Corynespora Leaf Spot Research Articles

Chemical control of Alternaria and Corynespora leaf spot complex in Bt cotton

Cotton is an important cash crop of India as well as Gujarat, enjoying the pre-eminent status as 'White gold' and 'King of Fibres'. Cotton is widely affected by many diseases including Alternaria leaf spot and target spot or Corynespora leaf spot caused by Alternaria alternata and Corynespora cassiicola, respectively. The main objective of the study was to investigate the effectiveness of fungicides against leaf spot complex (Alternaria leaf spot and target spot) of Bt cotton. The field experiment was conducted during Kharif 2022 at Plant Pathological Research Farm, Department of Plant Pathology, BA College of Agriculture, Anand Agricultural University Anand, Gujarat. Fungicides were applied two times at 15 days interval after initiation of disease and results showed that among the tested treatments including control, Azoxystrobin 18.2% + Difenoconazole 11.4% SC recorded significantly lowest disease intensity (18.50% and 17.92%) as compared to control (50.75% and 46.85%), with highest disease control (63.54% and 61.73%) for Alternaria leaf spot and target spot respectively. The highest yield (1968 kg ha ) and ICBR (1:10.62) was -1 recorded in Azoxystrobin 18.2% + Difenoconazole 11.4% SC. The next best treatment found in terms of disease control was Tebuconazole 50% + Trifloxystrobin 25% WG. So in nutshell it was found that readymix fungicides were found best and also cost effective in managing both the diseases.. KEYWORDS :Alternaria alternata, Corynespora cassiicola, leaf spot, fungicides

Characteristics of Corynespora cassiicola, the causal agent of tobacco Corynespora leaf spot, revealed by genomic and metabolic phenomic analysis

Corynespora cassiicola is a highly diverse fungal pathogen that commonly occurs in tropical, subtropical, and greenhouse environments worldwide. In this study, the isolates were identified as C. cassiicola, and the optimum growth and sporulation were studied. The phenotypic characteristics of C. cassiicola, concerning 950 different growth conditions, were tested using Biolog PM plates 1–10. In addition, the strain of C. cassiicola DWZ from tobacco hosts was sequenced for the using Illumina PE150 and Pacbio technologies. The host resistance of tobacco Yunyan 87 with different maturity levels was investigated. In addition, the resistance evaluation of 10 common tobacco varieties was investigated. The results showed that C. cassiicola metabolized 89.47% of the tested carbon source, 100% of the nitrogen source, 100% of the phosphorus source, and 97.14% of the sulfur source. It can adapt to a variety of different osmotic pressure and pH environments, and has good decarboxylase and deaminase activities. The optimum conditions for pathogen growth and sporulation were 25–30 °C, and the growth was better on AEA and OA medium. The total length of the genome was 45.9 Mbp, the GC content was 51.23%, and a total of 13,061 protein-coding genes, 202 non-coding RNAs and 2801 and repeat sequences were predicted. Mature leaves were more susceptible than proper mature and immature leaves, and the average diameter of diseased spots reached 17.74 mm at 12 days. None of the tested ten cultivars exhibited obvious resistance to Corynespora leaf spot of tobacco, whereby all disease spot diameters reached > 10 mm and > 30 mm when at 5 and 10 days after inoculation, respectively. The phenotypic characteristics, genomic analysis of C. cassiicola and the cultivar resistance assessment of this pathogen have increased our understanding of Corynespora leaf spot of tobacco.

GamaNNet: A Novel Plant Pathologist-Level CNN Architecture for Intelligent Diagnosis

Plant pathologies significantly jeopardise global food security, necessitating the development of prompt and precise diagnostic methods. This study employs advanced deep learning techniques to evaluate the performance of nine convolutional neural networks (CNNs) in identifying a spectrum of phytosanitary issues affecting the foliage of Solanum lycopersicum (tomato). Ten thousand RGB images of leaf tissue were subsampled in training (64%), validation (16%), and test (20%) sets to rank the most suitable CNNs in expediting the diagnosis of plant disease. The study assessed the performance of eight well-known networks under identical hyperparameter conditions. Additionally, it introduced the GamaNNet architecture, a custom-designed model optimised for superior performance on this specific type of dataset. The investigational results were most promising for the innovative GamaNNet and ResNet-152, which both exhibited a 91% accuracy rate, as evidenced by their confusion matrices, ROC curves, and AUC metrics. In comparison, LeNet-5 and ResNet-50 demonstrated lower assertiveness, attaining accuracies of 74% and 69%, respectively. GoogLeNet and Inception-v3 emerged as the frontrunners, displaying diagnostic preeminence, achieving an average F1-score of 97%. Identifying such pathologies as Early Blight, Late Blight, Corynespora Leaf Spot, and Septoria Leaf Spot posed the most significant challenge for this class of problem.

