Full-length transcriptome and RNA-Seq analyses reveal the resistance mechanism of sesame in response to Corynespora cassiicola.

BACKGROUND: Corynespora leaf spot is a common leaf disease occurring in sesame, and the disease causes leaf yellowing and even shedding, which affects the growth quality of sesame. At present, the mechanism of sesame resistance to this disease is still unclear. Understanding the resistance mechanism of sesame to Corynespora leaf spot is highly important for the control of infection. In this study, the leaves of the sesame resistant variety (R) and the sesame susceptible variety (S) were collected at 0-48 hpi for transcriptome sequencing, and used a combined third-generation long-read and next-generation short-read technology approach to identify some key genes and main pathways related to resistance. RESULTS: The gene expression levels of the two sesame varieties were significantly different at 0, 6, 12, 24, 36 and 48 hpi, indicating that the up-regulation of differentially expressed genes in the R might enhanced the resistance. Moreover, combined with the phenotypic observations of sesame leaves inoculated at different time points, we found that 12 hpi was the key time point leading to the resistance difference between the two sesame varieties at the molecular level. The WGCNA identified two modules significantly associated with disease resistance, and screened out 10 key genes that were highly expressed in R but low expressed in S, which belonged to transcription factors (WRKY, AP2/ERF-ERF, and NAC types) and protein kinases (RLK-Pelle_DLSV, RLK-Pelle_SD-2b, and RLK-Pelle_WAK types). These genes could be the key response factors in the response of sesame to infection by Corynespora cassiicola. GO and KEGG enrichment analysis showed that specific modules could be enriched, which manifested as enrichment in biologically important pathways, such as plant signalling hormone transduction, plant-pathogen interaction, carbon metabolism, phenylpropanoid biosynthesis, glutathione metabolism, MAPK and other stress-related pathways. CONCLUSIONS: This study provides an important resource of genes contributing to disease resistance and will deepen our understanding of the regulation of disease resistance, paving the way for further molecular breeding of sesame.

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.

Resistance risk assessment of mefentrifluconazole in Corynespora cassiicola and the control of cucumber target spot by a two-way mixture of mefentrifluconazole and prochloraz.

The cucumber target spot, caused by Corynespora cassiicola, is a major cucumber disease in China. Mefentrifluconazole, a new triazole fungicide, exhibits remarkable efficacy in controlling cucumber target spot. However, the resistance risk and mechanism remain unclear. In this study, the inhibitory activity of mefentrifluconazole against 101 C. cassiicola isolates was determined, and the results indicated that the EC50 values ranged between 0.15 and 12.85 µg/mL, with a mean of 4.76 µg/mL. Fourteen mefentrifluconazole-resistant mutants of C. cassiicola were generated from six parental isolates in the laboratory through fungicide adaptation or UV irradiation. The resistance was relatively stable after ten consecutive transfers on a fungicide-free medium. No cross-resistance was observed between mefentrifluconazole and pyraclostrobin, fluopyram, prochloraz, mancozeb, or difenoconazole. Investigations into the biological characteristics of the resistant mutants revealed that six resistant mutants exhibited an enhanced compound fitness index (CFI) compared to the parental isolates, while others displayed a reduced or comparable CFI. The overexpression of CcCYP51A and CcCYP51B was detected in the resistant mutants, regardless of the presence or absence of mefentrifluconazole. Additionally, a two-way mixture of mefentrifluconazole and prochloraz at a concentration of 7:3 demonstrated superior control efficacy against the cucumber target spot, achieving a protection rate of 80%. In conclusion, this study suggests that the risk of C. cassiicola developing resistance to mefentrifluconazole is medium, and the overexpression of CcCYP51A and CcCYP51B might be associated with mefentrifluconazole resistance in C. cassiicola. The mefentrifluconazole and prochloraz two-way mixture presented promising control efficacy against the cucumber target spot.

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 Corynespora cassiicola causing leaf spot on Nanhaia speciosa in China.

