Host-Induced Gene Silencing of a G Protein α Subunit Gene CsGpa1 Involved in Pathogen Appressoria Formation and Virulence Improves Tobacco Resistance to Ciboria shiraiana

Hypertrophy sorosis scleroteniosis caused by Ciboria shiraiana is the most devastating disease of mulberry fruit. However, few mulberry lines show any resistance to C. shiraiana. An increasing amount of research has shown that host-induced gene silencing (HIGS) is an effective strategy for enhancing plant tolerance to pathogens by silencing genes required for their pathogenicity. In this study, two G protein α subunit genes, CsGPA1 and CsGPA2, were identified from C. shiraiana. Silencing CsGPA1 and CsGPA2 had no effect on hyphal growth but reduced the number of sclerotia and increased the single sclerotium weight. Moreover, silencing CsGpa1 resulted in increased fungal resistance to osmotic and oxidative stresses. Compared with wild-type and empty vector strains, the number of appressoria was clearly lower in CsGPA1-silenced strains. Importantly, infection assays revealed that the virulence of CsGPA1-silenced strains was significantly reduced, which was accompanied by formation of fewer appressoria and decreased expression of several cAMP/PKA- or mitogen-activated protein-kinase-related genes. Additionally, transgenic Nicotiana benthamiana expressing double-stranded RNA targeted to CsGpa1 through the HIGS method significantly improved resistance to C. shiraiana. Our results indicate that CsGpa1 is an important regulator in appressoria formation and the pathogenicity of C. shiraiana. CsGpa1 is an efficient target to improve tolerance to C. shiraiana using HIGS technology.

An APSES Transcription Factor Xbp1 Is Required for Sclerotial Development, Appressoria Formation, and Pathogenicity in Ciboria shiraiana

Sclerotinia diseases are important plant fungal diseases that, causes huge economic worldwide losses every year. Ciboria shiraiana is the main pathogen that results in mulberry sclerotia diseases. Sclerotia and appressoria play important roles in long-term pathogen survival and in host infection during life and disease cycles. However, the molecular mechanisms of sclerotial development and appressoria formation in C. shiraiana have not been well studied. Here, an Asm1p, Phd1p, Sok2p, Efg1p and StuAp (APSES)-type transcription factor in C. shiraiana, CsXbp1, involved in sclerotial development and appressoria formation was functionally characterized. Bioinformatics analyses showed that CsXbp1 contained an APSES-type DNA binding domain. The expression levels of CsXbp1 were higher in sclerotia and during later stages of infection. Compared with wild-type strains, hyphal growth was slower, the number and weight of sclerotia were reduced significantly, and appressoria formation was obviously delayed in CsXbp1 RNA interference (RNAi) strains. Moreover, the CsXbp1 RNAi strains showed weakened pathogenicity owing to compound appressoria defects. Tobacco rattle virus-mediated host-induced gene silencing enabled Nicotiana benthamiana to increase its resistance to C. shiraiana by reducing the CsXbp1 transcripts level. Thus, CsXbp1 plays vital roles in sclerotial formation, appressoria formation, and pathogenicity in C. shiraiana. This study provides new insights into the infection mechanisms of C. shiraiana and plant resistance breeding.

In vitro antimicrobial activity of methanolic extracts from cutaneous secretions of Amazonian amphibians against phytopathogens of agricultural interest

ABSTRACT The biochemical defense mechanisms of amphibians involve cutaneous secretions of bioactive molecules with antimicrobial activity. This study evaluated the in vitro activity of methanolic extracts from cutaneous secretions of two amphibian species of the Bufonidae family, Rhaebo guttatus and Rhinella marina, in the control of the phytopathogens Fusarium udum, Fusarium solani, Colletotrichum truncatum, Aspergillus flavus, Rhizoctonia solani, Macrophomina phaseolina, and Calonectria pseudometrosideri. The R. guttatus extract decreased the mycelial growth of F. udum, F. solani, A. flavus, and M. phaseolina at some tested concentrations. The R. marina extract decreased the mycelial growth of C. truncatum at the concentration of 0.5 mg mL-¹, and inhibited the mycelial growth of A. flavus at concentrations of 0.1 and 0.5 mg mL-¹, which was similar to the inhibition by the positive control. The R. marina extract also decreased the microsclerotia production by R. solani at concentrations of 0.2 and 0.3 mg mL-¹. In addition, the extracts inhibited conidial sporulation and germination at varying degrees. The inhibition of appressoria formation in C. truncatum by the R. guttatus and R. marina extracts was 85-99% and 63-100%, respectively. Our results demonstrated that treatment with extracts from R. guttatus and R. marina cutaneous secretions showed antifungal activity against the studied phytopathogens.

