Pectobacterium brasiliense (Pbr) has resulted in severe economic losses of cucumber production in northern China. Quantitative reverse transcription PCR (RT-qPCR) is widely used to determine the fold change in the expression of genes of interest, and an appropriate reference gene played a critical role in the evaluation of genes expression. However, the suitable reference genes for transcript normalization during the interaction between cucumber and Pbr have not yet been systematically validated. In this study, we aimed to identify the suitable reference genes for accurate and reliable normalization of cucumber and Pbr RT-qPCR data. METHODS AND RESULTS: We selected 14 candidate reference genes for cucumber and 10 candidate reference genes for Pbr were analysed by using four algorithms (the deltaCt method, BestKeeper, NormFinder and geNorm). Furthermore, five genes in cucumber involved in plant resistance and five genes in Pbr related to the virulence were selected to confirm the reliability of the reference genes by RT-qPCR. CsARF (ADP-ribosylation factor 1) and pgi (glucose-6-phosphate isomerase) were suggested as the most suitable reference genes for cucumber and Pbr respectively. CONCLUSION: Our results suggested that CsARF (ADP-ribosylation factor 1) and pgi (glucose-6-phosphate isomerase) could be as the reference genes to normalize expression data for cucumber and Pbr during the process of pathogen-host interaction respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first systematic study of the optimal reference genes specific to cucumber and Pbr, which could help advance the molecular interactions research in Cucurbitaceae vegetables and Pectobacterium species pathosystems.

Rapidly Increasing Boscalid Resistance in Corynespora cassiicola in China.

Corynespora leaf spot caused by Corynespora cassiicola is an important foliar disease in cucumber. Succinate dehydrogenase inhibitors are the main fungicides used to control this disease. With the application of succinate dehydrogenase inhibitors (SDHIs) in the field, boscalid-resistant isolates have been continuously detected in the field. Resistance monitoring programs were performed to investigate the frequency and genotypes of resistant isolates. In our resistance monitoring, the frequency of resistant isolates rapidly increased from 9.68 to 85.88% in 2005 to 2020. Nine genotypes conferring SDHI resistance were found in resistant isolates, with different levels of resistance to SDHIs: B-H278R, B-H278L, B-H278Y, B-I280V, C-N75S, C-S73P, D-D95E, D-H105R, and D-G109V. The first sdh mutation was detected in Hebei Province in China, conferring an amino acid substitution at codon 278 in the sdhB subunit from histidine to tyrosine (B-H278Y), and it was the dominant resistance genotype in 2014 to 2015. Subsequently, other genotypes were gradually detected in the field, and the dominant mutations varied across years and across regions. The newest genotype (B-H278L) conferring SDHI resistance was found in 2020. To the best of our knowledge, this is the first report of C. cassiicola in cucumber. To date, multiple resistance to SDHIs, quinone outside inhibitors, benzimidazole fungicides, and dicarboximide fungicides have been detected, accounting for 75.64% of SDHI-resistant isolates. Therefore, the above four fungicides must be strictly restricted, and further monitoring work in other provinces with more isolates should be performed in the future.

First Report of Fusarium solani Species Complex Causing Vascular Wilt on Cauliflower in China.

