Evaluation of antifungal metabolites activity from bacillus licheniformis OE-04 against Colletotrichum gossypii.

Anthracnose disease in the cotton plant caused by fungal pathogen Colletotrichum gossypii. It is supposed to be most critical diseases in the cotton crop as it causes infection and leads to complete damaging of the cotton crop by infecting the leaves, stems, and bolls in the field. The disease control is challenging due to the absence of an effective fungicide without damaging the farmer health and environment. So the series of experiments were designed to assess the antagonistic activity of biosurfactant released by strain Bacillus licheniformis OE-04 against the anthracnose causing agent in cotton and this strain was screened out from forty eight strain of rhizobacteria. We also estimated the heat stability and pH range and toxicity of biosurfactant produced by strain 0E-04. The results showed that biosurfactant has maximum antifungal activity against C. gossypii. In vitro study concluded that the biosurfactant can reduce fungal activity by inhibiting the spore germination of C. gossypii. Moreover, the biosurfactant also has wide pH and temperature range. We observed Antifungal activity of biosurfactant at 5 to 10 pH range and temperature range was also wide from room temperature to 100 °C. We also observed the toxicity of biosurfactant produced by Bacillus licheniformis against zebra fish (Danio rerio). We were noticed that biosurfactant have least harmful effect with maximum concentration. The study confirmed that biosurfactant of Bacillus licheniformis have high pH and heat stability range with least harmful effects so it can be a good replacement of chemical pesticides for cotton anthracnose control.

The recombinant pea defensin Drr230a is active against impacting soybean and cotton pathogenic fungi from the genera Fusarium, Colletotrichum and Phakopsora.

Plant defensins are antifungal peptides produced by the innate immune system plants developed to circumvent fungal infection. The defensin Drr230a, originally isolated from pea, has been previously shown to be active against various entomopathogenic and phytopathogenic fungi. In the present study, the activity of a yeast-expressed recombinant Drr230a protein (rDrr230a) was tested against impacting soybean and cotton fungi. First, the gene was subcloned into the yeast expression vector pPICZαA and expressed in Pichia pastoris. Resulting rDrr230a exhibited in vitro activity against fungal growth and spore germination of Fusarium tucumaniae, which causes soybean sudden death syndrome, and against Colletotrichum gossypii var. cephalosporioides, which causes cotton ramulosis. The rDrr230a IC50 corresponding to inhibition of fungal growth of F. tucumaniae and C. gossypii var. cephalosporioides was 7.67 and 0.84 µM, respectively, demonstrating moderate activity against F. tucumaniae and high potency against C. gossypii var. cephalosporioides. Additionally, rDrr230a at 25 ng/µl (3.83 µM) resulted in 100 % inhibition of spore germination of both fungi, demonstrating that rDrr230a affects fungal development since spore germination. Moreover, rDrr230a at 3 µg/µl (460.12 µM) inhibited 100 % of in vitro spore germination of the obligatory biotrophic fungus Phakopsora pachyrhizi, which causes Asian soybean rust. Interestingly, rDrr230a substantially decreased the severity of Asian rust, as demonstrated by in planta assay. To our knowledge, this is the first report of a plant defensin active against an obligatory biotrophic phytopathogenic fungus. Results revealed the potential of rDrr230a as a candidate to be used in plant genetic engineering to control relevant cotton and soybean fungal diseases.

Variability of seed-borne Colletotrichum strains in cotton based on ITS1 and ITS2 ribossomal genes analysis / Variabilidade de estirpes de Colletotrichum provenientes de sementes de algodão com base na análise dos genes ribossomais ITS1 e ITS2

The use of DNA sequences analysis has been an important mean to distinguish and to identify populations of organisms at different levels. By molecular markers several complex organisms have been successful detected in plants for distinct aims. Ribosomal DNA (rDNA) has been used to evaluate genetic variability, microorganism phylogeny and to develop specific primers for detection of plant pathogens in plant tissues. In this study, the objective was to characterize isolates of Colletotrichum gossypii var. cephalosporioides and Colletotrichum gossypii, collected in different regions of Brazil, by analyzing the nucleotide sequence of rDNA regions. ITS1, ITS2, and the intervening 5.8S gene were amplified by PCR and their sequences compared to each other and to those from other species registered in the GenBank. The rDNA of isolates associated with Gossypium spp. showed sequence identities ranging from 96 to 100% in the ITS1 region, 98 to 100% in the 5.8S gene, and 97 to 100% in the ITS2 region. The sequences were submitted to UPGMA analysis, and according to the phylogenetic trees, the C. gossypii var. cephalosporioides and C. gossypii species clustered together along with isolates of Glomerella cingulata from mango and papaya, and thus no distinction could be made between isolates of those organisms.