Nanhaia speciosa, commonly known as Niudali, is a medicinal woody vine belonging to the Leguminosae family. Valued for its culinary and medicinal properties, it is extensively cultivated, covering approximately 5,973 hm2 in the Guangxi Zhuang Autonomous Region of China. The edible tubers of this plant are reported to possess antibacterial and antioxidant effects (Luo et al., 2023; Shu et al., 2020). In July 2021, a Niudali plantation in Yulin, Guangxi, China (22°64'N; 110°29'E) exhibited leaf spot symptoms, with an incidence rate exceeding 40% across a 46,690 m2 area. Initially, small circular, pale yellow spots appeared on the leaves, which subsequently evolved into dark brown lesions surrounded by yellow halos, ultimately leading to foliage wilting. Leaves exhibiting typical symptoms were collected for pathogen investigation. The leaves were thoroughly washed with sterile water and small tissue fragments (5×5 mm) were excised from the lesion periphery. These fragments were surface-sterilized with 75% ethanol and 1% NaClO, rinsed three times with sterile water, and subsequently cultured on potato dextrose agar (PDA) at 28 °C in darkness for 7 days. Through single-spore isolation, seven isolates with similar morphological traits were obtained. After 7 days of incubation on PDA at 28 °C in dark, the colonies exhibited a white to grey coloration on the upper surface with abundant aerial hyphae, while the underside appeared dark black. The conidia, cylindrical or obclavate in shape, were straight, pale brown, and measured 30.1-128.9 µm × 4.8-15.0 µm (n=50). The morphological characteristics matched those of Corynespora sp.(Wang et al. 2021). For molecular identification, the isolate N5-2 underwent DNA sequence analysis using genomic DNA and primers ITS1/ITS4 and EF1-688F/EF1-1251R. The sequences (ITS: OP550425; TEF1-α: OQ117118) were deposited in GenBank, exhibiting 98% identity to C. cassiicola (OP981637) for TEF1-α and 99% homology to C. cassiicola (OP957070) for ITS. Based on the concatenated ITS and TEF1-α, a maximum likelihood phylogenetic analyses using MEGA7.0 clustered the isolate with C. cassiicola. Consequently, the fungus was identified as C. cassiicola based on its morphological and molecular features. In the pathogenicity test on 1-year-old Nanhaia speciosa seedlings, leaves were gently scratched and inoculated with mycelial plugs (5 mm). Control seedlings received PDA plugs. Five leaves per plant and five plants per treatment were selected for assessment. All seedling were maintained in a greenhouse (12/12h light/dark cycle, 25 ± 2°C, 90% humidity). After a 7-day incubation period, all leaves subjected to fungal inoculation exhibited symptoms consistent with those observed in the field, while control plants remained symptom-free. The fungus was successfully reisolated from the infected leaves in three successive trials, fulfilling Koch's postulates. While C. cassiicola is well-documented for inducing leaf spots on various plant species, including Jasminum nudiflorum, Strobilanthes cusia, Acanthus ilicifolius, Syringa species (Hu et al., 2023; Liu et al., 2023; Xie et al., 2021; Wang et al., 2021), this study represents the first report of C. cassiicola causing leaf spots on Nanhaia speciosa in China. The identification of this pathogen in Nanhaia speciosa has significant implications for future epidemiological investigations and serves as a valuable reference for controlling leaf spot disease in Nanhaia speciosa.

Characterization of caffeoyl shikimate esterase gene family identifies CsCSE5 as a positive regulator of Podosphaera xanthii and Corynespora cassiicola pathogen resistance in cucumber.

KEY MESSAGE: CsCSE genes might be involved in the tolerance of cucumber to pathogens. Silencing of the CsCSE5 gene resulted in attenuated resistance of cucumber to Podosphaera xanthii and Corynespora cassiicola. Caffeoyl shikimate esterase (CSE), a key enzyme in the lignin biosynthetic pathway, has recently been characterized to play a key role in defense against pathogenic infection in plants. However, a systematic analysis of the CSE gene family in cucumber (Cucumis sativus) has not yet been conducted. Here, we identified eight CsCSE genes from the cucumber genome via bioinformatic analyses, and these genes were unevenly distributed on chromosomes 1, 3, 4, and 5. Results from multiple sequence alignment indicated that the CsCSE proteins had domains required for CSE activity. Phylogenetic analysis of gene structure and protein motifs revealed the conservation and diversity of the CsCSE gene family. Collinearity analysis showed that CsCSE genes had high homology with CSE genes in wax gourd (Benincasa hispida). Cis-acting element analysis of the promoters suggested that CsCSE genes might play important roles in growth, development, and stress tolerance. Expression pattern analysis indicated that CsCSE5 might be involved in regulating the resistance of cucumber to pathogens. Functional verification data confirmed that CsCSE5 positively regulates the resistance of cucumber to powdery mildew pathogen Podosphaera xanthii and target leaf spot pathogen Corynespora cassiicola. The results of our study provide information that will aid the genetic improvement of resistant cucumber varieties.