Cauliflower (Brassica oleracea var. botrytis L.), which belongs to the family Cruciferae, is a cool-season vegetable with green leaves around a large hard white head of flowers. China is the leading cauliflower and broccoli producing country in the world, with approximately 10.71 MT production (FAOSTAT 2019). During September 2018 to July 2019, wilting symptoms were observed on cauliflower in several commercial fields, with approximately 45% to 65% disease incidence in Shen county (115°48'E, 35°98N) of Liaocheng city, Shandong province, China. Plant stunting, leaves yellowing and wilting, and dark brown, hollow appearance of vascular stem tissues were the symptoms prominently observed. To isolate the causal organism, nine symptomatic tissues were collected and cut into small pieces (5 × 5 mm), disinfected in 75% ethanol for 30 s, rinsed three times in sterile water, transferred onto potato dextrose agar (PDA) medium. The plates were then incubated in air-conditioned room at 26°C with an artificial 12 h light-dark cycle provided by incandescent lamp. In total, 15 single-spore isolates were obtained and morphological characterization of 15 isolates was done on both PDA and carnation leaf agar (CLA; Leslie and Summerell 2006). The mycelia on PDA were initially white, fluffy, later becoming brown, and the underside of the colonies were light brown. Typical macroconidia were abundant on CLA. Macroconidia were hyaline, slightly curved, one to five septa, both ends were smooth, measuring 3.7 to 6.4 µm × 23.7 to 38.1 µm (n = 40). Microconidia were oval to cylindrical, hyaline, zero to one septate, measuring 2.0 to 4.1 × 4.3 to 10.3 µm (n = 40). Chlamydospores were terminal or intercalary, solitary or in pairs, globose to oval, thick wall, smooth or rough, 6.3 to 9.8 µm. Based on morphological characteristics, all of the 15 isolates were identified as Fusarium solani (Leslie and Summerell 2006). The isolates were further identified based on PCR amplification. The ITS, mtSSU, EF-1α and RPB2 genes were amplified using primers ITS1/ITS4, NSM1/NSM2 (Li et al. 1994), EF-1 Ha/EF-2Tb (O'Donnell et al. 1998) and RPB2-5F2/fRPB2-7cR (O'Donnell et al. 2008). BLAST analysis showed that 15 isolates were highly similar to F. solani species complex, with 100% similarity for ITS (AB470904.1), mtSSU (KF125009.1), EF-1α (KF372878.1), and RPB2 (MK048113.1), respectively. The sequences of isolate HYC1410080102 had been deposited in GenBank with accessions MT378292.1 for ITS, MT383122.1 for mtSSU, OK595059.1 for EF-1α and OK595060.1 for RPB2, respectively. Pathogenicity of the 15 isolates were conducted on 4-true-leaf seedlings cv. Jinsong by dipping the roots into a conidial suspension (107 conidia/mL) for 10 min. The conidial was prepared from 7-day old cultures grown on CLA at 26°C and suspended in sterilized water. Control plants were dipped in sterile distilled water. All treated seedlings were planted in 5.0 cm diameter plastic pots containing pasteurized soil matrix. Then the plants were kept in a greenhouse at 15°C (night)/26°C (day) and 80%RH with natural daylight. Twelve days later, brown lesions appeared on stem bases in all inoculated cauliflowers, and finally, the plants wilted, similar to those observed in the field. The control plants remained healthy. Re-isolation of the infected tissues showed same morphological characteristics of F. solani as the original isolates, which were verified using PCR. To our knowledge, this is the first report of F. solani causing cauliflower wilt in China and the world (Farr and Rossman 2021). F. solani is a destructive pathogen with a broad host range worldwide and is responsible for significant crop losses, prevention and control measures should be considered.

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.

Site-directed mutagenesis of the succinate dehydrogenase subunits B and D from Corynespora cassiicola reveals different fitness costs and sensitivities to succinate dehydrogenase inhibitors.

Carboxamide fungicides target succinate dehydrogenase (SDH). Recently published monitoring studies have shown that Corynespora cassiicola isolates are resistant to one or several SDH inhibitors (SDHIs) with amino acid substitutions in the SDH B and D subunits. We confirmed, by site-directed mutagenesis of the sdhB and sdhD genes, that each of the mutations identified in the field strains of C. cassiicola conferred resistance to boscalid and, in some cases, cross-resistance to other SDHIs (fluopyram, carboxin and penthiopyrad). Analyses of the enzyme activity and sdhB and sdhD gene expression show that modifications (SdhB_H278Y and SdhD_H105R) that result in a decline in SDH enzyme activity may be complemented by gene overexpression. The SdhB_H278Y, SdhB_I280V and SdhD_H105R mutants suffered large fitness penalties based on their biological properties, including conidia production and germination, mycelial growth, pathogenicity or survival abilities under environment stress. However, fitness cost was not found in the SdhB_H278R, SdhD_D95E and SdhD_G109V mutants. In the evaluation of resistance to boscalid in 2018 and 2019, the frequency of the SdhD_D95E and SdhD_G109V genotypes in the Liaoning and Shandong provinces changed dramatically compared with 2005-2017, from low resistance frequency (0.53% for D95E and 2.53% for G109V) to dominant resistance frequency (17.28% for D95E and 15.38% for G109V). Considering both the fitness and increased frequency of these genotypes, we may infer that the SdhD_D95E and SdhD_G109V mutants will be the dominant resistance mutants in field.

First Report of Cercospora Leaf Spot Caused by Cercospora cf. flagellaris on Okra in China.

