Biochemical changes during the development of witches' broom: the most important disease of cocoa in Brazil caused by Crinipellis perniciosa

Witches' broom disease (WBD) is caused by the hemibiotrophic basidiomycete fungus Crinipellis perniciosa, which is one of the most important diseases of cocoa in the western hemisphere. In this study, the contents of soluble sugars, amino acids, alkaloids, ethylene, phenolics, tannins, flavonoids, pigments, malondialdehyde (MDA), glycerol, and fatty acids were analysed in cocoa (Theobroma cacao) shoots during the infection and development of WBD. Alterations were observed in the content of soluble sugars (sucrose, glucose, and fructose), asparagine and alkaloids (caffeine and theobromine), ethylene, and tannins. Ethylene and tannins increased prior to symptom development and declined with the death of the infected tissues. Furthermore, MDA and glycerol concentrations were higher in infected tissue than in the controls, while fatty acid composition changed in the infected tissues. Chlorophylls a and b were lower throughout the development of the disease while carotenoids and xanthophylls dropped in the infected tissue by the time of symptom development. These results show co-ordinated biochemical alterations in the infected tissues, indicating major stress responses with the production of ethylene. Ethylene levels are hypothesized to play a key role in broom development. Some of the other biochemical alterations are directly associated with ethylene synthesis and may be important for the modification of its effect on the infected tissues.5641386587

Protein extraction for proteome analysis from cacao leaves and meristems, organs infected by Moniliophthora perniciosa, the causal agent of the witches' broom disease

Preparation of high-quality proteins from cacao vegetative organs is difficult due to very high endogenous levels of polysaccharides and polyphenols. in order to establish a routine procedure for the application of proteomic and biochemical analysis to cacao tissues, three new protocols were developed; one for apoplastic washing fluid (AWF) extraction, and two for protein extraction - under denaturing and nondenaturing conditions. The first described method allows a quick and easy collection of AWF - using infiltration-centrifugation procedure - that is representative of its composition in intact leaves according to the smaller symplastic contamination detected by the use of the hexose phosphate isomerase marker. Protein extraction under denaturing conditions for 2-DE was remarkably improved by the combination of chemically and physically modified processes including phenol, SDS dense buffer and sonication steps. With this protocol, high-quality proteins from cacao leaves and meristems were isolated, and for the first time well-resolved 1-DE and 2-DE protein patterns of cacao vegetative organs are shown. It also appears that sonication associated with polysaccharide precipitation using tert-butanol was a crucial step for the nondenaturing protein extraction and subsequent enzymatic activity detection. It is expected that the protocols described here could help to develop high-level proteomic and biochemical studies in cacao also being applicable to other recalcitrant plant tissues.29112391240

Biological control of cacao diseases

This chapter discusses the advances in biological control of cacao diseases over the last 15 years. Most attention has been focused on biological control of frosty pod rot (Moniliophthora roreri), witches' broom (Moniliophthora perniciosa) and black pod disease (Phytophthora spp.). Research on biocontrol of other diseases in the cacao phyllosphere or rhizosphere is scarce or in its infancy. There is, however, a steady increase in information regarding the factors influencing and the mechanisms underlying biological control of cacao diseases as well as practical aspects such as inoculum production, formulation and application. There has been a clear shift away from inundative approaches using epiphytic BCAs towards more classical biocontrol approaches using bacterial and fungal endophytes as well as vesicular arbuscular mycorrhiza. These have the advantage that they can permanently establish themselves in the cacao tree. Moreover, besides direct competition for space and nutrients, antibiosis and mycoparasitism, through induced resistance and growth promotion, endophytes have a larger arsenal of mechanisms through which they can help protect their host. Endophytic BCAs could thus provide more effective and sustainable disease control. Recent advances in our understanding of the mechanisms through which endophytic biocontrol agents can reduce pest and disease impact provide possibilities for innovative disease control strategies, including combination therapies together with natural or chemical products. Continued work on production, formulation and application is also necessary in order for biocontrol to become economically interesting. However, biological control will not become a stand-alone solution for disease control but should become part of integrated pest management strategies, with cultural management as a central and reinforcing pillar. (Résumé d'auteur