Codon Optimization Enables the Geneticin Resistance Gene to Be Applied Efficiently to the Genetic Manipulation of the Plant Pathogenic Fungus Botrytis cinerea.

Botrytis cinerea can infect almost all of the important horticultural crops and cause severe economic losses globally every year. Modifying candidate genes and studying the phenotypic changes are among the most effective ways to unravel the pathogenic mechanism of this crop killer. However, few effective positive selection markers are used for B. cinerea genetic transformation, which limits multiple modifications to the genome, especially genes involving redundant functions. Here, we optimized a geneticin resistance gene, BcNPTII, based on the codon usage preference of B. cinerea. We found that BcNPTII can greatly increase the transformation efficiency of B. cinerea under G418 selection, with approximately 30 times higher efficiency than that of NPTII, which is applied efficiently to transform Magnaporthe oryzae. Using the gene replacement method, we successfully knocked out the second gene BOT2, with BcNPTII as the selection marker, from the mutant ΔoahA, in which OAHA was first replaced by the hygromycin resistance gene HPH in a field strain. We obtained the double knockout mutant ΔoahA Δbot2. Our data show that the codon-optimized BcNPTII is an efficient positive selection marker for B. cinerea transformation and can be used for various genetic manipulations in B. cinerea, including field wild-type strains.

Plant defensin MtDef4-derived antifungal peptide with multiple modes of action and potential as a bio-inspired fungicide.

Chemical fungicides have been instrumental in protecting crops from fungal diseases. However, increasing fungal resistance to many of the single-site chemical fungicides calls for the development of new antifungal agents with novel modes of action (MoA). The sequence-divergent cysteine-rich antifungal defensins with multisite MoA are promising starting templates for design of novel peptide-based fungicides. Here, we experimentally tested such a set of 17-amino-acid peptides containing the γ-core motif of the antifungal plant defensin MtDef4. These designed peptides exhibited antifungal properties different from those of MtDef4. Focused analysis of a lead peptide, GMA4CG_V6, showed that it was a random coil in solution with little or no secondary structure elements. Additionally, it exhibited potent cation-tolerant antifungal activity against the plant fungal pathogen Botrytis cinerea, the causal agent of grey mould disease in fruits and vegetables. Its multisite MoA involved localization predominantly to the plasma membrane, permeabilization of the plasma membrane, rapid internalization into the vacuole and cytoplasm, and affinity for the bioactive phosphoinositides phosphatidylinositol 3-phosphate (PI3P), PI4P, and PI5P. The sequence motif RRRW was identified as a major determinant of the antifungal activity of this peptide. While topical spray application of GMA4CG_V6 on Nicotiana benthamiana and tomato plants provided preventive and curative suppression of grey mould disease symptoms, the peptide was not internalized into plant cells. Our findings open the possibility that truncated and modified defensin-derived peptides containing the γ-core sequence could serve as promising candidates for further development of bio-inspired fungicides.

Molecular identification and characterization of Botrytis cinerea associated to the endemic flora of semi-desert climate in Chile.

Botrytis cinerea is a phytopathogenic fungus that infects over 200 plant species and can cause significant crop losses in local and worldwide agricultural industries. However, its presence in the endemic flora in the Coquimbo Region and its impact on local flora have not been studied yet. In order to determine whether Botrytis spp is present in the native plant in the Coquimbo Region, fifty-two field-samples were analysed. A total of 30 putative Botrytis spp were isolated and phenotypic and genetically characterized. The internal transcribed spacer (ITS) analysis of these isolates revealed that it corresponded to genus Botrytis. For further confirmation, nuclear protein-coding genes (G3PDH, HSP60, and RPB2) were sequenced and showed 100% identity against B. cinerea. Complementary to this, Botrytis can also be clustered in two different groups, group I (B. pseudocinerea) and group II (B. cinerea), based on DNA polymorphism, the Botrytis isolates were identified as member of group II. On the order hand, we investigated the presence and frequency distribution of the transposable elements boty and flipper in the isolates obtained. The results indicate that 83.3% of the isolates presented both transposable elements, boty and flipper, indicating that the most prevalent genotype was transpose. In addition, 16.6% of the isolates showed substantially reduced virulence in apple fruit in comparison to B05.10 strain. According to fungicide resistance studies, the results indicate that resistance to Fenhexamid or Boscalid was observed in the 22.6% of isolates. The results show for the first time that B. cinerea has not been described before in fourteen new host plants and contributes to our fundamental understanding of the presence of B. cinerea in the native plant in the Coquimbo Region and the possible ecological impact of this disease on native and endemic plants.