Okra [Abelmoschus esculentus (L.) Moench], which belongs to the family Malvaceae, is widely grown in the tropics, sub-tropics and warmer areas of the temperate zones for its immature seed pods which are consumed as a vegetable. In China, okra pods are consumed as not only vegetables but also as a traditional medicine to cure dental diseases and gastric ulcers. During September 2018 to June 2019, extensive spots on okra leaves were observed in several commercial fields (approximately 2.0 hectares), with disease incidence of approximately 25%~50% in the Yanqing District (115°98'E, 40°46'N) of Beijing, China. Symptoms of the disease initially appeared as small pale brown spots with yellow haloes. As the disease progressed, some spots gradually coalesced, forming larger irregular dark brown lesions. The centers of the lesions became grayish white. A total of 13 small fragments (3 to 5 mm) excised from the lesion margins were sterilized in 1% sodium hypochlorite (NaClO) for 1 min, followed by three washes with sterile distilled water, and then placed on potato dextrose agar (PDA) and incubated at 25°C in the dark for 5 days. In total, 21 cultures were obtained and purified by single-spore subcultures on PDA for morphological identification. The colonies on PDA were whitish to gray, with cottony aerial mycelium. Conidiophores were fasciculate, olivaceous brown, straight or geniculate, uniform in width, multiseptate, and ranged from 286/span> to 711 µm (avg. = 578 µm, n = 50). Conidia were hyaline, slightly curved or straight, needle shaped, truncate at the base, and terminal at the tip, 3-17-septate, and measuring 52 to 231 µm (avg. = 182 µm, n = 50). The morphological features were consistent with Cercospora cf. flagellaris Ellis & G. Martin (Groenewald et al. 2013). Pathogenicity tests were conducted on potted okra plants cv. 'Jiayuan'. Twenty four healthy okra plants at the true leaf stage were sprayed with conidial suspensions (1 × 106 conidia/mL), incubated at a glass cabinet maintained at 25°C and 90% relative humidity (RH). To each leaf approximately 10 mL of conidial suspension was applied. Plants sprayed with water were used as controls. Seven days later, dark brown spot, which were identical to those observed in the fields, were observed on inoculated leaves, whereas the control plants remained healthy. C. cf. flagellaris was reisolated from symptomatic leaves, confirming Koch's Postulates. Genomic DNA was extracted from fungal mycelium using the Plant Genomic DNA Kit (Tiangen Biotech Co. Ltd., Beijing, China). The nuclear ribosomal internal transcribed spacer region (ITS), and portions of the actin (ACT), histone H3 (HIS3), and translation elongation factor 1-α (TEF1) genes were amplified using primers ITS1/ITS4 (Groenewald et al. 2013), ACT-512F/ACT-783R (Carbone & Kohn 1999), CYLH3F/CYLH3R (Crous et al. 2006), and EF1-728F/EF1-986R (Carbone & Kohn 1999). The resulting 542 bp ITS, 226 bp ACT, 410 bp HIS3 and 306 bp TEF1 sequences of isolate QK14091813 were deposited in GeneBank (Accession nos. MT949700, MT949701, MT949702 and MT949703, respectively). The ITS, ACT, HIS3 and TEF1 sequences shared 99.42% to 100% identities to previously published sequences of C. cf. flagellaris (Accession nos. MN633275 for ITS, MF680960 for ACT, MK991295 for HIS3, and MK991292.1 for TEF1, respectively). Multi-locus phylogenetic analyses (ITS, ACT, HIS3, and TEF1) were performed by neighbor-joining method using MEGA 7.0. The resulting trees showed that C. cf. flagellaris isolate QK14091813 (this study) nested within the clade that includes other isolates of C. cf. flagellaris with a 99% confidence level. To our knowledge, this is the first report of C. cf. flagellaris causing leaf spot on okra (Farr and Rossman 2020). The pathogen has a worldwide distribution and an unusually broad host range, which can be of great significance, and the plant protection policy of priority to prevention and synthetical prevention should be followed.

First Report of Fusarium oxysporum Causing Wilt on Coriander in China.