Risk assessment for resistance to fludioxonil in Corynespora cassiicola in Liaoning China.

Cucumber corynespora leaf spot, caused by Corynespora cassiicola, is the primary disease of cucumber leaves in greenhouses in China. Fludioxonil is a phenylpyrrole fungicide that inhibits C. cassiicola growth. We studied the sensitivity of 170 isolates of C. cassiicola to fludioxonil and evaluated resistance risk. All of the isolates were sensitive to fludioxonil. The EC50 values ranged from 0.082 to 0.539 µg/mL with a mean of 0.207 ± 0.0053 µg/mL. Laboratory-created mutants with a high resistance factor to fludioxonil were genetically stable after 10 transfers and showed positive cross-resistance to iprodione and procymidone but not to azoxystrobin, carbendazim, pydiflumetofen, and prochloraz. There was no significant difference in mycelial growth and temperature adaptation between the mutant s and the sensitive isolates, except for pathogenicity and sporulation. The resistant isolates accumulated less glycerol than their parental isolates and were more sensitive to osmotic stress. The histidine kinase activity of the sensitive isolates was significantly inhibited compared to that of the resistant mutants. Sequence alignment of the histidine kinase gene CCos revealed that the mutants RTL4, RXM5, and RFS102 had point mutations at different sites that resulted in amino acid changes at G934E, S739F, and A825P in the CCos protein. The mutant RFS102 had an alanine deletion at site 824. After fludioxonil treatment, CCos expression by RFS20 was significantly lower than that of the parental isolate. Our findings demonstrate that C. cassiicola exhibits moderate resistance to fludioxonil.

First report of target spot disease of strawberry caused by Corynespora cassiicola in Taiwan.

Strawberry (Fragaria x ananassa Duch.) is an important crop worldwide. Tontonaka, Aroma and Benihoppe, are most popular cultivars in Taiwan, especially cv. Aroma is dominated in the market. In September 2021, the target spot outbreak occurred on the leaves of cv. Aroma and Benihoppe in Nantou County. In a greenhouse, the target spot incidents were estimated at 90-100% and 40-50% in Aroma and Benihoppe, respectively, and caused 2~5% plants lost. Between April and June of 2022, the target spot occurred in another greenhouse where the target spot incidents were 90% and 5-8% in Aroma and Benihoppe, respectively. Early symptoms were small and circular to irregular brown spot on the leaves with its diameter at 1-2 mm. Then the brown lesion expanded to 2-5 mm in diameter with pale green halo. Some lesions appeared with gray center, and 2-3 spots might merge into one lesion, and some lesions were surrounded with yellow tissues later. The round to oval brown spots were also observed on stems. Ten symptomatic leaves and stems each were collected for pathogen isolation. Pieces of tissue from the edge of the brown lesion on leaf were cut and disinfested with 0.6% NaOCl for 30 sec, and rinsed three times with sterile distilled water (SDW) followed by being placed on 2% water agar. The isolates obtained from symptomatic leaves/stems of Aroma and Benihoppe showed same colonies with 100% isolation rate. Isolates from cv. Benihoppe (Cos21-1) and Aroma (Cos21-2) were selected for further observation and tests. Colonies on potato dextrose agar exhibited gray aerial mycelium at 28 °C in dark after 7-day. Conidiophores were brown, single or in clustered, unbranched, 2 to 11 septa. Conidia were 5.6-6.7× 28.1-270.0 µm (n=50) in size with obclavate to cylindrical shape, 1 to 16 septa, and olivaceous to dark brown. Based on the morphology, two fungal isolates were identified as Corynespora cassiicola. Four regions, internal transcribed spacer (ITS), ß-tubulin, translation elongation factor (TEF), and actin, were used to confirm the two isolates. Sequences of ITS and ß-tululin shared 100% identity to ITS (MZ093622) and ß-tululin (MW961419) of C. cassiicola in GenBank. Sequences of TEF and actin shared 99.60% and 99.70% identity to C. cassiicola (MK882240 and FJ853005), respectively. For the pathogenicity test, the conidial suspension (1x105 spores/ ml) of Cos21-1 and Cos21-2 was sprayed on leaves of two-month-old strawberry cv. Benihoppe and Aroma without wounds, respectively. Three plants each with more than two leaves were spray-inoculated with the selected isolates whereas three plants with SDW as controls and the test was repeated once. Inoculated plants were covered with plastic bags in the greenhouse, then removed when the initial symptoms were observed on leaves 5 days after inoculation whereas symptoms on stems were observed within 7 days. Re-isolation of the pathogens from the symptomatic leaves/stems demonstrated that the pathogen was C. cassiicola. The leaf spot or target spot caused by C. cassiicola on strawberry has been reported in Mainland China and North America. To our knowledge, this is the first report of target spot disease of strawberry caused by C. cassiicola in Taiwan.

