Genome-Wide Association Studies in Sunflower: Towards Sclerotinia sclerotiorum and Diaporthe/Phomopsis Resistance Breeding.

Diseases caused by necrotrophic fungi, such as the cosmopolitan Sclerotinia sclerotiorum and the Diaporthe/Phomopsis complex, are among the most destructive diseases of sunflower worldwide. The lack of complete resistance combined with the inefficiency of chemical control makes assisted breeding the best strategy for disease control. In this work, we present an integrated genome-wide association (GWA) study investigating the response of a diverse panel of sunflower inbred lines to both pathogens. Phenotypic data for Sclerotinia head rot (SHR) consisted of five disease descriptors (disease incidence, DI; disease severity, DS; area under the disease progress curve for DI, AUDPCI, and DS, AUDPCS; and incubation period, IP). Two disease descriptors (DI and DS) were evaluated for two manifestations of Diaporthe/Phomopsis: Phomopsis stem canker (PSC) and Phomopsis head rot (PHR). In addition, a principal component (PC) analysis was used to derive transformed phenotypes as inputs to a univariate GWA (PC-GWA). Genotypic data comprised a panel of 4269 single nucleotide polymorphisms (SNP), generated via genotyping-by-sequencing. The GWA analysis revealed 24 unique marker-trait associations for SHR, 19 unique marker-trait associations for Diaporthe/Phomopsis diseases, and 7 markers associated with PC1 and PC2. No common markers were found for the response to the two pathogens. Nevertheless, epistatic interactions were identified between markers significantly associated with the response to S. sclerotiorum and Diaporthe/Phomopsis. This suggests that, while the main determinants of resistance may differ for the two pathogens, there could be an underlying common genetic basis. The exploration of regions physically close to the associated markers yielded 364 genes, of which 19 were predicted as putative disease resistance genes. This work presents the first simultaneous evaluation of two manifestations of Diaporthe/Phomopsis in sunflower, and undertakes a comprehensive GWA study by integrating PSC, PHR, and SHR data. The multiple regions identified, and their exploration to identify candidate genes, contribute not only to the understanding of the genetic basis of resistance, but also to the development of tools for assisted breeding.

Efficient Agrobacterium tumefaciens-mediated transformation system of Diaporthe caulivora.

This is the first report describing the genetic transformation of Diaporthe caulivora, the soybean stem canker fungus. A simple and 100% efficient protocol of Agrobacterium tumefaciens-mediated transformation used mycelium as starting material and the hygromycin B resistance and green fluorescent protein (GFP) as a selection and reporter agents, respectively. All transgenic isolates were mitotically stable in two independent experiments and polymerase chain reaction with hygromycin B resistance primers confirmed successful T-DNA integration into the fungal genome. Plant-fungus interaction studies, including pathogenicity, latency, and endophytism, as well as further studies of random and targeted mutagenesis will be possible with GFP-expressing isolates of D. caulivora and other species in the Diaporthe / Phomopsis complex.

Specific PCR-based detection of Phomopsis heveicola the cause of leaf blight of Coffee (Coffea arabica L.) in China.

Coffee (Coffea arabica L.) is currently grown in many tropical and subtropical areas countries and is a major traded commodity for the developing world. Coffee leaf blight, caused by Phomopsis heveicola, is one of the most important fungal diseases dangerous to coffee crops in China. This study aimed to develop a PCR-based diagnostic method for detecting P. heveicola in planta. Specific primers (CPHF/CPHR) were designed based on sequence data of region of internal transcribed spacer (ITS1 and ITS4) of P. heveicola. The efficiency and specificity of CPHF/CPHR were established by PCR analysis of DNA from P. heveicola strains isolated from China and fungal isolates of other genera. A single amplification product of 318 bp was detected from DNA P. heveicola isolates. No amplification product was observed with any of the other fungal isolates tested. The specific primers designed and employed in PCR detected P. heveicola up to 3 pg from DNA isolated. This is the first report on the development of a species-specific PCR assay for identification and detection of P. heveicola. Thus, the PCR-based assay developed was very specific, rapid and sensitive tool for the detection of pathogen P. heveicola.

The disruption of the MAPKK gene triggering the synthesis of flavonoids in endophytic fungus Phomopsis liquidambaris.

Flavonoids, which are mainly extracted from plants, are important antioxidants and play an important role in human diseases. However, the growing market demand is limited by low productivity and complex production processes. Herein, the flavonoids biosynthesis pathway of the endophytic fungus Phomopsis liquidambaris was revealed. The mitogen-activated protein kinase kinase (MAPKK) of the strain was disrupted using a newly constructed CRISPR-Cas9 system mediated by two gRNAs which was conducive to cause plasmid loss. The disruption of the MAPKK gene triggered the biosynthesis of flavonoids against stress and resulted in the precipitation of flavonoids from fermentation broth. Naringenin, kaempferol and quercetin were detected in fed-batch fermentation with yields of 5.65 mg/L, 1.96 mg/L and 2.37 mg/L from P. liquidambaris for dry cell weigh using the mixture of glucose and xylose and corn steep powder as carbon source and nitrogen source for 72 h, respectively. The biosynthesis of flavonoids was triggered by disruption of MAPKK gene in P. liquidambaris and the mutant could utilize xylose.