O uso de sequências de fragmentos de DNA tem sido importante ferramenta para distinguir e identificar populações de organismos em diferentes níveis de variação. Por meio de marcadores moleculares alguns organismos com variações taxonômicas complexas têm sido detectados com sucesso em tecidos vegetais. O DNA ribossomal tem sido utilizado para avaliar variabilidade genética, filogenia de micro-organismo e para desenvolver oligonucleotídeos específicos visando à detecção de fitopatógenos. Nesse estudo o objetivo foi comparar isolados do complexo Colletotrichum, incluindo C. gosssypii var. cephalosporioides e C. gossypii, coletados em diferentes regiões do Brasil, todos associados às sementes de algodão, pela análise de sequências de nucleotídeos de regiões de rDNA. ITS1, ITS2 e o gene 5.8S que foram amplificados por PCR e suas sequências comparadas entre si com outras sequências depositadas no GenBank. O rDNA de isolados associados com Gossypium spp. mostraram identidades de sequências na faixa de 96 to 100% na região ITS1, 98 to 100% na região de 5.8S, e 97 a 100% na região ITS2. As sequências foram submetidas a análise UPGMA, e de acordo com as árvores filogenéticas , C. gossypii var. cephalosporioides e C. gossypii fizeram parte de um mesmo cluster junto com isolados de Glomerella cingulata de manga e mamão, e assim nenhuma distinção pode ser feita entre os isolados destes organismos.

Rizobactérias no controle da mancha angular do algodoeiro / Rhizobacteria to control cotton bacterial blight

Avaliou-se o potencial de rizobactérias na indução de resistência do algodoeiro à Xanthomonas axonopodis pv. malvacearum. Após o isolamento das rizobactérias, foram selecionados os isolados capazes de reduzir os sintomas da mancha angular bacteriana em casa de vegetação, os quais foram aplicados espacialmente separados do patógeno desafiador. Os melhores isolados foram testados quanto à capacidade de reduzir os sintomas da ramulose e da murcha de Verticillium e de inibir diretamente os patógenos in vitro. Do total de 123 isolados de rizobactérias foram selecionados cinco, L2-1 (Bacillus cereus), MT5-6 (Bacillus cereus), L2-2 (Achromobacter xylosoxidans), MT5-5 (Bacillus cereus) e MT5-11 (Brevibacterium sp.), os quais apresentaram controle da mancha angular acima de 40 por cento, em relação à testemunha. Nenhum isolado reduziu a severidade da ramulose e da murcha de Verticillium em relação à testemunha, nem apresentou efeito inibitório direto in vitro a X. axonopodis pv. malvacearum e Colletotrichum gossypii var. cephalosporioides. Para V. dahliae, apenas o isolado L2-1 apresentou efeito inibitório.

The potential of rhizobacteria was evaluated for resistance induction against Xanthomonas axonopodis pv. malvacearum. After isolation, the rhizobacteria were screened for the reduction of angular leaf spot severity under greenhouse conditions. They were spatially separated from the challenging pathogen. The best isolates were tested for the capacity to reduce ramulose and Verticillium wilt severity and directly inhibit pathogens in vitro. From a total of 123 rhizobacterial isolates, five were selected, L2-1 (Bacillus cereus), MT5-6 (Bacillus cereus), L2-2 (Achromobacter xylosoxidans), MT5-5 (Bacillus cereus) and MT5-11 (Brevibacterium sp.), which showed angular leaf spot control above 40 percent as compared to the control. The tested isolates neither reduced the severity of ramulose and verticillium wilt compared to the control nor showed in vitro direct inhibition to X. axonopodis pv. malvacearum and Colletotrichum gossypii var. cephalosporioides. For V. dahliae, only isolate L2-1 showed direct inhibition.