Sensitivity to 12 Fungicides and Resistance Mechanism to Trifloxystrobin, Carbendazim, and Succinate Dehydrogenase Inhibitors in Cucumber Corynespora Leaf Spot (Corynespora cassiicola).

Corynespora cassiicola is the causal agent of cucumber Corynespora leaf spot, which affects many economically important plant species. Chemical control of this disease is hampered by the common development of fungicide resistance. In this study, 100 isolates from Liaoning Province were collected, and their sensitivity to 12 fungicides was determined. All the isolates (100%) were resistant to trifloxystrobin and carbendazim, and 98% were resistant to fluopyram, boscalid, pydiflumetofen, isopyrazam, and fluxapyroxad. However, none were resistant to propiconazole, prochloraz, tebuconazole, difenoconazole, and fludioxonil. The Cytb gene of trifloxystrobin-resistant isolates encoded the G143A mutation, whereas the ß-tubulin gene of carbendazim-resistant isolates encoded the E198A and E198A and M163I mutations. Mutations in SdhB-I280V, SdhC-S73P, SdhC-H134R, SdhD-D95E, and SdhD-G109V were associated with resistance to the succinate dehydrogenase inhibitors (SDHIs). Trifloxystrobin, carbendazim, and fluopyram were barely effective on the resistant isolates, whereas fludioxonil and prochloraz were effective on the isolates that were resistant to the quinone outside inhibitors (QoIs), SDHIs, and benzimidazoles. Ultimately, this study demonstrates that fungicide resistance seriously threatens the effective control of Corynespora leaf spot.

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.

Comparison and Correlation of Corynespora cassiicola Populations from Kiwifruit and Other Hosts Based on Morphology, Phylogeny, and Pathogenicity.

Corynespora leaf spot, which is caused by Corynespora cassiicola (Berk. & M. A. Curtis) C.T. Wei (C. cassiicola), has been globally reported in many plant species. 'Hongyang' was reported as highly sensitive kiwifruit cultivar to C. cassiicola. This cultivar is an important germplasm resource in the Actinidiaceae family and is widely cultivated throughout China. Even though C. cassiicola has been identified as the pathogen associated with kiwifruits in China, the C. cassiicola population from kiwifruit has not been characterized based on morphology, phylogeny, and pathogenicity. In this study, 133 and 48 representative C. cassiicola isolates from kiwifruit and 11 other hosts, respectively, recovered from symptomatic leaves were classified into eight morphological subgroups based on host origins. Using three loci (rDNA ITS, caa5, and act1), a phylogenetic tree showed that C. cassiicola isolates in Sichuan Province were grouped into three clades. All kiwifruit isolates were genetically identical to the rubber isolates from different countries. However, most isolates from other hosts in this study were genetically identical to the cucumber, soybean, and cowpea isolates in China, Brazil, and the United States, and two strawberry isolates clustered with isolates from tomato and other hosts in China, Brazil, and the United States. Furthermore, we confirmed host shift of C. cassiicola among different plant species in this study. Although 51 isolates from kiwifruit and different hosts were pathogenic to kiwifruit, blueberry, cucumber, and soybean, virulence levels of the pathogen were diverse for four hosts. Kiwifruit isolates exhibited host specificity with regards to the original host in degree. In addition, those isolates revealed a correlation between morphology and pathogenicity. The results suggest that C. cassiicola in Sichuan Province were derived from three different phylogenetic lineages. Promotion of the susceptible 'Hongyang' cultivar led to the emergence of a regnant C. cassiicola population from kiwifruit. In conclusion, rapid development of the C. cassiicola-sensitive crop in agricultural systems led to the emergence of a regnant C. cassiicola population. In some dominant populations (e.g., the C. cassiicola population from kiwifruit in this study), host origin was found to be a key factor influencing the morphologic, genetic, and pathogenic characterization of C. cassiicola.