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 Ccβ-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.

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

BackgroundCorynespora 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.ResultsThe 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.ConclusionsThis 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.

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.

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 β-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.

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.

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.

Corynespora leaf spot of Ligustrum quihoui caused by Corynespora cassiicola in China

Ligustrum quihoui is a perennial shrub that has been cultivated in China as a landscape plant. In November 2020, leaf spots on L. quihoui were observed in several residential areas in the east of Anqing city. Symptoms first appeared as small dark brown dots on the leaf surface which then became larger, forming necrotic lesions and resulting in intense defoliation. To identify the pathogen, infected leaves were collected and cultured on potato dextrose agar (PDA). Three cultures were isolated from the diseased leaves, which grew to form olive green colonies after 5 days of incubation on PDA. Based on colony characteristics, morphology phylogenetic analysis of ribosome transcriptional spacers (rDNA-ITS), and pathogenicity tests, the causal agent of leaf spot on L. quihoui was identified as Corynespora cassiicola. This is the first report to show that C. cassiicola can cause leaf spot on L. quihoui.

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.

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.

Changes in the sensitivity of &lt;i&gt;Corynespora cassiicola&lt;/i&gt; to fungicides on cucumber plants in Okayama Prefecture, Japan, since the 2000s

In 2014, we collected 234 isolates of cucumber Corynespora leaf spot fungus (Corynespora cassiicola Berkeley & M.A. Curtis C.T. Wei) from 24 commercial cucumber fields in Okayama Prefecture, Japan and assessed their sensitivity to several fungicides. Among the tested strains, the proportion of thiophanate-methyl (T) highly resistant/dietofencarb (D) weakly and moderately resistant strains were 37% and 49%, respectively. Azoxystrobin (A)-resistant strains were 95%, whereas boscalid (B)-resistant strains were 39%. TD- and A-resistant strains were more widespread and more frequent than in 2000. B-resistant strains have only recently emerged.

Relationship between Weather Variables and the Corynespora Leaf Spot Disease in Soybean

The present investigation on weather and diseases relationships in soybean to establish forewarning procedures for timely preventive measures was conducted at the Main Agricultural Research Station (MARS), University of Agricultural Sciences, Dharwad during kharif 2017. Due to changing climatic scenario, biotic and abiotic threats are more prominent. It is well known that the biotic stress factors like insect pests and diseases are affected by abiotic factors. Further, the severity and degree of virulence of diseases over a geographical area is dependent on weather factors. Hence, in this context, there is need to study the role of weather parameters in diseases. In this endeavour, the present study has been undertaken with a new concept called lead-time concept for plant disease forecasting helps controlling early before they reach to economic injury level (EIL), and reducing cost of cultivation in use of low use of pesticides and insecticide. Corynespora leaf spot (CLS) is a disease of soybean, which infects mainly to leaves. The weather data was collected from the MARS Dharwad observatory and micrometeorological data collected in crop field and they are collected at three days interval. The Correlation coefficients between disease grade of Corynespora Leaf Spot and 29 weather variables were presented in the data show that the disease grade ratings were positively correlated, with r =0.69 for PRHT (X14), r = 0.69 for PRHM (X17), r = 0.69 for PRHB (X20), and r = 0.28 for PRHO (X26). And remining were negative correlated. Hence, the weather plays an important role in the disease incidence and development.

Evaluation of fungicides and bacterial antagonists for management of Corynespora leaf spot on mungbean (Vigna radiata L. Wilczek)

To minimize losses likely to occur due to Corynespora leaf spot, systemic and contact fungicides and formulation of bacterial antagonists like Bacillus subtilis DTBS-5, B. amyloliquefaciens DSBA-11, and Pseudomonas fluorescens DTPF-3 talc-based formulations were evaluated to manage the disease. Though mancozeb has the lowest EC50 value of 1250.54 ppm it was not efficient in the field experiment. In addition, dual culture assay to determine the efficacy of bacterial antagonists against C. cassiicola, B. amyloliquefaciens DSBA-11 effectiovely reduced growth of C. cassiicola. In the field experiments conducted during Kharif 2019 and 2020, two sprays with Azoxystrobin 23 SC@ 0.5 ml lit−1 were found most effective in reducing disease severity by 57.7% and 67.7% respectively during Kharif 2019 and 2020. Azoxystrobin 23 SC treatment resulted in a maximum increase of 281.5 kg in grain yield followed by Pseudomonas fluorescens DTPF-3 264.7 kg and Bacillus subtilis DTBS-5 248.9 kg yield.

