Double Mutations in Succinate Dehydrogenase Are Involved in SDHI Resistance in Corynespora cassiicola

With the further application of succinate dehydrogenase inhibitors (SDHI), the resistance caused by double mutations in target gene is gradually becoming a serious problem, leading to a decrease of control efficacy. It is important to assess the sensitivity and fitness of double mutations to SDHI in Corynespora cassiicola and analysis the evolution of double mutations. We confirmed, by site-directed mutagenesis, that all double mutations (B-I280V+D-D95E/D-G109V/D-H105R, B-H278R+D-D95E/D-G109V, B-H278Y+D-D95E/D-G109V) conferred resistance to all SDHI and exhibited the increased resistance to at least one fungicide than single point mutation. Analyses of fitness showed that all double mutations had lower fitness than the wild type; most of double mutations suffered more fitness penalties than the corresponding single mutants. We also further found that double mutations (B-I280V+D-D95E/D-G109V/D-H105R) containing low SDHI-resistant single point mutation (B-I280V) exhibited higher resistance to SDHI and low fitness penalty than double mutations (B-H278Y+D-D95E/D-G109V) containing high SDHI-resistant single mutations (B-H278Y). Therefore, we may infer that a single mutation conferring low resistance is more likely to evolve into a double mutation conferring higher resistance under the selective pressure of SDHI. Taken together, our results provide some important reference for resistance management.

High dilution of Belladonna affect the mycelial growth of Corynespora cassiicola in vitro

Introduction: The target spot is a disease caused by fungus Corynespora cassiicola (Berk. & Curt.) Wei. This disease has occurred in several states of Brazil. It is a late season disease and causes economic losses in various crops such as soybeans [1]. Currently there is no adequate treatment for the control of C. cassiicola in organic cultivation of soybeans, since the application of fungicides for the control and management of diseases is not allowed by Brazilian legislation [2]. Thus, the purpose of this experiment was to test the effectiveness of high dilutions of Belladonna in vitro on mycelial growth of Corynespora cassiicola. Materials and Methods: The preliminary tests were conducted at the Laboratory of Plant Pathology, State University of Maringá (UEM). The fungal isolate of C. cassicola was obtained from Embrapa Soja. The fungus was peaked and grown on PDA (potato dextrose agar) maintained at 25°C ± 2 and 12h photoperiod. Belladonna dilutions (6, 12, 18, 24 and 30dH) were obtained according to the Brazilian Homeopathic Pharmacopoeia [3]. PDA culture medium plus Belladonna dilutions (6, 12, 24 and 30dH) beyond the control containing distilled water were placed in petri dishes after filtration through a Millipore membrane (pore diameter of 0.45µm ). After medium solidification, a disc of mycelium (4 mm diameter) of C. cassiicola was peaked towards the center of each plate and sealed with plastic wrap and then incubated at 25°C with 12h photoperiod. The mycelial growth was measured daily for 8 days. The control consisted of distilled water. The data were analyzed by ANOVA and means were compared by Scott-Knott test (P ≤ 0.05). Results and Discussion: All dilutions of Belladonna (6, 12, 24, 30dH) were effective (p

Complete genome sequence of a novel fusarivirus from the phytopathogenic fungus Corynespora cassiicola

Corynespora cassiicola is an important phytopathogenic fungus and it has severely impaired the production of crops. In this study, we report on the molecular characterization of a novel (+) ssRNA mycovirus, Corynespora cassiicola fusarivirus 1 (CcFV1) isolated from C. cassiicola strain 20200826-3-1. Excluding a poly (A) tail, the genome of the virus is 6491 nt containing three putative open reading frames. The large ORF1 encodes a polypeptide of 1524aa with a conserved RNA-dependent RNA polymerase (RdRp) domain, a helicase (Hel) domain, and a Phage-holin-3-6 (Phage-holin) domain. ORF2 encodes a polypeptide with a conserved Chromosome segregation ATPase (Smc) domain. The smallest ORF3 encodes a putative protein with an unknown function. Phylogenetic analysis based on the ORF1 and ORF2 of CcFV1 encoded polypeptide showed that CcFV1 is phylogenetically related to the newly proposed family Fusariviridae. Thus, we suggest that CcFV1 might be a novel member of the family Fusariviridae and is also the first discovered in C. cassiicola.