ACTIVIDAD ENZIMÁTICA Y PROCESO DE INFECCIÓN INICIAL DE LA PLANTA DE GUARANÁ (Paullinia cupana) POR CEPAS PATOGÉNICAS Y ENDOFÍTICAS DE Colletotrichum guaranicola

Anthracnose of the guarana plant (Paullinia cupana) is a disease caused by Colletotrichum guaranicola Albuq. Few studies have analyzed the origin, the form of penetration and the colonization of endophytic fungi in the guarana plant. This study aimed to determine differences in the enzymatic activities of endophytic and pathogenic strains of C. guaranicola during the initial infection process in leaves of clones sensitive to guarana anthracnose. Fourteen pathogenic and nine endophytic strains of C. guaranicola were isolated from guarana leaves with and without anthracnose symptoms, respectively. The enzymatic activities of protease, amylase, polyphenol oxidase, lipase, pectinase, and cellulolytic activities were evaluated for each fungal strain. The experimental design for each enzymatic assay was completely randomized with five replicates. The enzymatic evaluation was based on the measurement of halo produced by the enzyme reaction in each assay. Conidia were evaluated during germination and appressoria formation. Results did not detect enzymatic difference between pathogenic and endophytic strains. Data prior to germination showed differences between endophytic and pathogenic strains. Colonization of plant tissues by pathogenic strain was evidenced after the appearance of disease symptoms 48 hours after inoculation (hai), while for endophytic strains the presence of appressoria was observed on the surface of the epidermis 48 hai, and colonization of cells was not observed.

Oligomycins Inhibit Magnaporthe oryzae Triticum and suppress wheat blast disease

Oligomycins are macrolide antibiotics, produced by Streptomyces spp., show biological activities to several microorganisms like bacteria, fungi, nematodes and peronosporomycetes. Conidiogenesis, germination of conidia and formation of appressoria are crucial for a successful disease cycle and pathogenicity of the filamentous fungal phytopathogen. The goal of this research was to evaluate the effects of two oligomycins, oligomycin B and oligomycin F along with a commercial fungicide Nativo® 75WG on hyphal growth, conidiogenesis, conidia germination, appressoria formation, and disease development of a worrisome wheat blast fungus, Magnaporthe oryzae Triticum (MoT) pathotype. Both oligomycins suppressed the growth of MoT mycelia depending on the dose. Between the two natural products, oligomycin F displayed the maximum inhibition of MoT hyphal growth accompanied by oligomycin B with minimum inhibitory concentrations (MICs) of 0.005 and 0.05 μg/disk, respectively. The application of the compounds also completely halted the conidia formation in the MoT mycelia in agar medium. A further bioassay showed that these compounds significantly inhibited MoT conidia germination and induced lysis; if germinated, induced abnormal germ tube and suppressed appressoria formation. Interestingly, the application of these macrolides significantly inhibited wheat blast disease on detached leaves of wheat. This is a first report on the inhibition of mycelial growth, process of conidia formation, germination of conidia, morphological changes in germinated conidia, and suppression of blast disease of wheat by oligomycins from Streptomyces spp. A further study is needed to evaluate the mode of action and field trials of these natural compounds to consider them as biopesticides for controlling this devastating wheat killer.

