First Report of Bacterial Leaf Spot on Muskmelon Caused by Pseudomonas syringae pv. syringae in China.

Muskmelon (Cucumis melo L.) is one of the most widely cultivated and economically important fruit crops in the world. In January 2023, muskmelon leaves of cultivar 'Sheng Gu' were observed with irregularly shaped spots in four nurseries in Wanxiang Village, Pudong District of Shanghai, China. Initial symptoms were irregular soaking on the leaves, which progressed to rotting and necrotic spots. The disease incidence of melon seedlings in different nurseries ranged from 10 to 25%. To isolate and identify the causal agent, the small pieces of lesion tissues (5×5 mm) from symptomatic leaves were sterilized in 75% ethanol for 30 s and rinsed three times with sterile water. Following that, tissues were crushed with sterile glass rod in a sterile 2.0 mL centrifuge tube containing 100 µl of sterile water. The suspension was serially diluted before being spread on Luria-Bertani (LB) medium. After 48 h of incubation at 28°C, the cream-colored bacterial colonies from the 10-4 dilution were tiny and purified by streaking on new LB plates. To confirm the species identity of the bacterial isolates, genomic DNA was extracted from four independent representative colonies from different diseased plants, and several conserved genes were amplified and sequenced, including the 16S rRNA gene with primers 27F/1492R, gyrB gene with primers gyrBFor2/gyrBRev2, and rpoD gene with primers rpoDFor2/rpoDRev2 (Lelliot et al. 1966; Murillo et al. 2011). The results showed that the four colonies were identical. Using BLAST analysis in GenBank, the 16S rDNA (accession no. OQ659765, 1,402 bp), the gyrB (accession no. OQ708618, 911 bp), and rpoD sequences (accession no. OQ708619, 798 bp) showed 99.86-100% homology with 99-100% coveage as the corresponding gene sequences in the P. syringae pv. syringae strain HS191 (accession no. CP006256.1). The bacterial isolate was designated as P. syringae pv. syringae strain PDTG. Phylogenetic tree analysis of 16S rDNA, gyrB and rpoD genes further verified that the bacteria isolate was in close proximity to P. syringae pv. syringae. Additionally, all four isolates were detected in PCR with P. syringae pv. syringae specific primers, PsyF/ PsyR (Borschinger et al. 2016; Guilbaud et al. 2016). Ten two weeks old healthy 'Sheng Gu' muskmelon seedlings were inoculated by spraying with a bacterial suspension of 108 CFU/ml, and ten additional healthy plants treated with sterilized water served as the control. The inoculated plants were maintained at 25°C and 75% relative humidity for 7 days in artificial climate room. Water-soaked rot, similar as those seen in the nurseries, appeared on leaves 7 days after inoculation (dai), while the leaves of control plants remained healthy. The bacteria were re-isolated from rot of inoculated leaves and confirmed as the original pathogen by PCR with the PsyF/ PsyR primers and the 16S rRNA gene sequences. To our knowledge, this is the first report of P. syringae pv. syringae causing bacterial leaf spot on muskmelon in China, and this report expands the host range of P. syringae pv. syringae.

Occurrence of Tomato Leaf Curl New Delhi Virus in Cucurbit Plants in China.