Field efficacy of metiram 70 wg against fungal diseases in cotton

Metiram 70 WG @ 750, 1000, 1250 and 1500 g ha−1 in comparison with carbendazim 50 WP (125 g ha−1) and propiconazole 25 EC (500 mL ha−1) along with untreated control were evaluated in randomized block design. Treatments replicated thrice were imposed at 21 days interval with first spray starting immediately after the appearance of Alternaria leaf spot. Metiram 70 WG @ 3000 g ha−1 was tested for phytotoxicity alone. Highest reduction in Alternaria blight and Corynespora leaf spot was recorded with metiram 70 WG @ 1500 g ha−1 (84.1 and 77.9%) followed by 1250 g ha−1 (80.1 and 72.1%). Carbendazim 50 WP @ 250 g ha−1 resulted in reduction of anthracnose (84.1%) and grey mildew (80.9%) followed by metiram 70 WG 1500 g ha−1 (65.2% and 78.7%). Rust was reduced to the tune of 68.6% with metiram 70 WG @ 1500 g ha−1 followed by propiconazole 25 EC @ 500 mL ha−1 (63.6%). Highest yield increase of 23.1% was obtained with metiram 70 WG 1500 g ha−1 followed by propiconazole (22.8%) and metiram 70 WG 1250 g ha−1 (21.6%). Highest dose did not induce any phytotoxic symptoms. These results indicate broad spectrum activity of metiram 70 WG against fungal diseases in cotton.

First report of leaf spot disease caused by Corynespora cassiicola on American sweetgum (Liquidambar styraciflua L.) in China.

American sweetgum (Liquidambar styraciflua L.) is a forest plant native to North America, which has been introduced into other countries due to its ornamental and medicinal values. In June 2019, symptoms of leaf spots on sweetgum were observed in a field (5 ha) located in Xuzhou, Jiangsu Province, China. On this field, approximately 45% of 1,000 trees showed the same symptoms. Symptoms were observed showing irregular or circular dark brown necrotic lesions approximately 5 to 15 mm in diameter with a yellowish margin on the leaves. To isolate the pathogen, diseased leaf sections (4×4mm) were excised from the margin of the lesion, surface-sterilized with 0.1% NaOCl for 90 s, rinsed 4 times in sterile distilled water, air dried and then transferred on potato dextrose agar (PDA) medium at 25°C in the dark. Pure cultures were obtained by monospore isolation after subculture. Ten purified isolates, named FXI to FXR, were transferred to fresh PDA and incubated as above to allow for morphological and molecular identification. After 7 days, the aerial mycelium was abundant, fluffy and exhibited white to greyish-green coloration. The conidia were dark brown or olive, solitary or produced in chains, obclavate, with 1 to 15 pseudosepta, and measured 45 to 200µm  10 to 18µm. Based on morphological features, these 10 isolates were identified as Corynespora cassiicola (Ellis et al. 1971). Genomic DNA of each isolate was extracted from mycelia using the cetyltrimethylammonium bromide (CTAB) method. The EF-1α gene and ITS region were amplified and sequenced with the primer pairs rDNA ITS primers (ITS4/ITS5) (White et al. 1990) and EF1-728F/EF-986R (Carbone et al.1999) respectively. The sequences were deposited in GenBank. BLAST analysis revealed that the ITS sequence had 99.66% similarity to C. cassiicola MH255527 and that the EF-1α sequence had 100% similarity to C. cassiicola KX429668A. maximum likelihood phylogenetic analysis based on EF-1α and ITS sequences using MEGA 7 revealed that ten isolates were placed in the same clade as C. cassiicola (Isolate: XQ3-1; accession numbers: MH572687 and MH569606, respectively) at 98% bootstrap support. Based on the morphological characteristics and phylogenetic analyses, all isolates were identified as C. cassiicola. For the pathogenicity test, a 10 µl conidial suspension (1×105 spores/ml) of each isolate was dripped onto healthy leaves of 2-year-old sweetgum potted seedlings respectively. Leaves inoculated with sterile water served as controls. Three plants (3 leaves per plant) were conducted for each treatment. The experiment was repeat twice. All seedlings were enclosed in plastic transparent incubators to maintain high relative humidity (90% to 100%) and incubated in a greenhouse at 25°C with a 12-h photoperiod. After 10 days, leaves inoculated with conidial suspension of each isolate showed symptoms of leaf spots, similar to those observed in the field. Control plants were remained healthy. In order to reisolate the pathogen, surface-sterilized and monosporic isolation was conducted as described above. The same fungus was reisolated from the lesions of symptomatic leaves, and its identity was confirmed by molecular and morphological approaches, thus fulfilling Koch's postulates. Chlorothalonil and Boscalid can be used to effectively control Corynespora leaf spot (Chairin T et al.2017). To our knowledge, this is the first report of leaf spot caused by C. cassiicola on L. styraciflua in China.

