Genomic-proteomic analysis of a novel Bacillus thuringiensis strain: toxicity against two lepidopteran pests, abundance of Cry1Ac5 toxin, and presence of InhA1 virulence factor.

Bacillus thuringiensis (Bt) is a biological alternative to the indiscriminate use of chemical insecticides in agriculture. Due to resistance development on insect pests to Bt crops, isolating novel Bt strains is a strategy for screening new pesticidal proteins or strains containing toxin profile variety that can delay resistance. Besides, the combined genomic and proteomic approaches allow identifying pesticidal proteins and virulence factors accurately. Here, the genome of a novel Bt strain (Bt TOL651) was sequenced, and the proteins from the spore-crystal mixture were identified by proteomic analysis. Toxicity bioassays with the spore-crystal mixture against larvae of Diatraea saccharalis and Anticarsia gemmatalis, key pests of sugarcane and soybean, respectively, were performed. The toxicity of Bt TOL651 varies with the insect; A. gemmatalis (LC50 = 1.45 ng cm-2) is more susceptible than D. saccharalis (LC50 = 73.77 ng cm-2). Phylogenetic analysis of the gyrB gene indicates that TOL651 is related to Bt kenyae strains. The genomic analysis revealed the presence of cry1Aa18, cry1Ac5, cry1Ia44, and cry2Aa9 pesticidal genes. Virulence factor genes such as phospholipases (plcA, piplc), metalloproteases (inhA), hemolysins (cytK, hlyIII, hblA, hblC, hblD), and enterotoxins (nheA, nheB, nheC) were also identified. The combined use of the genomic and proteomic data indicated the expression of Cry1Aa18, Cry1Ac5, and Cry2Aa9 proteins, with Cry1Ac5 being the most abundant. InhA1 also was expressed and may contribute to Bt TOL651 pathogenicity. These results provide Bt TOL651 as a new tool for the biocontrol of lepidopteran pests.

Occurrence of Colletotrichum musicola causing anthracnose on Manihot esculenta.

Cassava (Manihot esculenta) is one of the main food sources of energy in developing countries owing to its starch-rich roots (Pinweha et al., 2015). Anthracnose is considered the most destructive disease of the aerial part of this crop (Bragança et al., 2016; Liu et al., 2019), and it is caused by species such as Colletotrichum plurivorum, C. karstii, C. fructicola, C. siamense (Liu et al., 2019), and C. theobromicola (Oliveira et al, 2016). In 2019, leaves with irregular necrotic spots, typical symptoms of anthracnose, were collected in Pará, Brazil. Commercial sampled fields showed 20% of incidence of anthracnose. Colletotrichum strains were isolated and cultured on potato dextrose agar at 25 ºC with a 12-h light photoperiod from surface-disinfected (70% alcohol and 1% sodium hypochlorite) lesion transition area. Five of the obtained isolates exhibited brown colonies on the upper and lower surfaces. Conidia were hyaline, cylindrical and aseptate, 12.82-15.23 µm × 3.52-5.25 µm in size. These phenotypic characters were similar to those belonging to C. orchidearum sensu lato (Damm et al. 2019). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ß-tubulin (TUB2), chitin synthase 1(CHS-1), and histone HIS3 partial gene were amplified and sequenced for one representative isolate (UFT/Coll89). Sequences were deposited in GenBank [Accession numbers: MT396235 (GAPDH), MT800856 (TUB2), MT800870 (CHS-1), and MT856672 (HIS3)]. BLASTn searches of CHS-1 and HIS3 sequences showed 100% identity to C. musicola. Maximum Likelihood Phylogenetic analysis, including previously published sequences of closely related species, placed the isolate from Cassava in the C. musicola clade with 100% support, and confidently it assigned to this species. Pathogenicity was proven with inoculations by spraying a conidial suspension (106 conida mL-1) on 3-month-old cassava plants (three unwounded leaves per plant). The plants were placed in a humid chamber at 25 °C for 48h, and a 12-h photoperiod. The negative control was represented by plants inoculated with sterile distilled water. The experiment was repeated twice. The same symptoms observed in the field were reproduced only in inoculated leaves, from which the pathogen was reisolated lesions fulfilling Koch's postulates. No symptoms were observed on the negative control. To our knowledge, this is the first report of C. musicola joining a group of new and emergent species of Colletotrichum causing anthracnose in cassava producing regions around the world. The identification of this species causing cassava anthracnose is crucial to improve the disease control strategies and resistance breeding programs.