In August 2022, melon (Cucumis melo), cucumber (Cucumis sativus) and luffa (Luffa aegyptiaca) plants showed virus-like symptoms characteristic of geminiviruses (yellowish green, mosaic patterns and severe curling of leaves, short internodes, and stunting) in 10 greenhouses in Fengxian district and 20 greenhoues in Jiading district of Shanghai, China. Fifty symptomatic leaf samples were randomly collected: 28 from melon, 17 from cucumber, and 5 from luffa. To investigate the etiology of the observed disease, total DNA and RNA was extracted via a DNA extraction kit (Tiangen, Not: DP350) and TRIzol reagent (Sigma-Aldrich, Not: T9424), respectively. Healthy melon plants grown in a growth chamber served as negative control. The DNA and RNA samples were screened for the presence of geminiviruses, Cucurbit chlorotic yellow virus (CCYV), Melon yellow spot virus (MYSV), Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV) and Cucumber green mottle mosaic virus (CGMMV) through PCR or RT‒PCR with geminiviruses (Deng et al. 1994), CCYV, MYSV, CMV, ZYMV and CGMMV (Zeng et al. 2011, 2019) primers. The PCR results showed that 28 melon leaves and 17 cucumber leaves were positive for geminivirus and CCYV, respectively, 5 luffa samples were infected with only geminivirus, and virus was not detected from the healthy plants. These results indicate that these two viruses are widely distributed throughout cucurbit crops in Shanghai, China. All the geminiviruses sequences (approximately 510 bp) were quite similar to each other and were most similar (99.4%) to the Tomato leaf curl New Delhi virus (ToLCNDV) sequence (GenBank Accession No. OP356207) (Li et al. 2022). To confirm the presence of geminiviruses, the segments of DNA-A and DNA-B were amplified by PCR with 4 ToLCNDV-specific primer sets (Mizutani et al. 2011) and sequenced from 10 samples (4 melon, 4 cucumber and 2 luffa). Both DNA-A and DNA-B of the ToLCNDV sequences and features were deposited in GenBank under the accession numbers OQ190939-OQ190948 (DNA-A, 2739 nt) and OQ190949-OQ190958 (DNA-B, 2693 nt). BLASTn analysis of Shanghai isolates of ToLCNDV (DNA-A and DNA-B) showed that the sequences shared nucleotide identities ranged from 99.3% to 100% among them and with values of more than 99.4% nucleotide identity with ToLCNDV isolates from tomato in China (OP356207 and OP356208) (Li et al. 2022). To confirm the virus infection, we have successfully constructed an infectious clone for 0823-1 isolate in the binary plasmid and inoculated melon with and without an infectious clone. The melon plants inoculated with ToLCNDV 15 dpi showed the high accumulation of the virus and displayed symptoms similar to viruses in greenhouse. Based on the complete sequences, results of the molecular phylogenetic analysis (Fig. 2) and infectious clone, these geminiviruses were identified as ToLCNDV. ToLCNDV has been reported to occur and spread by the whitefly (Bemisia tabaci) in many Asian countries (Sohrab et al. 2003; Sohrab et al. 2011; Aamir et al. 2020) and Europe (Juárez et al. 2014; Ruiz et al. 2015; Luigi et al. 2019). Large populations of whiteflies were also present in all our surveyed areas. However, to our knowledge, this is the first report of the occurrence of ToLCNDV in cucurbit plants in China. The presence of ToLCNDV and CCYV can cause severe losses in crop yields or even crop failure. In addition to TYLCV, ToLCNDV is another major geminivirus-induced disease threatening cucurbit and other vegetable production in China.

Resistance Profiles of Botrytis cinerea to Fluxapyroxad from Strawberry Fields in Shanghai, China.

Fluxapyroxad, a type of succinate dehydrogenase inhibitor fungicide, has been used to control the growth of gray mold on strawberry for more than 5 years in China. Selection for resistance to the causal agent Botrytis cinerea became a threat to the efficacy of fluxapyroxad. In total, 160 B. cinerea isolates collected from Shanghai during 2020 and 2021 were tested for their resistance to fluxapyroxad using mycelial growth inhibition. The results indicated that the curve of baseline sensitivity was unimodal, with an approximately normal distribution and a mean effective concentration of the fungicide that inhibited growth by 50% (EC50) of 0.18 ± 0.01 µg/ml. In total, 27.78 and 48.57% of isolates were resistant to fluxapyroxad in 2020 and 2021, respectively, where the lowest EC50 value of mycelial growth inhibition was 0.03 µg/ml and the highest value was 51.3 µg/ml. Resistance mechanism analysis showed that the succinate dehydrogenase subunit B (SdhB) N230I mutation could lead to resistance and P225F mutation could lead to higher resistance. These data suggest that the resistance frequency of B. cinerea isolates to fluxapyroxad increased in 2021 compared with 2020, which requires appropriate fungicide rotation strategies to be implemented in order to control gray mold on strawberry in the future.

First Report of Leaf Spot and Blight on Crown Daisy Caused by Cercospora apii in China.