Coriander (Coriandrum sativum L), which belongs to the family Apiaceae, is a medicinal and aromatic plant. In China, coriander is widely cultivated in several parts as a vegetable crop. During August 2019 to June 2020, wilting symptoms were observed on coriander (cv. 'Tiegan') in a commercial plantation, with disease incidence of approximately 25 to 40% in Xiajiawang village (118°88'E, 35°46'N) of Linyi city, Shandong province, China. Symptoms included wilting and leaf yellowing, plant stunting, root rot, and vascular discoloration of the stem bases and roots. A total of eight symptomatic plants were uprooted and collected from three fields. To determine the cause of the disease, symptomatic root tissues were excised, surface disinfected with 75% ethanol for 30s, followed by three washes with sterile distilled water, and then placed on potato dextrose agar (PDA) and incubated at 28°C for 6 days. In total, 10 cultures were obtained and purified by single-spore subcultures on PDA for morphological identification. The morphology of multiple colonies was consistent and originally white, later becoming light to dark purple in color with abundant aerial hyphae. Macroconidia were hyaline and falcate, straight to slightly curved, 3-4 septate, 27.86 to 34.23 × 4.07 to 6.13 µm (n = 30), with apical cells curved and basal cells foot-shaped. Microconidia were hyaline, oval or ellipsoid, 0-1 septate, with a flat base, measuring 5.67 to 9.37 × 3.66 to 5.40 µm (n = 30). These morphological characteristics resembled those of Fusarium oxysporum (Leslie and Summerell 2006). Genomic DNA was extracted from fungal mycelium using the Plant Genomic DNA Kit (Tiangen, China). The nuclear ribosomal internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α) and mitochondrial small subunit (mtSSU) genes were amplified with primer pairs ITS1/ITS4 (White et al. 1990), EF1Ha/EF2Tb (O' Donnell et al. 1998) and NMS1a/NMS2b (Li et al. 1994). The resulting ITS (550-bp), TEF1-α (681-bp) and mtSSU (692-bp) sequences of isolate QC20091601 were deposited in GenBank (accession nos. MW900439, MW692008 and MW711738, respectively). BLAST analysis demonstrated 100% identities to the ITS, TEF-1α and mtSSU sequences of F. oxysporum (MN856370.1, MN507110.1 and MN386808.1), respectively. According to the morphological and molecular identification, the fungus was identified as F. oxysporum. In the pathogenicity test, healthy coriander plants (cv. 'Tiegan') at the 4-true-leaf stage were inoculated by dipping the roots into a conidial suspension of 1 × 107 conidia/mL for 10 min. Plants dipped in sterile distilled water served as controls. All treated plants were placed in a greenhouse maintained at temperature 30°C and 80% relative humidity. Ten days later, inoculated plants developed typical symptoms of leaf yellowing, wilting and vascular discoloration, which were identical to those observed in the fields, whereas the control plants remained healthy. F.oxysporum was reisolated from the symptomatic roots, and its identity was confirmed by PCR with the primes described above, fulfilling Koch's postulates. To our knowledge, this is the first report of F. oxysporum as a pathogen on coriander in China. F. oxysporum is a destructive plant pathogen with an unusually broad host range and worldwide distribution, prevention and control measures should be taken in advance.

The complete genome sequence of Rahnella aquatilis ZF7 reveals potential beneficial properties and stress tolerance capabilities.

Rahnella aquatilis ZF7 is a plant beneficial strain isolated from Sakura tree soil with potential for biocontrol. Here, we present the complete genome sequence of R. aquatilis ZF7, which consists of one 4.49 Mb circular chromosome and a 54-kb plasmid named pRAZF7. Phylogenetic analyses revealed that R. aquatilis ZF7 is much similar to the strains Rahnella sp. Y9602 and R. aquatilis HX2 than others evaluated. In this study, multiple genes encoding functions that likely contribute to plant growth promotion, biocontrol and stress tolerance were identified by comparative genome analyses, including IAA production, phosphate solubilization, antibiotic resistance and formation of Se nanoparticles (SeNPs). In addition, these functions were also confirmed by in vitro experiments. Considering its ability to form SeNPs, strain R. aquatilis ZF7 will contribute to nano-agriculture. Overall, the features of R. aquatilis ZF7 make it a high potential and competitive strain in biocontrol, and the genome data will help further studies on the mechanisms of plant growth promotion and biocontrol.

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.

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.

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.

Comparative genomic analysis of Pseudomonas amygdali pv. lachrymans NM002: Insights into its potential virulence genes and putative invasion determinants.

Pseudomonas amygdali pv. lachrymans is currently of important plant pathogenic bacteria that causes cucumber angular leaf spot worldwide. The pathogen has been studied for its roles in pathogenicity and plant inheritance resistance. To further delineate traits critical to virulence, invasion and survival in the phyllosphere, we reported the first complete genome of P. amygdali pv. lachrymans NM002. Analysis of the whole genome in comparison with three closely-related representative pathovars of P. syringae identified the conservation of virulence genes, including flagella and chemotaxis, quorum-sensing systems, two-component systems, and lipopolysaccharide and antiphagocytosis. It also revealed differences of invasion determinants, such as type III effectors, phytotoxin (coronatine, syringomycin and phaseolotoxin) and cell wall-degrading enzyme, which may contribute to infectivity. The aim of this study was to derive genomic information that would reveal the probable molecular mechanisms underlying the virulence, infectivity and provide a better understanding of the pathogenesis of the P. syringae pathovars.