Molecular characterization of a novel victorivirus infecting Corynespora cassiicola.

A novel victorivirus was detected in an isolate of Corynespora cassiicola strain 20180909-03 and was named "Corynespora cassiicola victorivirus 1" (CcVV1). The complete genome sequence of this virus is 5140 bp in length and contains 57% GC with two large open reading frames (ORFs) overlapping at the tetranucleotide AUGA. The ORFs were predicted to encode a coat protein (CP) and an RNA-dependent RNA polymerase (RdRp), respectively, which are conserved in dsRNA fungal viruses of the family Totiviridae. Comparison and phylogenetic analysis of the deduced amino acid sequences of RdRp and CP showed that CcVV1 is a new member of the genus Victorivirus. This is the first report of a genomic sequence of a victorivirus infecting Corynespora cassiicola.

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.

SDH mutations confer complex cross-resistance patterns to SDHIs in Corynespora cassiicola.

Succinate dehydrogenase inhibitors (SDHIs) are one of the most frequently used fungicides in cucumber fields in China. Our previous studies indicated that the sensitivity profile of Corynespora cassiicola, the causal agent of Corynespora leaf spot, to different SDHIs varied greatly; however, the underlying mechanism remains unclear. The 50% effective concentration (EC50) values of boscalid, fluopyram, fluxapyroxad and isopyrazam in C. cassiicola collected from 2017 to 2020 shifted, with resistance frequencies of 79.83%, 78.43%, 83.19% and 49.86%, respectively. The sequence alignment of sdhB/C/D of resistant strains revealed that eight single amino acid mutations (B-H278Y/L, B-I280V, C-S73P, C-N75S, C-H134R, D-D95E and D-G109V), and three dual-mutations (B-I280V&C-S73P, B-I280V&C-N75S and C-S73P&C-N75S) conferred various SDHI resistance levels and cross-resistance profiles. The expression level of the sdhB/C/D gene and succinate dehydrogenase (SDH) activity in the mutants were significantly altered by the presence of SDHIs, compared with the wild type strain. Additionally, molecular docking results suggested that the missense mutation influenced the crystal structure of SDH and subsequently interfered with the interaction bonds and bond distances among the target protein and chemicals. In brief, amino acid mutations altered the fungicide response of target gene expression, SDH activity and the binding features of SDH-ligand complexes and subsequently conferred multiple resistance levels and complex cross-resistance patterns to SDHIs in C. cassiicola. The evaluation of C. cassiicola resistance to SDHIs provided a significant foundation for efficient chemical development and integrated CLS management strategies.

First Report of Corynespora cassiicola Causing Leaf spot of Strobilanthes cusia in China.