Crown daisy (Glebionis coronaria L.), also known as chrysanthemum greens, is a popular vegetable in Asia, especially in China. The leaves have been used in folk medicine as a tonic for the liver, blood, intestines and to control anemia and high blood pressure. In November 2020, severe leaf spot and blight was observed with 80% to 95% incidence on crown daisy growing in greenhouses in Fengxian, Shanghai, China (121°22'E, 30°53'N). Irregular rounded spots appeared with a light gray center and water-soaked margins. Round lesions enlarged and merged with age, followed by the development of a necrotic area resulting in the typical "frog-eye" and causing a continuous deterioration of crown daisy. Diseased leaves were washed in running water for 30 min. Small fragments (5 × 5 mm) taken from the margin of lesions were disinfected with 1% NaClO for 3 min, rinsed three times with sterile water, cultured on potato sucrose agar (PSA) augmented with 50 mg streptomycin/liter at 26 oC,and incubated in the dark. Colonies had identical morphology, and TH11290202 was selected and deposited in the plant pathology lab of Shanghai Academy of Agricultural Sciences. Mycelium was initially cottony and white and became appressed to the medium and dark brown with time. Conidia did not form on any media, including PSA, PDA, V8 agar (V8A), maize leaf carbonate agar (MLPCA), pepper leaf carbonate agar (PLPCA), etc. To confirm the identity of the pathogen, genomic DNA was extracted from TH11290202 with the cetyltrimethylammonium ammonium bromide (CTAB) method from the mycelia. Five loci were PCR amplified, namely, the internal transcribed spacer (ITS), translation elongation factor (TEF), calmodulin (cmdA), histone (H3) and actin (ACT), using primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Jaklitsch et al. 2005), CAL-F/CAL-R (O'Donnell et al. 2000), cylh3f/cylh3r (Glass and Donaldson 1995), and ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. The resulting sequences were deposited in GenBank (MW819910, MW981277, MW981278, ON798723, and MW981279). Analysis of the ITS, TEF, cmdA, H3 and ACT gene sequences of isolate TH11290202 revealed that it was a member of the genus Cercospora, sharing 99.79%, 99.66%, 98.10% 99.74% and 100% sequence similarity with type strain of Cercospora apii CBS 116455. A multilocus phylogenetic analysis was performed using sequences from other closely related taxa obtained from GenBank. Based on morphological and molecular characteristics, TH11290202 was identified as C. apii (Crous and Braun 2003; Groenewald et al. 2006; Milosavljevic et al. 2014). To confirm pathogenicity, Koch's postulates were fulfilled on 30 mature plants, which were maintained in a growth chamber (at 26 °C, relative humidity 90%, 12/12 h light/dark). Surface-sterilized leaves were sprayed with a mycelial suspension. Brown lesions were formed 7 days after inoculation on 15 plants, whereas the noninoculated controls remained asymptomatic on the other 15 plants. To our knowledge, this is the first report of C. apii causing leaf spot and blight on G. coronaria in China and will provide useful information for developing effective control strategies.

First Report of Pectobacterium brasiliense causing Soft Rot on Conophytum luiseae in China.

