Requirement of Simultaneous Assessment of Crystal- and Supernatant-Related Entomotoxic Activities of Bacillus thuringiensis Strains for Biocontrol-Product Development

Abstract

Bioinsecticides with lower concentrations of endospores/crystals and without loss of efficiency are economically advantageous for pest biocontrol. In addition to Cry proteins, other Bacillus thuringiensis (Bt) toxins in culture supernatants (SN) have biocontrol potential (e.g., Vip3A, Cry1I, Sip1), whereas others are unwanted (β-exotoxins), as they display widespread toxicity across taxa. A strain simultaneously providing distinct toxin activities in crystals and SN would be desirable for bioinsecticides development; however, strains secreting β-exotoxins should be discarded, independently of other useful entomotoxins. Entomotoxicity of crystals and SN from a Brazilian Bt tolworthi strain (Btt01) was tested against Spodoptera frugiperda to assess the potential for biocontrol-product development based on more than one type of toxin/activity. Tests showed that 107 endospores mL−1 caused >80% of larvae mortality, suggesting Btt01 may be used in similar concentrations as those of other Bt-based biopesticides. When it was applied to cornfields, a significant 60% reduction of larvae infestation was observed. However, bioassays with Btt01 SN revealed a thermostable toxic activity. Physicochemical characterization strongly suggests the presence of unwanted β-exotoxins, with isolate-specific temporal variation in its secretion. Knowledge of the temporal pattern of secretion/activity in culture for all forms of toxins produced by a single strain is required to both detect useful activities and avoid the potential lack of identification of undesirable toxins. These findings are discussed in the contexts of commercial Bt product development, advantages of multiple-activity strains, and care and handling recommended for large-scale fermentation systems.

Immunodetection of the toxic portion of Vip3A reveals differential temporal regulation of its secretion amongBacillus thuringiensisstrains

“…However, isolate‐specific physiological variations in culture can affect screening procedures for these secreted toxins, which may underestimate the true biocontrol potential of various isolates under scrutiny (Argôlo‐Filho et al . , ). Immunochemical methods are helpful in this context, since they provide practical and efficient tools for detection of specific proteotoxins within a heterogeneous set of compounds; they are advantageous over PCR methods, as developed antibodies can directly detect the products of expressing genes of interest.…”

“…Second, the physiological variations among strains in the temporal secretion of the protein (Argôlo‐Filho et al . , ) may have interfered in its detection by the antibody, either by Vip3A secretion being initiated after the last culture time evaluated (28 h) or by being secreted and degraded (e.g. Milne et al .…”

“…As previously evaluated and discussed, physiological variations among Bt isolates can significantly affect entomotoxin screening processes, so that the use of a single collection/assessment time for various strains grown simultaneously can lead to premature discard of useful strains (Argôlo‐Filho et al . , ). A variation among strains in the maximum amount of secreted Vip3A toxin detected was observed, including OD readings (HD11 and HD29) that were even higher than the average readings for the positive control.…”

“…, ; Argôlo‐Filho et al . , ). The most common strategies used to screen Bt strains usually include PCR‐based assays (Rice ) associated with another confirmatory procedure (Masson et al .…”

Anthropogenic action shapes the evolutionary ecology of Bacillus thuringiensis: response to Ruan et al

“…To us, however, this process is, in fact, currently ongoing everywhere largely because bacteria are among the fastest evolving organisms on Earth (with generation times in hours), and because Bt's genome changes rapidly through mutation and incorporation of new genes by conjugation, transformation, and transduction. Such a high genetic and physiologic diversity in Bt (the raw material of evolution) was evidenced by significant temporal variation in protein/ insect-toxicity secretion in culture and the presence of alternative toxins among isolates from a single subspecies [8,9]. It is noteworthy that the metabolic expense of producing insecticidal crystal proteins does not seem to significantly alter the fitness of Bt, as suggested by the similar levels of plant-colonization ability shown by strains lacking those ICPs [10].…”