Three cystatin open reading frames named TcCys1, TcCys2 and TcCys3 were identified in cDNA libraries from compatible interactions between Theobroma cacao (cacao) and Moniliophthora perniciosa. In addition, an ORF named TcCys4 was identified in the cDNA library of the incompatible interaction. The cDNAs encoded conceptual proteins with 209, 127, 124, and 205 amino acid residues, with a deduced molecular weight of 24.3, 14.1, 14.3 and 22.8 kDa, respectively. His-tagged recombinant proteins were purified from Escherichia coli expression, and showed inhibitory activities against M. perniciosa. The four recombinant cystatins exhibited K(i) values against papain in the range of 152-221 nM. Recombinant TcCYS3 and TcCYS4 immobilized in CNBr-Sepharose were efficient to capture M. perniciosa proteases from culture media. Polyclonal antibodies raised against the recombinant TcCYS4 detected that the endogenous protein was more abundant in young cacao tissues, when compared with mature tissues. A ~85 kDa cacao multicystatin induced by M. perniciosa inoculation, MpNEP (necrosis and ethylene-inducing protein) and M. perniciosa culture supernatant infiltration were detected by anti-TcCYS4 antibodies in cacao young tissues. A direct role of the cacao cystatins in the defense against this phytopathogen was proposed, as well as its involvement in the development of symptoms of programmed cell death.

The collective impact of several environmental factors on the biocontrol activity of Trichoderma stromaticum (Ts) against Moniliophthora perniciosa (Mp), the cause of cacao witches' broom disease, was assessed under field conditions of shaded cacao (Theobroma cacao) in south-eastern Bahia, Brazil. Biocontrol experiments were performed adjacent to an automated weather station, with sensors and Ts-treated brooms placed at different canopy heights. Sporulation occurred at the same dates for all Ts isolates, but in different quantities. Broom moisture >30%, air temperature of approximately 23 ± 3°C, relative humidity >90%, solar radiation intensities <0AE12 KW m )2 and wind speed near zero were the key environmental parameters that preceded Ts sporulation events. A multiple logistic regression indicated that these weather variables combined were capable of distinguishing sporulation from non-sporulation events, with a significant effect of wind speed. Analyses of environmental factors at ground level indicated similar pre-sporulation conditions, with a soil moisture content above a threshold of 0AE34 m 3 m )3 preceding all sporulation events. The sporulation of five selected Ts isolates was compared at four different canopy heights. Isolates responded differently to weather variation in terms of sporulation and antagonism to Mp at different canopy levels, indicating that different microclimates are established along the vertical profile of a shaded cacao plantation. The potential of these findings for development of predictive mathematical models and disease-management approaches is discussed.