Conophytum luiseae is native to the Namaqualand region of Cape, South Africa. It is a lovely plant with many short succulent spines on ingot-shaped fleshy leaf surfaces, and a high-value ornamental plant in China. In August to October 2021, a serious soft rot disease on Conophytum luiseae plants was observed in four greenhouses at a horticultural farm in Songjiang District, Shanghai, China. 70% of Conophytum luiseae plants on this farm had severe rot symptoms. Initially, wilting and soft rot symptoms appeared on fleshy leaves, then progressed into browning and withering symptoms of all fleshy leaves. To isolate and identify the causal agent, small pieces of lesion tissues were sterilized by 75% ethanol for 30 s, and rinsed three times with sterile water. Later, the tissues were crushed in sterile 2.0 mL centrifuge tube with 100 µl of sterile water. The suspension was serially diluted and spread on Luria-Bertani agar (LB) medium. After incubation at 28°C for 48 h, the bacterial colonies were tiny and streaked on LB plate for purification. After purification, five independent representative colonies were used for further confirmation. Genomic DNA from the bacterial isolate was extracted and used as the template to amplify 16s rDNA with primers 27F/1492R (Ying et al. 2012) and the housekeeping genes, dnaX with primers dnaXF/ dnaXR (Slawiak et al. 2009), and leuS with primers leuSF/ leuSR (Portier et al. 2019), respectively, by a polymerase chain reaction (PCR). The 16S rRNA sequences of one bacterial isolate was deposited in GenBank (GenBank accession OM333246) and showed a 99% similarity to that of Pectobacterium brasiliense (syn. Pectobacterium carotovorum subsp. brasiliense, Pcb) strains HG1501090309 (KU997683), BC1(CP009769), KC08 (KY021029). The dnaX (OM320998) and leuS (OM321306) sequences showed high similarity (> 99%) to P. brasiliense sequences. To further validate this identification, Pcb-specific primers BR1f/L1r was used for PCR, and it produced a predicted amplicon of 322 bp expected for P. brasiliense (Duarte et al. 2004). All five isolates could be detected by BR1f/L1r primer. To fulfill Koch's postulates, five healthy Conophytum luiseae were inoculated by spraying bacterial inoculum (108 CFU/ml), meanwhile five additional healthy Conophytum luiseae were implemented with sterilized distilled water as a negative control. The plants were then kept at 70% humidity and 25ºC. Seven days after inoculation, the inoculated plants showed serious soft rot, while the control samples remained healthy. Bacteria were re-isolated from rot of inoculated tissues, and the isolates were identified as the original pathogen by the 16S rRNA gene sequences. P. Brasiliense has been reported to cause soft rot on diverse plant hosts, like sweet potato, radish, tobacco (Liu et al. 2019; Voronina et al., 2019; Wang et al., 2017). Best to our knowledge, this is the first report that P. Brasiliense causes soft rot on Conophytum luiseae in China.

Baseline sensitivity of Rhizoctonia solani to four DMI fungicides.

A set of 78 Rhizoctonia solani isolates from Shanghai, China, were examined for their sensitivity in vitro to four demethylase inhibitor (DMI) fungicides, epoxiconazole, difenoconazole, hexaconazole, and tebuconazole, by the mycelial growth rate method. The isolates were very sensitive to the four DMI fungicides. The ranges and means of the EC50 values were 0.004-0.045 and 0.014 ± 0.007 µg ml-1 for epoxiconazole, 0-0.103 and 0.010 ± 0.016 µg ml-1 for difenoconazole, 0.001-0.078 and 0.011 ± 0.013 µg ml-1 for hexaconazole, and 0.001-0.210 and 0.034 ± 0.035 µg ml-1 for tebuconazole, respectively. The frequency distribution of the EC50 values showed a normal curve for epoxiconazole and an approximately unimodal curve for difenoconazole, hexaconazole, and tebuconazole. There was no correlation between the sensitivities of the R. solani population to any two of the four fungicides (r < 0.3). In field trials, in 2015 and 2016, the control efficacies of hexaconzole, epoxiconazole, and tebuconazole were >90.0% at 75 g.a.i. ha-1 , 80.0%-90.0% at 75 g.a.i. ha-1 and 80.0%-90.0% at 97 g.a.i. ha-1 , respectively; these values are significantly higher (p < 0.05) than those obtained in this study for difenoconazole (65.0%-70.0% at 80 g.a.i. ha-1 ) and jinggangmycin (68.0%-71.0% at 150 g.a.i. ha-1 ). These results show that sensitivity data can be used to monitor and detect any variations in the sensitivity of the R. solani population to these four DMI fungicides, and demonstrate that epoxiconazole, hexaconazole, and tebuconazole are likely to be good alternatives to jinggangmycin for controlling rice sheath blight.

First Report of Strawberry Crown Rot Caused by Xanthomonas fragariae in China.

