Biosurfactants synthesized by endophytic Bacillus strains as control of Moniliophthora perniciosa and Moniliophthora roreri

Abstract

Ecuador is responsible for up to 70 % of the world's production of fine flavor cocoa.Although fungal diseases have reduced cocoa production, eco-friendly alternatives have been implemented to control the spread of phytopathogens. This study aimed to explore the potential of endophytic strains isolated from fine flavor Nacional cocoa trees to synthetize compounds with antimicrobial and surface active properties. It was determined that under in vitro conditions, seven strains (DS03, DS07, DS18, DS23, DS31, DS34 and DS50) exhibited antifungal activity that inhibited the mycelial growth of Moniliophthora roreri and Moniliophthora perniciosa. The strains were identified based on their morphological (pattern forming Bacilli) and genetic characteristics using the 16S SSU rRNA gene sequences. Furthermore, we determined that the endophytic strains contained genes that encoded the lipopeptides Fengycin (fenA, fenB, fenC, fenE), Iturin (ituB, ituC, ituD), and Bacyllomicin D (bamC, bamD). The production of biosurfactant compounds was determined by a reduction in surface tension in the mineral medium. Additionally, the biosurfactant crude extract (BCE) was tested for its inhibitory activity against M. perniciosa and M. roreri. The minimal inhibitory concentration (MIC) was determined to be 0.07 and 0.035 mg mL -1 , respectively, in both cases in a fungistatic action mode. The swelling granulation and fragmentation of both phytopathogens' hyphae were observed using the BCE treatment.These results suggested the biocontrol capacity of the endophytic strains, which under in vitro conditions, have the ability to synthesize antifungal and surface active compounds that might be used as an ecological alternative in cocoa plants to prevent infection by phytopathogens.

Chemical Characterization of Trichoderma spp. Extracts with Antifungal Activity against Cocoa Pathogens

“…On the other hand, the best PGI against M. roreri was observed for the extract of T. spirale (C10) at 1000 µg/mL (26.20 ± 2.06%) and 100 µg/mL (17.10 ± 1.23%). Higher PGI values (48%) have been reported for culture filtrates from Brazil [ 31 ] and comparable data (15.90–37.10%) for isolates from Mexico [ 20 ] against M. roreri . Differences among results could be attributed to the technique employed since the referenced studies used a dual-culture assay, through which the inoculum size, agar volume, well size, and other variables possibly affect the fungal interactions [ 17 ], isolates, culture filtrates, extracts, or compounds assessed.…”

“…perniciosa interaction are limited, higher inhibitory activity (75%) affecting mycelial growth and even spore germination of M. perniciosa has been reported for T. harzianum [ 28 ], and significant mycoparasitism levels against M. roreri have been achieved by T. spirale [ 29 ]. In addition, remarkably, antifungal activity against M. perniciosa and M. roreri has been found for foliar endophytic fungi [ 30 ] and endophytic Bacillus [ 31 ] isolated from cocoa cultivars. Additionally, T. spirale has been shown to inhibit the growth of Fusarium oxysporum during in vitro and greenhouse experiments, demonstrating the relevance of the metabolites produced by this strain, which also indicates plant protection against fungal disease [ 32 ].…”

Biosurfactant from Bacillus subtilis DS03: Properties and Application in Cleaning Out Place System in a Pilot Sausages Processing

“…In previous work, we isolated, identified, and selected B. subtillis DS03 from cocoa crops for producing an organic extract with high surface activity. A preliminary evaluation showed that BCE production was affected by carbon source (e.g., C 6 H 12 O 6 ), nitrogen source (e.g., NaNO 3 ), and phosphate salts (e.g., Na 2 HPO 4 and KH 2 PO 4 ) [ 22 ]. This study evaluated three alternative carbon sources for BCE economic production (i.e., cassava starch, whey, and molasses) according to a new sustainable approach to the biotechnology process.…”

“…Bacillus subtilis DS03 strain was isolated from cocoa crops, producing the microbial surfactant [ 22 ]. The medium used for BS production was MMI (20 g/L dextrose, 1 g/L yeast extract, 8.5 g/L NaNO 3 , 5.7 g/L Na 2 HPO 4 , 4 g/L KH 2 PO 4 , 0.21 g/L MgSO 4 7H 2 O, 0.01 g/L FeSO 4 7H 2 O, and 7 × 10 −6 g/L CaCl 2 ).…”

“…The BCE was collected by centrifugation for 20 min at 4 °C, 11,000× g , and washed twice with acid distilled water at pH 2 to eliminate any impurities. The BCE was dried in an oven (Thermo Scientific 6963, Waltham, MA, USA) at 40 °C until obtaining a constant weight and then quantified by gravimetry (g/L) [ 22 ].…”