Strobilanthes cusia (Nees) Kuntze is a vital medicinal and industrial herb, planted extensively in southern China (Hu, et al. 2011.). In July and August of 2021, leaf spot incidence on >60% plants and reduced yields >20% for fresh leaves were observed in S. cusia cultivar 'Malan No.1' across the Shufeng whole Township, Xianyou County, Fujian province. Initial symptoms on leaves were observed as small, dark-brown, spots surrounded by a yellow halo, expanding irregularly or into semicircular spots. As symptoms developed, the spots became dark brown, thin and fragile, forming small holes. In severe cases plants were defoliated. The pathogen was isolated from the margin of 60 symptomatic leaf lesions， surfacesterilized with 75% ethanol for 45 s, rinsed three times with sterile water, air dried, and cultured on PDA at 25°C in the dark. Pure cultures were obtained by single-spore isolation after subculture. Ten representative single-spore isolates (MY-1 to MY-10) from 154 pathogens in 10 sampling points were selected for morphological characterization and identification. After 7 days, mycelial colonies were gray to dark gray with few aerial hyphae. Conidia (32.3 to 132.8 × 5.8 to 8.4 µm, average 81.4 × 6.3 µm, n=50) were pale to brown, erect or curved, solitary or in chains, with 0 to 15 pseudosepta. Based on morphological characteristics, the isolates were preliminarily identified as Corynespora cassiicola. Genomic DNA of isolate MY-2 (randomly selected from 10 isolates as representative) was extracted from mycelia using the Ezup DNA extraction kit (Sangon Biotech Co., Ltd. Shanghai, China). The ITS (internal transcribed spacer) region of rDNA, TEF1-α (translation elongation factor 1 alpha) and TUB2 (beta-tubulin) genes were amplified and sequenced with primers ITS4/ITS5, EF1-728F/EF-986R (Wang et al. 2021) and Bt2a/Bt2b (Glass et al. 1995), respectively. BLASTN sequence analyses of ITS (538 bp), TEF1-α (302 bp) and TUB2 (436 bp) of isolate MY-2 (GenBank accessions OK355515, OM339443, OM339442) showed 100%, 97.6%, 100% identity with C. cassiicola in GenBank (Accession numbers JX908713, MW961421, AB539228). A neighbor-joining phylogenetic analysis based on ITS and TEF1-α sequences using MEGA7 showed that MY-2 clustered in the same clade with C. cassiicola. For pathogenicity tests, five S. cusia plants were inoculated onto the adaxial surface of leaves with mycelial plugs from ten isolates of 8-day-oldcultures on PDA. Five leaves per plant were inoculated, covered with wet cotton, and kept in a controlled greenhouse (26~33 °C, RH 80% ~ 90%). Leaves inoculated with sterile PDA plugs served as a negative control. At 3-5 days post inoculation, all 25 inoculated leaves of each isolate showed leaf spot lesions similar to those observed in the field, and control leaves were symptomless. C. cassiicola was successfully reisolated from the diseased leaves. The pathogenicity tests were repeated three times under the same conditions and similar results were observed. In view of morphology, pathogenicity and sequence results, the isolates were identified as C. cassiicola, a pathogen reported from many important crops (Lu et al. 2021). This is the first report of C. cassiicola as a pathogen in China which poses a potential threat to leaf production and S. cusia processing. References: Glass, N. L., et al. 1995. Appl. Environ. Microb. 61:1323 Hu, J.Q., et al. 2011. Flora of China. Science Press, Beijing, China. Volume 19: 407 Li, Q.L., et al. 2013. Plant Dis. 97 (5): 690 Lu, P. et al. 2021. Plant Dis. 105:3753 Wang S. H., et al. 2021.Forest Pathology, 51(2):1 Keywords: fungal disease, Strobilanthes cusia, medicinal plants, etiology, leaf spot.

Isolation and Identification of Secondary Metabolites Produced by Phytopathogenic Fungus Corynespora cassiicola from Hevea brasiliensis.

The secondary metabolites of the phytopathogenic fungus Corynespora cassiicola CC01 from Hevea brasiliensis were investigated. As a result, two new compounds, 5-acetyl-7-hydroxy-6- methoxybenzofuran-2(3H)-one (1) and (S)-2-(2,3-dihydrofuro [3,2-c]pyridin-2-yl)propan-2-ol (2), together with seven known compounds, 4,6,8-trihydroxy-3,4-dihydronaphthalen-1(2H)-one (3), 3,6,8-trihydroxy-3,4-dihydronaphthalen-1(2H)-one (4), curvulin acid (5), 2-methyl-5-carboxymethyl- 7-hydroxychromone (6), tyrosol (7), p-hydroxybenzoic acid (8) and cerevisterol (9), were isolated from the fermentation extract by comprehensive silica gel, reverse phase silica gel, Sephadex-LH20 column chromatography and high-performance liquid chromatography (HPLC). The structures of these compounds were identified by using high-resolution electrospray mass spectrometry (HRESIMS), nuclear magnetic resonance spectroscopy (NMR), optical rotation, ultraviolet and infrared spectroscopy techniques and a comparison of NMR data with those reported in the literature. Compounds 1 and 2 were new compounds, and compounds 3-9 were discovered from this phytopathogenic fungus for the first time. Compounds 1-9 were tested for phytotoxicity against the fresh tender leaf of Hevea brasiliensis, and the results show that none of them were phytotoxic. Additionally, these compounds were subjected to an antimicrobial assay against three bacteria (E. coli, methicillin-resistant Staphylococcus aureus and Micrococcus luteus), but they showed no activity.

Subcutaneous phaeohyphomycosis caused by plant pathogenic Corynespora cassiicola: A case report.

Corynespora cassiicola is a common plant pathogen responsible for leaf-spotting diseases in the tropical and subtropical areas. C. cassiicola seldom causes human infections. Here we describe a case of subcutaneous phaeohyphomycosis caused by C. cassiicola in a 76-year-old Chinese man, who presented to our hospital with a purulent discharge and painful sensation on his right leg. Skin biopsy revealed an abscess, and culture confirmed C. cassiicola to be the causative agent. The result was further identified by sequence analysis of the internal transcribed spacer region. The patient was successfully treated with systemic voriconazole and wound debridement: the lesion disappeared after 20 days.