Strawberry (Fragaria × ananassa Duch.) is a kind of fruit with great economic importance and widely cultivated in the world. From 2019 to 2020, a serious crown rot disease was sporadically observed in several strawberry cultivars including 'Zhang Ji', 'Hong Yan' and 'Yue Xiu' in Shanghai, China. Initially, water-soaked rot appeared in inner tissue of strawberry crown, then progressed into browning and hollowing symptoms accompanied with yellow discolorations of young leaves. To isolate and identify the causal agent, small pieces of tissue taken from ten diseased crowns were sterilized by 70% alcohol. The cut-up pieces were macerated and serially diluted. The dilutions were placed on nutrient agar (NA) medium. After incubation at 25°C for 4-5 days, the yellow bacterial colonies were tiny and were streaked on NA plate for purification. The colonies were yellow, mucoid, smooth-margined, and five independent representative colonies were used for further confirmation. To confirm the species identity of the bacterial, genomic DNA was extracted from the five representative isolates, and 16S rRNA gene was amplified and sequenced using universal primers 27F/1492R. The 16S rRNA sequence was deposited in GenBank (MW725235) and showed 99% nucleotide similarity with Xanthomonas fragariae strain LMG 708 (NR_026318). The isolate's identity was further confirmed by X. fragariae-specific primers XF9/XF12 (Roberts et al. 1996). All five isolates could be detected by XF9/XF12 primer. To confirm Koch's postulates, five healthy strawberry plants were placed in 1000 ml glass beakers by submerging the cutting wound in 50 ml the bacterial suspension of 108 CFU/ml. Five additional strawberry plants treated with sterilized water served as a control. The beakers containing inoculated plants were sealed with plastic film at 25°C. Water-soaked rot appeared on internal tissue of crown similar to those observed in the field within 10-12 days after inoculation, while the control samples remained healthy. The bacteria were re-isolated from rot of inoculated crowns, and confirmed by X. fragariae-specific primers XF9/XF12. X. fragariae has been reported to cause angular leaf spot on strawberry in China (Wang et al. 2017; Wu et al., 2020). It's also found that X. fragariae could systematically infect crown tissue (Milholland et al. 1996; Mahuku and Goodwin, 1997). To our knowledge, this is the first report of X. fragariae causing strawberry crown rot in China. This report increased our understanding of X. fragariae, and showed that the spread of this disease might seriously threaten the development of strawberry industry in the future.

Genome sequence and spore germination-associated transcriptome analysis of Corynespora cassiicola from cucumber.

BACKGROUND: Corynespora cassiicola, as a necrotrophic phytopathogenic ascomycetous fungus, can infect hundreds of species of plants and rarely causes human diseases. This pathogen infects cucumber species and causes cucumber target spot, which has recently caused large cucumber yield losses in China. Genome sequence and spore germination-associated transcriptome analysis will contribute to the understanding of the molecular mechanism of pathogenicity and spore germination of C. cassiicola. RESULTS: First, we reported the draft genome sequences of the cucumber-sampled C. cassiicola isolate HGCC with high virulence. Although conspecific, HGCC exhibited distinct genome sequence differences from a rubber tree-sampled isolate (CCP) and a human-sampled isolate (UM591). The proportion of secreted proteins was 7.2% in HGCC. A total of 28.9% (4232) of HGCC genes, 29.5% (4298) of CCP genes and 28.6% (4214) of UM591 genes were highly homologous to experimentally proven virulence-associated genes, respectively, which were not significantly different (P = 0.866) from the average (29.7%) of 10 other phytopathogenic fungi. Thousands of putative virulence-associated genes in various pathways or families were identified in C. cassiicola. Second, a global view of the transcriptome of C. cassiicola spores during germination was evaluated using RNA sequencing (RNA-Seq). A total of 3288 differentially expressed genes (DEGs) were identified. The majority of KEGG-annotated DEGs were involved in metabolism, genetic information processing, cellular processes, the organismal system, human diseases and environmental information processing. CONCLUSIONS: These results facilitate the exploration of the molecular pathogenic mechanism of C. cassiicola in cucumbers and the understanding of molecular and cellular processes during spore germination.

Pathogen Infection and Host-Resistance Interactively Affect Root-Associated Fungal Communities in Watermelon.

