Reexamining the Mycovirome of Botrytis spp.

Botrytis species cause gray mold disease in more than 200 crops worldwide. To control this disease, chemical fungicides are usually applied. However, more sustainable control alternatives should be explored, such as the use of hypovirulent mycovirus-infected fungal strains. To determine the mycovirome of two Botrytis species, B. cinerea and B. prunorum, we reanalyzed RNA-Seq and small RNA-Seq data using different assembly programs and an updated viral database, aiming to identify new mycoviruses that were previously not described in the same dataset. New mycoviruses were identified, including those previously reported to infect or be associated with B. cinerea and Plasmopara viticola, such as Botrytis cinerea alpha-like virus 1 and Plasmopara viticola lesion-associated ourmia-like virus 80. Additionally, two novel narnaviruses, not previously identified infecting Botrytis species, have been characterized, tentatively named Botrytis cinerea narnavirus 1 and Botrytis narnavirus 1. The analysis of small RNAs suggested that all identified mycoviruses were targeted by the antiviral fungal mechanism, regardless of the viral genome type. In conclusion, the enlarged list of newly found viruses and the application of different bioinformatics approaches have enabled the identification of novel mycoviruses not previously described in Botrytis species, expanding the already extensive list.

In Planta Gene Expression Analysis and Colonization of Fusarium oxysporum.

In planta gene expression analysis and GFP-based confocal microscopy are two powerful techniques that may be coupled to assess the extent and dynamics of plant colonization by a fungal pathogen. Here we describe methods to prepare common bean plants for inoculation with a highly virulent strain of Fusarium oxysporum f. sp. phaseoli, quantify the extent of colonization by RT-qPCR, and visualize the colonized tissues by confocal microscopy.

Nitric Oxide Metabolism Affects Germination in Botrytiscinerea and Is Connected to Nitrate Assimilation.

Nitric oxide regulates numerous physiological processes in species from all taxonomic groups. Here, its role in the early developmental stages of the fungal necrotroph Botrytis cinerea was investigated. Pharmacological analysis demonstrated that NO modulated germination, germ tube elongation and nuclear division rate. Experimental evidence indicates that exogenous NO exerts an immediate but transitory negative effect, slowing down germination-associated processes, and that this effect is largely dependent on the flavohemoglobin BCFHG1. The fungus exhibited a "biphasic response" to NO, being more sensitive to low and high concentrations than to intermediate levels of the NO donor. Global gene expression analysis in the wild-type and ΔBcfhg1 strains indicated a situation of strong nitrosative and oxidative stress determined by exogenous NO, which was much more intense in the mutant strain, that the cells tried to alleviate by upregulating several defense mechanisms, including the simultaneous upregulation of the genes encoding the flavohemoglobin BCFHG1, a nitronate monooxygenase (NMO) and a cyanide hydratase. Genetic evidence suggests the coordinated expression of Bcfhg1 and the NMO coding gene, both adjacent and divergently arranged, in response to NO. Nitrate assimilation genes were upregulated upon exposure to NO, and BCFHG1 appeared to be the main enzymatic system involved in the generation of the signal triggering their induction. Comparative expression analysis also showed the influence of NO on other cellular processes, such as mitochondrial respiration or primary and secondary metabolism, whose response could have been mediated by NmrA-like domain proteins.

A Major Effect Gene Controlling Development and Pathogenicity in Botrytis cinerea Identified Through Genetic Analysis of Natural Mycelial Non-pathogenic Isolates.

Botrytis cinerea is a necrotrophic plant pathogenic fungus with a wide host range. Its natural populations are phenotypically and genetically very diverse. A survey of B. cinerea isolates causing gray mold in the vineyards of Castilla y León, Spain, was carried out and as a result eight non-pathogenic natural variants were identified. Phenotypically these isolates belong to two groups. The first group consists of seven isolates displaying a characteristic mycelial morphotype, which do not sporulate and is unable to produce sclerotia. The second group includes one isolate, which sporulates profusely and does not produce sclerotia. All of them are unresponsive to light. Crosses between a representative mycelial non-pathogenic isolate and a highly aggressive field isolate revealed that the phenotypic differences regarding pathogenicity, sporulation and production of sclerotia cosegregated in the progeny and are determined by a single genetic locus. By applying a bulked segregant analysis strategy based on the comparison of the two parental genomes the locus was mapped to a 110 kb region in chromosome 4. Subcloning and transformation experiments revealed that the polymorphism is an SNP affecting gene Bcin04g03490 in the reference genome of B. cinerea. Genetic complementation analysis and sequencing of the Bcin04g03490 alleles demonstrated that the mutations in the mycelial isolates are allelic and informed about the nature of the alterations causing the phenotypes observed. Integration of the allele of the pathogenic isolate into the non-pathogenic isolate fully restored the ability to infect, to sporulate and to produce sclerotia. Therefore, it is concluded that a major effect gene controlling differentiation and developmental processes as well as pathogenicity has been identified in B. cinerea. It encodes a protein with a GAL4-like Zn(II)2Cys6 binuclear cluster DNA binding domain and an acetyltransferase domain, suggesting a role in regulation of gene expression through a mechanism involving acetylation of specific substrates.