Nanopore/Illumina Hybrid Genome Sequence Resource for Corynespora cassiicola Strain XJ Infecting Rubber Tree in China.

Corynespora cassiicola is a ubiquitous pathogenic fungus that can infect a broad range of plant hosts. Corynespora leaf fall, caused by C. cassiicola, is one of the major diseases on rubber tree in China. This disease is having an increasing affect on natural rubber production worldwide. In this study, by combining the Nanopore and Illumina sequencing technologies, we present the chromosome-scale genome sequence of the rubber tree-sampled C. cassiicola strain XJ collected in the subtropical region of China. The assembly consists of 23 scaffolds (N50 = 4.62 Mb) with an estimated genome size of 44.42 Mb (only 166 non-ATCG bases) and 16,108 protein-coding genes. The genome will provide a valuable resource for further research on the pathogenesis and comparative genomics of C. cassiicola on rubber tree and other hosts.

First Report of Corynespora cassiicola Causing Target Spot on Soybean in Taiwan.

Soybean (Glycine max [L.] Merr.) is an important crop in Taiwan. In October 2020, an unknown leaf spot disease was counted (n = 100) to occur over 70% of soybean cultivar 'Hualien No.1' in the Shoufeng Township of Hualien County, eastern Taiwan. Initial symptoms on leaves as tiny lesions approximately 3 mm in diameter, which later enlarged and developed into round, irregular, and reddish-brown spots with concentric rings surrounded by a yellowish halo. The symptoms appeared on both young and old leaves, but rarely on the stem or pods. The lesions at the margin of healthy and infected tissues were surface-disinfested in 1% NaOCl for 30 seconds, washed twice in sterilized distilled water, dissected and plated on potato dextrose agar (PDA) to isolate the potential pathogen. Colonies on PDA exhibited light to dark brown color at 24°C with 12-hours light after 7-days incubation. The average growth rate was 3 mm per day. Conidia were light brown in color and obclavate to cylindrical in shape. The size of a conidium was measured with an average of 110.8 ± 28.2 µm in length and 15.2 ± 2.8 µm in width, typically with 3 to 18 septa (n = 50). To confirm the pathogenicity of this fungus, conidial suspension (104 conidia/mL) of two isolates, HL_GM-6 and HL_GM-7, were sprayed on the healthy leaves of 4-weeks-old soybean. Plants sprayed with sterile distilled water were used as a control. After inoculation, the plants were covered with plastic bags to maintain a high humidity for 24 hours before moving into a greenhouse with a condition of 20 to 25°C and relative humidity of 75 to 80%. After 7 days of inoculation, foliar symptoms began to appear and which were identical with the field observations. To complete the Koch's postulates, pathogen isolation was attempted and the identical fungus was retrieved from the foliar spots of the inoculated leaves. The foliar symptoms as well as the morphology of the conidiophores and conidia suggested the pathogen to be Corynespora cassiicola (Ellis et al. 1971). Molecular characterization was performed using the sequences of internal transcribed spacer (ITS) region of rDNA, actin (act1), tubulin, and translation elongation factor 1 alpha (tef1) genes after a PCR with ITS1/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn, 1999), BT2a/Bt2b (Udayanga et al. 2012), EF1-728F/EF1-986R (Udayanga et al. 2012), respectively. BLASTN sequence analyses of the ITS, act1, tubulin, and tef1 genomic regions of the isolate HL_GM-7 (GenBanK accessions MW548097 MW961420, MW961419 and MW961421) showed high similarity with the isolates of C. cassiicola including 99.58% with sequence KF810854 (Deon et al. 2014), 99.11% with FJ853005 (Dixon et al. 2009), 99.34% with MH763700 (Duan et al. 2019), and 99.33% with KY112719 (Zhang et al. 2018) respectively. Based on the morphology, pathogenicity, and sequence results, this study becomes the first report of C. cassiicola causing target spot on soybean in Taiwan. C. cassiicola is known to infect a broad host range (Dixon et al. 2009; Lopezet al. 2018), and it has been found to infect tomato, cucumber, papaya, and Salvia miltiorrhiza in Taiwan (Lu et al. 2019; Tsai et al. 2015). Therefore, the emergence of soybean target spot should be aware to avoid potential damage to soybean production in Taiwan.

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.