Edição gênica de Metarhizium Anisopliae Brm 2335 por Crispr/Cas9 “Marker-Free” e seu endofitismo em plantas de soja

Abstract

The cosmopolitan fungus of the genus Metarhizium exhibits a versatile lifestyle, being effective in controlling various insect pests and also as a plant endophyte. The advent of CRISPR/Cas technology has revolutionized genome editing, offering simplicity, precision and cost-effectiveness. Although its application in entomopathogenic fungi has been limited, including M. anisopliae, recent advances show promising results. However, the integration of Cas9 and the selection of markers in fungal genomes present challenges, such as reducing their efficiency and toxicity and effects on non-target pests. Therefore, markerless genetic engineering through a recyclable CRISPR system presents a viable solution, allowing the efficient generation of mutants without compromising fitness and virulence. In the first chapter, this pioneering study constructed marker-free strains of M. anisopliae using a recyclable CRISPR/Cas9 system. The precise deletion of albA and ku70, together with the insertion of the gfp (green fluorescent protein) cassette confirmed the efficiency of the system. Furthermore, the M. anisopliae Δku70 and gfp constructs maintained their original fitness, killing adults of the brown soybean bug Euschistus heros with the same efficiency as the wild-type (WT) isolate. In the second chapter, the endophytic colonization of BRM 2335 in soybean plants after seed treatment was determined. Seed vigor and germination were not affected at doses of 1 × 108 and 5 × 108 conidia/mL. Seed treatment with 1× 108 conidia/mL significantly increased the dry mass of soybean plant roots only after seven days, but not after 14 days. On the other hand, the dry mass of the aerial part of the plants was similar for both untreated seeds and those treated with the fungus, at seven and 14 days. The length and height of soybean plants were also not affected by seed treatment with the fungus. In agar/water M. anisopliae was isolated in all stem samples and in all roots of the distal part. In soil, M. anisopliae was isolated endophytically in a few plant samples (14) and was found in the distal part of the root, stem, cotyledon and non-expanded primary leaves of soybean. Gfptagged Metarhizium facilitated the location and confirmation of isolate BRM 2335 in soybean plants. Isolation of BRM 2335 gfp in soybean plants was similar when seeds were treated with 1 × 108 , 5 × 108 and 1 × 109 conidia/mL. There was also no difference in the extraction of Metarhizium gfp by cutting part of the plants compared to macerating parts of the plants. In this study, endophytic colonization by Metarhizium was confirmed in a few samples of soybean plants, possibly due to competition from other fungi present in the soil. This innovative approach, CRISPR/Cas9, has great potential to facilitate in-depth molecular studies, understand their ecological roles in agricultural systems, and improve the effectiveness of biocontrol against insect pests through genetic improvements.