Abiotic stress resistance, plant growth promotion and antifungal potential of halotolerant bacteria from a Tunisian solar saltern.

The uses of halotolerant bacteria isolated from naturally saline habitats have the potential to be useful crop protection agents for plants in stressful conditions. These beneficial microbes generate several plant growth regulators and bioactive molecules, which enhance plant protection from adversities, such as plant pathogens, salts and metals stresses. In this study, 15 halotolerant bacterial strains endowed with important antimicrobial activities were isolated from Sfax solar saltern (Tunisia). All of these strains were characterized by biochemical and molecular tools aiming to investigate their in-vitro and in-vivo antifungal potentialities, plant growth promotion capabilities and metal tolerance abilities under saline stress condition. The 16S rRNA gene sequencing showed that the isolated strains were affiliated to different phylum and three species were described for the first time as plant growth promoting strains (Idiomarina zobelli FMH6v, Nesterenkonia halotolerans FMH10 and Halomonas janggokensis FMH54). The tested strains exhibited several potentialities: to tolerate high salt and heavy metal concentrations, to produce biosurfactants, exopolysaccharides and extracellular hydrolytic enzymes, to form biofilms and to liberate plant promoting substances. Eight strains were able to protect tomatoes fruits from the proliferation of the fungal disease caused by Botrytis cinerea and six strains improved plant vigor indexes. Principal component analysis showed an important correlation between in-vitro and in-vivo potentialities and two strains Bacillus velezensis FMH2 and Bacillus subtilis subsp. spizizenii FMH45 were statistically considered as the most effective strains in protecting plants from fungal pathogens attack and promoting the growth of tomatoes seedlings under saline and multi heavy-metals stress conditions.

[Gray mold disease of Paris polyphylla var. yunnanensis and its pathogen identification].

Gray mold disease is one of the most important diseases of planted Paris polyphylla var. yunnanensis, the disease appeared primarily as blossom blights and fruit rots, but also as stem rots, leaf rots.In this study, the pathogenetic fungi was isolated from plant tissue or sclerotia that covering the fruit of diseased P. polyphylla var. yunnanensis, the pathogen was certified according to Koch's Postulation. The pathogen produced abundant black, irregular sclerotia on surface of diseased plants and potato dextrose agar. The conidiophores and clusters of oval conidia resembled a grape-like cluster, the size of conidia was 9.70-13.70 µm [average of (11.32±0.82)µm]×7.05-9.12 µm [average of (8.24±0.48)µm], the microconidia produced on potato dextrose agar were spherical,and the size was （3.34±0.31） µm,the pathogen was identified as Botrytis sp based on morphological characteristics. The DNA sequence analysis of the G3PDH, HSP60, RPB2 genes placed the pathogen in a single clade that outside defined species of Botrytis, so the pathogen could be identified as a new species of Botrytis. The pathogen requires 20 °C, pH 8, darkness or low light condition for the best growth.

Gray mold disease of Paris polyphylla var. yunnanensis and its pathogen identification / 中国中药杂志

Gray mold disease is one of the most important diseases of planted Paris polyphylla var. yunnanensis, the disease appeared primarily as blossom blights and fruit rots, but also as stem rots, leaf rots.In this study, the pathogenetic fungi was isolated from plant tissue or sclerotia that covering the fruit of diseased P. polyphylla var. yunnanensis, the pathogen was certified according to Koch's Postulation. The pathogen produced abundant black, irregular sclerotia on surface of diseased plants and potato dextrose agar. The conidiophores and clusters of oval conidia resembled a grape-like cluster, the size of conidia was 9.70-13.70 μm [average of (11.32±0.82)μm]×7.05-9.12 μm [average of (8.24±0.48)μm], the microconidia produced on potato dextrose agar were spherical,and the size was （3.34±0.31） μm,the pathogen was identified as Botrytis sp based on morphological characteristics. The DNA sequence analysis of the G3PDH, HSP60, RPB2 genes placed the pathogen in a single clade that outside defined species of Botrytis, so the pathogen could be identified as a new species of Botrytis. The pathogen requires 20 °C, pH 8, darkness or low light condition for the best growth.