Molecular Characterization of A Novel Victorivirus Infecting Corynespora Cassiicola

One victorivirus was detected in the isolate of Corynespora cassiicola strains 20180909-03, which was named Corynespora cassiicola victorivirus 1 (CcVV1). The whole-genome sequence of the virus was sequenced and identified. The CcVV1 genome is 5140 nt and contains 56.87%GC with two large open reading frames (ORFs) overlapping at the tetranucleotide AUGA. The two ORFs were predicted to encode coat protein (CP) and RNA-dependent RNA polymerase (RdRp) respectively, which were conservative in dsRNA fungal viruses of the family Totiviridae. Conservative domains comparison and phylogenetic analysis of the deduced amino acid sequence of RdRp and CP showed that CcVV1 was a new virus of the Victorivirus genus. As far as we know, it is the first report of a genomic sequence of the genus Victorivirus infecting Corynespora cassiicola.

Morphological Characterization of Corynespora cassiicola Isolates in Culture Media

Corynespora cassiicola threatens soybean and cotton production in Brazil. The objective of this study was to evaluate cultural and morphological aspects of C. cassiicola isolated from soybean and cotton of different Brazilian regions, in culture media. The isolates were grown in PDA (Potato Dextrose Agar) and V8 juice agar media. The characteristics evaluated were: color, aspect, and growth rate of mycelia, as well as production and dimension of conidia, and number of septa per conidium. Culture media and isolates were compared using the Kruskal-Wallis or Tukey’s test at 5% significance level. The mycelia of the isolates were predominantly dark gray and light brown. C. cassiicola isolates grew better in V8 juice agar medium, presenting a higher mycelial growth rate. In PDA medium, the production of conidia was higher in isolates from cotton, compared with soybean isolates. There was great variation in the production of conidia in V8 juice agar medium, regardless of the host origin. Conidia length and width varied for isolate and culture medium. The isolates of C. cassiicola coming from cotton presented a higher number of septa per conidium when grown in PDA medium. The morphological aspects of C. cassiicola vary depending on the host of origin and the culture medium.

Quantifying Airborne Dispersal Route of Corynespora cassiicola in Greenhouses

Target leaf spot (TLS), caused by Corynespora cassiicola, is an emerging and high-incidence disease that has spread rapidly on the global scale. Aerospores released by infected plants play a significant role in the epidemiology of cucumber TLS disease; however, no data exist concerning the infectiousness and particle size of C. cassiicola aerospores, and the experimental evidence for the aerospores transmission was lacking. In the present study, highly effective approaches to collect and quantify aerospores were developed for exposure chamber and greenhouse studies. Quantifiable levels of C. cassiicola aerospores were detected in 27 air samples from nine naturally infested greenhouses, ranging from 198 to 5,969 spores/m3. The C. cassiicola strains isolated from air samples were infective to healthy cucumber plants. Exposure chambers were constructed to study the characteristics of C. cassiicola aerospores released by artificially infested cucumber plants. The particle size of C. cassiicola ranged predominately from 2.1 to 4.7 μm, accounting for 71.97% of the total amount. In addition, the transmission dynamics of C. cassiicola aerospores from donor cucumber plants to recipient cucumber plants were confirmed in exposure chambers and greenhouses. The concentration of C. cassiicola aerospores was positively associated with cucumber TLS disease severity. This study suggested that aerospore dispersal is an important route for the epidemiology of plant fungal disease, and these data will contribute to the development of new strategies for the effective alleviation and control of plant diseases.