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Theobromine exerts deleterious effects on animal physiology. Removal of theobromine from the millions of metric tons of cocoa pod husks (CPH) discarded annually could allow for the production of cheap, CPH-based animal feed. The aim of this study was to evaluate safety and nutritional value of bio-detheobrominated CPH in Sprague-Dawley rats. Theobromine was removed from CPH by treatment with an isolate of Talaromyces verruculosus (TvTD). Substituted feeds containing CPH were formulated by replacing 30% or 50% of the maize content of regular rat feed with TvTD-treated or inactivated TvTD-treated CPH. Feeding groups included control groups without or with theobromine administration. Effects of the feed formulations on water and feed intake, weight gain, blood biochemistry and organ-specific toxicity were assessed. Rats ingesting theobromine in inactivated TvTD-treated CPH-based diet or by oral gavage variably exhibited marked deleterious effects, mainly evident in body weight, thymus wet weight and tissue histology. In contrast, substitution with TvTD-treated CPH caused significant increase in body weight. Substitution at 30% did not cause mortality or organ-specific toxicity with reference to the testes, kidneys, spleen or liver, unlike substitution at 50%. The data demonstrate that detheobrominated CPH may safely replace up to 30% of maize in animal feed formulations.

Pythium-induced damping-off disease is a major disease limiting cucumber and tomato production in different parts of the world. The current study investigated the efficiency of Talaromyces variabilis and its bioactive metabolites in suppressing Pythium-induced damping-off of cucumbers and tomatoes. T. variabilis inhibited the in vitro growth of P. aphanidermatum in solid and liquid media. In addition, abnormalities in P. aphanidermatum hyphae were observed as a result of T. variabilis. Extracts from T. variabilis induced cellular leakage and suppressed oospore production of P. aphanidermatum. Biochemical analyses of T. variabilis metabolites showed that T. variabilis produces glucanase, cellulase and siderophores, suggesting the contribution of these metabolites in the inhibition of P. aphandermatum growth and in hyphal abnormalities. Treating cucumber seeds with spore and mycelial suspension of T. variabilis isolates led to a significant improvement in the seedling survival of P. aphanidermatum-inoculated seedlings from 18 to 52% (improvement by 34%) for isolate 48 P and from 30-66% (improvement by 36%) for isolate 28 R. Similarly, treating tomato seeds with spore and mycelial suspension of T. variabilis isolates led to a significant improvement in the seedling survival of P. aphanidermatum-inoculated seedlings from 7 to 36% (improvement by 29%) for isolate 28 R and from 20 to 64% (improvement by 44%) for isolate 48 P. Differences in the percent improvement in seedling survival between experiments may be related to difference in the efficacy of the two different isolates or their interaction with the hosts and pathogen. The use of T. variabilis in the biocontrol of Pythium-induced diseases may offer alternatives to the currently used chemical control.

In microbial cultures the production of secondary metabolites is affected by experimental conditions, and the discovery of novel compounds is often prevented by the re-isolation of known metabolites. To limit this, it is possible to cultivate microorganisms by simulating naturally occurring interactions, where microbes co-exist in complex communities. In this work, co-culturing experiments of the biocontrol agent Trichoderma harzianum M10 and the endophyte Talaromyces pinophilus F36CF have been performed to elicit the expression of genes which are not transcribed in standard laboratory assays. Metabolomic analysis revealed that the co-culture induced the accumulation of siderophores for both fungi, while production of M10 harzianic and iso-harzianic acids was not affected by F36CF. Conversely, metabolites of the latter strain, 3-O-methylfunicone and herquline B, were less abundant when M10 was present. A novel compound, hereby named harziaphilic acid, was isolated from fungal co-cultures, and fully characterized. Moreover, harzianic and harziaphilic acids did not affect viability of colorectal cancer and healthy colonic epithelial cells, but selectively reduced cancer cell proliferation. Our results demonstrated that the co-cultivation of plant beneficial fungi may represent an effective strategy to modulate the production of bioactive metabolites and possibly identify novel compounds.