The Bioactivity and Efficacy of Benzovindiflupyr Against Corynespora cassiicola, the Causal Agent of Cucumber Corynespora Leaf Spot.

Corynespora cassiicola, which causes Corynespora leaf spot, results in considerable yield loss of cucumber grown in greenhouses. Frequent reports of reduced efficacy and control failure of fungicides warrant new, efficient alternative chemistries. In this study, the sensitivity of C. cassiicola to benzovindiflupyr was evaluated using a collection of 81 isolates collected from Shandong, China. The mean EC50 values for mycelial growth, spore germination, and germ tube elongation of C. cassiicola were 0.69 ± 0.44, 0.12 ± 0.063, and 0.13 ± 0.076 µg ml-1, respectively. Benzovindiflupyr treatment led to a reduced respiration rate and ATP production of C. cassiicola and decreased spore pathogenicity by 21.9% on average. Additionally, detached cucumber leaves sprayed with fungicides before or after inoculation were used to assess the efficacy of benzovindiflupyr against C. cassiicola. Benzovindiflupyr (150 µg ml-1) exhibited preventive and curative efficacies of 86.9 and 77.1%, respectively. Benzovindiflupyr at 150 g a.i. ha-1 provided over 70% efficacy in field trials performed in 2018 and 2019, which was significantly higher than that of the reference fungicides fluopyram and fluxapyroxad at the same dose. Furthermore, the yield of commercial cucumber increased as disease incidence decreased. Our findings pave the way for the introduction of benzovindiflupyr in the integrated management of Corynespora leaf spot.

Cell‐wall‐degrading enzymes produced by sesame leaf spot pathogen Corynespora cassiicola

AbstractCorynespora leaf spot (CLS) is one of the most important diseases in sesame and is caused by a fungal pathogen, Corynespora cassiicola. In this study, we investigated the role of cell‐wall‐degrading enzymes (CWDEs) in the process of pathogen infection and aimed to identify the enzyme that exerts positive effect. Corynespora cassiicola was inoculated in liquid culture and in sesame leaf. At 7 days postinoculation, CWDEs including polygalacturonase (PG), cellulase (CL), neutral xylanase (NEX) and laccase (LC) were detected in the supernatant and leaf extracts inoculated with Corynespora cassiicola. The result showed that the activities of LC and NEX were higher in liquid culture, while the activities of CL and PG were lower. In the sesame leaf infected by Corynespora cassiicola, the activity of NEX was the highest, those of CL and LC were the second highest and that of PG was the weakest, which indicated that NEX was the key enzyme in the process of sesame infection by Corynespora cassiicola. Secondly, the activities of the CWDEs secreted by Corynespora cassiicola were disparate in different sesame varieties. CWDEs activities of cultivar Jz were generally higher than cultivar Hn, and the activity change curve of Jz was also steeper than that of Hn. This indicates that the resistance mechanism of Hn may inhibit the activities of CWDEs and that the appearance and expansion of leaf necrosis spot is related to the comprehensive accumulation and action of various CWDEs. This is the first report investigating the association between CWDEs and pathogenesis of Corynespora cassiicola, causing Corynespora leaf spot in sesame.

Evaluation of Sensitivity and Resistance Risk of Corynespora cassiicola to Isopyrazam and Mefentrifluconazole.

Necrotic lesions on leaves caused by Corynespora leaf spot (CLS) seriously threaten the quality and yield of cucumber in China. Corynespora cassiicola has developed different degrees of sensitivity to various fungicides due to its long-term and extensive application. In our work, the effect of isopyrazam and mefentrifluconazole on different life stages of C. cassiicola was examined. To determine the optimal effect of binary mixtures of isopyrazam and mefentrifluconazole, the two fungicides were mixed at different proportions. Furthermore, the disease suppression of isopyrazam, mefentrifluconazole, and their compound mixture against CLS was evaluated in greenhouse experiments. Ultraviolet (UV) mutagenesis and fungicide-selection methods were performed to assess the risk of resistance development. Among the three life stages tested, isopyrazam showed the weakest inhibition on mycelial growth, and mefentrifluconazole showed the strongest inhibition of germ tube elongation. According to Wadley's and cotoxicity coefficient methods, the optimal proportion of the two-component mixture of isopyrazam and mefentrifluconazole was 1:1. Isopyrazam, mefentrifluconazole, and their binary mixture at 1:1 reduced the disease severity of CLS on potted cucumber plants, with protective effects of 31.11, 24.65, and 42.12% and curative effect of 33.90, 37.48, and 42.84%, respectively. Compared with isopyrazam or mefentrifluconazole alone, the binary mixture of the two fungicides at 1:1 did not exert significant influence on the change of C. cassiicola sensitivity. Undoubtedly, such data will greatly facilitate the screening of new fungicides for CLS and resistance management.