GWAS reveals a rapidly evolving candidate avirulence effector in the Cercospora leaf spot pathogen.

The major resistance gene BvCR4 recently bred into sugar beet hybrids provides a high level of resistance to Cercospora leaf spot caused by the fungal pathogen Cercospora beticola. The occurrence of pathogen strains that overcome BvCR4 was studied using field trials in Switzerland conducted under natural disease pressure. Virulence of a subset of these strains was evaluated in a field trial conducted under elevated artificial disease pressure. We created a new C. beticola reference genome and mapped whole genome sequences of 256 isolates collected in Switzerland and Germany. These were combined with virulence phenotypes to conduct three separate genome-wide association studies (GWAS) to identify candidate avirulence genes. We identified a locus associated with avirulence containing a putative avirulence effector gene named AvrCR4. All virulent isolates either lacked AvrCR4 or had nonsynonymous mutations within the gene. AvrCR4 was present in all 74 isolates from non-BvCR4 hybrids, whereas 33 of 89 isolates from BvCR4 hybrids carried a deletion. We also mapped genomic data from 190 publicly available US isolates to our new reference genome. The AvrCR4 deletion was found in only one of 95 unique isolates from non-BvCR4 hybrids in the United States. AvrCR4 presents a unique example of an avirulence effector in which virulent alleles have only recently emerged. Most likely these were selected out of standing genetic variation after deployment of BvCR4. Identification of AvrCR4 will enable real-time screening of C. beticola populations for the emergence and spread of virulent isolates.

Genome-Wide Identification of B-Box Gene Family and Expression Analysis Suggest Its Roles in Responses to Cercospora Leaf Spot in Sugar Beet (Beta Vulgaris L.).

The B-box (BBX) protein, which is a zinc-finger protein containing one or two B-box domains, plays a crucial role in the growth and development of plants. Plant B-box genes are generally involved in morphogenesis, the growth of floral organs, and various life activities in response to stress. In this study, the sugar beet B-box genes (hereafter referred to as BvBBXs) were identified by searching the homologous sequences of the Arabidopsis thaliana B-box gene family. The gene structure, protein physicochemical properties, and phylogenetic analysis of these genes were systematically analyzed. In this study, 17 B-box gene family members were identified from the sugar beet genome. A B-box domain can be found in all sugar beet BBX proteins. BvBBXs encode 135 to 517 amino acids with a theoretical isoelectric point of 4.12 to 6.70. Chromosome localization studies revealed that BvBBXs were dispersed across nine sugar beet chromosomes except chromosomes 5 and 7. The sugar beet BBX gene family was divided into five subfamilies using phylogenetic analysis. The gene architectures of subfamily members on the same evolutionary tree branch are quite similar. Light, hormonal, and stress-related cis-acting elements can be found in the promoter region of BvBBXs. The BvBBX gene family was differently expressed in sugar beet following Cercospora leaf spot infection, according to RT-qPCR data. It is shown that the BvBBX gene family may influence how the plant reacts to a pathogen infection.

Genome-wide association study reveals novel loci and a candidate gene for resistance to frogeye leaf spot (Cercospora sojina) in soybean.

Frogeye leaf spot, caused by the fungus Cercospora sojina, is a threat to soybeans in the southeastern and midwestern United States that can be controlled by crop genetic resistance. Limited genetic resistance to the disease has been reported, and only three sources of resistance have been used in modern soybean breeding. To discover novel sources and identify the genomic locations of resistance that could be used in soybean breeding, a GWAS was conducted using a panel of 329 soybean accessions selected to maximize genetic diversity. Accessions were phenotyped using a 1-5 visual rating and by using image analysis to count lesion number and measure the percent of leaf area diseased. Eight novel loci on eight chromosomes were identified for three traits utilizing the FarmCPU or BLINK models, of which a locus on chromosome 11 was highly significant across all model-trait combinations. KASP markers were designed using the SoySNP50K Beadchip and variant information from 65 of the accessions that have been sequenced to target SNPs in the gene model Glyma.11g230400, a LEUCINE-RICH REPEAT RECEPTOR-LIKE PROTEIN KINASE. The association of a KASP marker, GSM990, designed to detect a missense mutation in the gene was the most significant with all three traits in a genome-wide association, and the marker may be useful to select for resistance to frogeye leaf spot in soybean breeding.

Enhanced cercosporin production by co-culturing Cercospora sp. JNU001 with leaf-spot-disease-related endophytic bacteria.

BACKGROUND: Owing to the excellent properties of photosensitization, cercosporin, one of naturally occurring perylenequinonoid pigments, has been widely used in photodynamic therapy, or as an antimicrobial agent and an organophotocatalyst. However, because of low efficiency of total chemical synthesis and low yield of current microbial fermentation, the limited production restricts its broad applications. Thus, the strategies to improve the production of cercosporin were highly desired. Besides traditional optimization methods, here we screened leaf-spot-disease-related endophytic bacteria to co-culture with our previous identified Cercospora sp. JNU001 to increase cercosporin production. RESULTS: Bacillus velezensis B04 and Lysinibacillus sp. B15 isolated from leaves with leaf spot diseases were found to facilitate cercosporin secretion into the broth and then enhance the production of cercosporin. After 4 days of co-culture, Bacillus velezensis B04 allowed to increase the production of cercosporin from 128.2 mg/L to 984.4 mg/L, which was 7.68-fold of the previously reported one. Lysinibacillus sp. B15 could also enhance the production of cercosporin with a yield of 626.3 mg/L, which was 4.89-fold higher than the starting condition. More importantly, we found that bacteria B04 and B15 employed two different mechanisms to improve the production of cercosporin, in which B04 facilitated cercosporin secretion into the broth by loosening and damaging the hyphae surface of Cercospora sp. JNU001 while B15 could adsorb cercosporin to improve its secretion. CONCLUSIONS: We here established a novel and effective co-culture method to improve the production of cercosporin by increasing its secretion ability from Cercospora sp. JNU001, allowing to develop more potential applications of cercosporin.