Interactions of pathogen infection, host plant resistance, and fungal communities are poorly understood. Although the use of resistant watermelon cultivars is an effective control measure of watermelon wilt disease, fungal communities may also have significant effects on the development of the soil-borne pathogen complexes. We characterized the root and rhizosphere fungal communities associated with healthy and diseased watermelons of three different cultivars with different susceptibilities toward wilt disease by paired-end Illumina MiSeq sequencing. Thirty watermelon plants including highly wilt-resistant, moderately resistant, and susceptible cultivars were collected from a greenhouse, half of which showing clear wilt symptoms and the other half with no symptoms. Patterns of watermelon wilt disease and the response of the fungal communities varied among the three cultivars. The amount of the pathogen Fusarium oxysporum f. sp. niveum was higher in diseased root and rhizosphere samples, particularly in the susceptible cultivar, and was significantly positively correlated with the disease index of Fusarium wilt. Plant health had significant effects on root-associated fungal communities, whereas only the highly resistant cultivar had significant effects only on the rhizosphere fungal communities. Co-occurrence networks revealed a higher complexity of fungal communities in the symptom-free roots compared to diseased roots. In addition, networks from roots of the highly resistant plants showing symptoms had a higher complexity compared to the susceptible cultivars. Keystone species were identified for the plants with different symptom severity and the different cultivars in the root and rhizosphere, such as Fusarium oxysporum, Monosporascus cannonballus, and Mortierella alpina. Overall, the most important factor determining fungal communities in the roots was plant symptom severity, whereas in the rhizosphere, plant genotype was the most important factor determining fungal communities.

Rapid Detection of SdhB P225F and SdhB H272R Mutations in Boscalid Resistant Botrytis cinerea Strains by ARMS-PCR.

SdhB P225Fand SdhB H272R mutations have been found associated with boscalid resistance in Botrytis cinerea from strawberry in Shanghai, China. For rapid detection of two mutations, tetra-primers were designed and optimized to gain the relatively high accuracy and specificity based on the ARMS-PCR technique, by which resistance can be identified with different lengths of products on agarose gels. The tetra-primer ARMS-PCR systems for SdhB P225F and SdhB H272R were validated by 9 SdhB-squenced strains repeatedly. Then, sensitivity of 30 more strains were also tested by the methods, which were accordant with genotypes by sequencing and the sensitivity of conidial germination to boscalid by 100%. Thus, the methods developed in this study are proved to be rapid, inexpensive, accurate and practical for resistance detection of Botrytis cinerea caused by SdhB P225F and SdhB H272R mutations.

Correction to: Prediction of pathogenesis-related secreted proteins from Stemphylium lycopersici.

Following publication of the original article [1], we have been notified.

Rapid detection of Cucumber green mottle mosaic virus by reverse transcription recombinase polymerase amplification.

Reverse transcription recombinase polymerase amplification (RT-RPA) for detection of Cucumber green mottle mosaic virus (CGMMV) was developed in this study. It was found to be specific, with a limit of detection of 0.5 pg of total CGMMV RNA. CGMMV on inoculated leaves was tested using RT-RPA assay, suggesting that this method is suitable for CGMMV detection from leaves.

Prediction of pathogenesis-related secreted proteins from Stemphylium lycopersici.

BACKGROUND: Gray leaf spot is a devastating disease caused by Stemphylium lycopersici that threatens tomato-growing areas worldwide. Typically, many pathogenesis-related and unrelated secreted proteins can be predicted in genomes using bioinformatics and computer-based prediction algorithms, which help to elucidate the molecular mechanisms of pathogen-plant interactions. RESULTS: S. lycopersici-secreted proteins were predicted from 8997 proteins using a set of internet-based programs, including SignalP v4.1 TMHMM v2.0, big-PI Fungal Predictor, ProtComp V9.0 and TargetP v1.1. Analysis showed that 511 proteins are predicted to be secreted. These proteins vary from 51 to 600 residues in length, with signal peptides ranging from 14 to 30 residues in length. Functional analysis of differentially expressed proteins was performed using Blast2GO. Gene ontology analysis of 305 proteins classified them into 8 groups in biological process (BP), 6 groups in molecular function (MF), and 10 groups in cellular component (CC). Pathogen-host interaction (PHI) partners were predicted by performing BLASTp analysis of the predicted secreted proteins against the PHI database. In total, 159 secreted proteins in S. lycopersici might be involved in pathogenicity and virulence pathways. Scanning S. lycopersici-secreted proteins for the presence of carbohydrate-active enzyme (CAZyme)-coding gene homologs resulted in the prediction of 259 proteins. In addition, 12 of the 511 proteins predicted to be secreted are small cysteine-rich proteins (SCRPs). CONCLUSIONS: S. lycopersici secretory proteins have not yet been studied. The study of S. lycopersici genes predicted to encode secreted proteins is highly significant for research aimed at understanding the hypothesized roles of these proteins in host penetration, tissue necrosis, immune subversion and the identification of new targets for fungicides.