CgSCD1 Is Essential for Melanin Biosynthesis and Pathogenicity of Colletotrichum gloeosporioides

Colletotrichum gloeosporioides, an important phytopathogenic fungus, mainly infects tropical fruits and results in serious anthracnose. Previous studies have shown that melanin biosynthesis inhibitor can inhibit the melanization of the appressoria of Magnaporthe grisea and Colletotrichum orbiculare, resulting in limited infection of the hosts. In this study, we identified and characterized a scytalone dehydratase gene (CgSCD1) from C. gloeosporioides which is involved in melanin synthesis. The CgSCD1 gene deletion mutant ΔCgscd1 was obtained using homologous recombination. The ΔCgscd1 mutant showed no melanin accumulation on appressoria formation and vegetative hyphae. Furthermore, the virulence of ΔCgscd1 was significantly reduced in comparison with the wild-type (WT) strain. Further investigations showed that the growth rate as well as germination and appressorium formation of ΔCgscd1 displayed no difference compared to the wild-type and complemented transformant Cgscd1com strains. Furthermore, we found that the appressorial turgor pressure in the ΔCgscd1 mutant showed no difference compared to that in the WT and Cgscd1com strains in the incipient cytorrhysis experiment. However, fewer infectious hyphae of ΔCgscd1 were observed in the penetration experiments, suggesting that the penetration ability of nonpigmented appressoria was partially impaired. In conclusion, we identified the CgSCD1 gene, which is involved in melanin synthesis and pathogenicity, and found that the melanization defect did not affect appressorial turgor pressure in C. gloeosporioides.

Host-induced silencing of the Colletotrichum gloeosporioides conidial morphology 1 gene (CgCOM1) confers resistance against Anthracnose disease in chilli and tomato

Anthracnose disease is caused by the ascomycetes fungal species Colletotrichum, which is responsible for heavy yield losses in chilli and tomato worldwide. Conventionally, harmful pesticides are used to contain anthracnose disease with limited success. In this study, we assessed the potential of Host-Induced Gene Silencing (HIGS) approach to target the Colletotrichum gloeosporioides COM1 (CgCOM1) developmental gene involved in the fungal conidial and appressorium formation, to restrict fungal infection in chilli and tomato fruits. For this study, we have developed stable transgenic lines of chilli and tomato expressing CgCOM1-RNAi construct employing Agrobacterium-mediated transformation. Transgenic plants were characterized by molecular and gene expression analyses. Production of specific CgCOM1 siRNA in transgenic chilli and tomato RNAi lines was confirmed by stem-loop RT-PCR. Fungal challenge assays on leaves and fruits showed that the transgenic lines were resistant to anthracnose disease-causing C. gloeosporioides in comparison to wild type and empty-vector control plants. RT-qPCR analyses in transgenic lines revealed barely any CgCOM1 transcripts in the C. gloeosporioides infected tissues, indicating near complete silencing of CgCOM1 gene expression in the pathogen. Microscopic examination of the Cg-challenged leaves of chilli-CgCOM1i lines revealed highly suppressed conidial germination, germ tube development, appressoria formation and mycelial growth of C. gloeosporioides, resulting in reduced infection of plant tissues. These results demonstrated highly efficient use of HIGS in silencing the expression of essential fungal developmental genes to inhibit the growth of pathogenic fungi, thus providing a highly precise approach to arrest the spread of disease.

Medicago lupulina lines with defects in the development of efficient arbuscular mycorrhiza

Background. The work is aimed to solve actual problems in biology of arbuscular mycorrhiza (AM). Currently, a lot of mutants had been obtained in various plant model objects with defects in genes controlling AM development, however, the mechanisms controlling development of effective AM symbiosis are still unclear. Materials and methods. The authors conducted a mutagenesis in Medicago lupulina, a new convenient model plant for molecular-genetic studies. High mycotrophic M. lupulina line have early and high response to mycorrhization, high seed production, as well as signs of dwarfism under conditions without of AM and low level of phosphorus available for plants. This method allows visually to identify plant lines with defects in AM symbiosis. Results. 14 modes for mutagenesis by ethylmethanesulfonate were conducted. Usage of 3 mutagenesis modes allowed to obtain: productive M1 progeny with high part of viable seedlings (73.3%–86.0%); 1405 plants in M2 progeny. Conclusion. According to population analysis for mutant plants in M2 progeny (up to M9 generation) 15 plant lines were selected: one Myc– plant line unable to form AM symbiosis, 4 Pen– plant lines unable to form AM symbiosis, but characterized by appressoria formation; 3 Rmd– plant lines forming low-activity ineffective AM symbiosis; 3 Rmd– plant lines forming low-activity effective AM and 4 Rmd++ plant lines forming effective AM with high abundance of symbiotic structures (mycelium/arbuscules/vesicles) in the roots.