Population genetic structure and migration patterns of the maize pathogenic fungus, Cercospora zeina in East and Southern Africa.

Cercospora zeina is a causal pathogen of gray leaf spot (GLS) disease of maize in Africa. This fungal pathogen exhibits a high genetic diversity in South Africa. However, little is known about the pathogen's population structure in the rest of Africa. In this study, we aimed to assess the diversity and gene flow of the pathogen between major maize producing countries in East and Southern Africa (Kenya, Uganda, Zambia, Zimbabwe, and South Africa). A total of 964 single-spore isolates were made from GLS lesions and confirmed as C.zeina using PCR diagnostics. The other causal agent of GLS, Cercospora zeae-maydis, was absent. Genotyping all the C.zeina isolates with 11 microsatellite markers and a mating-type gene diagnostic revealed (i) high genetic diversity with some population structure between the five African countries, (ii) cryptic sexual recombination, (iii) that South Africa and Kenya were the greatest donors of migrants, and (iv) that Zambia had a distinct population. We noted evidence of human-mediated long-distance dispersal, since four haplotypes from one South African site were also present at five sites in Kenya and Uganda. There was no evidence for a single-entry point of the pathogen into Africa. South Africa was the most probable origin of the populations in Kenya, Uganda, and Zimbabwe. Continuous annual maize production in the tropics (Kenya and Uganda) did not result in greater genetic diversity than a single maize season (Southern Africa). Our results will underpin future management of GLS in Africa through effective monitoring of virulent C.zeina strains.

Identification and characterization of Cercospora beticola necrosis-inducing effector CbNip1.

Cercospora beticola is a hemibiotrophic fungus that causes cercospora leaf spot disease of sugar beet (Beta vulgaris). After an initial symptomless biotrophic phase of colonization, necrotic lesions appear on host leaves as the fungus switches to a necrotrophic lifestyle. The phytotoxic secondary metabolite cercosporin has been shown to facilitate fungal virulence for several Cercospora spp. However, because cercosporin production and subsequent cercosporin-initiated formation of reactive oxygen species is light-dependent, cell death evocation by this toxin is only fully ensured during a period of light. Here, we report the discovery of the effector protein CbNip1 secreted by C. beticola that causes enhanced necrosis in the absence of light and, therefore, may complement light-dependent necrosis formation by cercosporin. Infiltration of CbNip1 protein into sugar beet leaves revealed that darkness is essential for full CbNip1-triggered necrosis, as light exposure delayed CbNip1-triggered host cell death. Gene expression analysis during host infection shows that CbNip1 expression is correlated with symptom development in planta. Targeted gene replacement of CbNip1 leads to a significant reduction in virulence, indicating the importance of CbNip1 during colonization. Analysis of 89 C. beticola genomes revealed that CbNip1 resides in a region that recently underwent a selective sweep, suggesting selection pressure exists to maintain a beneficial variant of the gene. Taken together, CbNip1 is a crucial effector during the C. beticola-sugar beet disease process.

Comparative transcriptome analysis of two Cercospora sojina strains reveals differences in virulence under nitrogen starvation stress.

BACKGROUND: Cercospora sojina is a fungal pathogen that causes frogeye leaf spot in soybean-producing regions, leading to severe yield losses worldwide. It exhibits variations in virulence due to race differentiation between strains. However, the candidate virulence-related genes are unknown because the infection process is slow, making it difficult to collect transcriptome samples. RESULTS: In this study, virulence-related differentially expressed genes (DEGs) were obtained from the highly virulent Race 15 strain and mildly virulent Race1 strain under nitrogen starvation stress, which mimics the physiology of the pathogen during infection. Weighted gene co-expression network analysis (WGCNA) was then used to find co-expressed gene modules and assess the relationship between gene networks and phenotypes. Upon comparison of the transcriptomic differences in virulence between the strains, a total of 378 and 124 DEGs were upregulated, while 294 and 220 were downregulated in Race 1 and Race 15, respectively. Annotation of these DEGs revealed that many were associated with virulence differences, including scytalone dehydratase, 1,3,8-trihydroxynaphthalene reductase, and ß-1,3-glucanase. In addition, two modules highly correlated with the highly virulent strain Race 15 and 36 virulence-related DEGs were found to contain mostly ß-1,4-glucanase, ß-1,4-xylanas, and cellobiose dehydrogenase. CONCLUSIONS: These important nitrogen starvation-responsive DEGs are frequently involved in the synthesis of melanin, polyphosphate storage in the vacuole, lignocellulose degradation, and cellulose degradation during fungal development and differentiation. Transcriptome analysis indicated unique gene expression patterns, providing further insight into pathogenesis.