A Multi-Omics Characterization of the Natural Product Potential of Tropical Filamentous Marine Cyanobacteria

Microbial natural products are important for the understanding of microbial interactions, chemical defense and communication, and have also served as an inspirational source for numerous pharmaceutical drugs. Tropical marine cyanobacteria have been highlighted as a great source of new natural products, however, few reports have appeared wherein a multi-omics approach has been used to study their natural products potential (i.e., reports are often focused on an individual natural product and its biosynthesis). This study focuses on describing the natural product genetic potential as well as the expressed natural product molecules in benthic tropical cyanobacteria. We collected from several sites around the world and sequenced the genomes of 24 tropical filamentous marine cyanobacteria. The informatics program antiSMASH was used to annotate the major classes of gene clusters. BiG-SCAPE phylum-wide analysis revealed the most promising strains for natural product discovery among these cyanobacteria. LCMS/MS-based metabolomics highlighted the most abundant molecules and molecular classes among 10 of these marine cyanobacterial samples. We observed that despite many genes encoding for peptidic natural products, peptides were not as abundant as lipids and lipopeptides in the chemical extracts. Our results highlight a number of highly interesting biosynthetic gene clusters for genome mining among these cyanobacterial samples.     

Comparative Metabologenomics Analysis of Polar Actinomycetes

Biosynthetic and chemical datasets are the two major pillars for microbial drug discovery in the omics era. Despite the advancement of analysis tools and platforms for multi-strain metabolomics and genomics, linking these information sources remains a considerable bottleneck in strain prioritisation and natural product discovery. In this study, molecular networking of the 100 metabolite extracts derived from applying the OSMAC approach to 25 Polar bacterial strains, showed growth media specificity and potential chemical novelty was suggested. Moreover, the metabolite extracts were screened for antibacterial activity and promising selective bioactivity against drug-persistent pathogens such as Klebsiella pneumoniae and Acinetobacter baumannii was observed. Genome sequencing data were combined with metabolomics experiments in the recently developed computational approach, NPLinker, which was used to link BGC and molecular features to prioritise strains for further investigation based on biosynthetic and chemical information. Herein, we putatively identified the known metabolites ectoine and chrloramphenicol which, through NPLinker, were linked to their associated BGCs. The metabologenomics approach followed in this study can potentially be applied to any large microbial datasets for accelerating the discovery of new (bioactive) specialised metabolites.     

Structure-Activity Relationships of the Bioactive Thiazinoquinone Marine Natural Products Thiaplidiaquinones A and B

In an effort to more accurately define the mechanism of cell death and to establish structure-activity relationship requirements for the marine meroterpenoid alkaloids thiaplidiaquinones A and B, we have evaluated not only the natural products but also dioxothiazine regioisomers and two precursor quinones in a range of bioassays. While the natural products were found to be weak inducers of ROS in Jurkat cells, the dioxothiazine regioisomer of thiaplidiaquinone A and a synthetic precursor to thiaplidiaquinone B were found to be moderately potent inducers. Intriguingly, and in contrast to previous reports, the mechanism of Jurkat cell death (necrosis vs. apoptosis) was found to be dependent upon the positioning of one of the geranyl sidechains in the compounds with thiaplidiaquinone A and its dioxothiazine regioisomer causing death dominantly by necrosis, while thiaplidiaquinone B and its dioxothiazine isomer caused cell death via apoptosis. The dioxothiazine regioisomer of thiaplidiaquinone A exhibited more potent in vitro antiproliferative activity against human tumor cells, with NCI sub-panel selectivity towards melanoma cell lines. The non-natural dioxothiazine regioisomers were also more active in antiplasmodial and anti-farnesyltransferase assays than their natural product counterparts. The results highlight the important role that natural product total synthesis can play in not only helping understand the structural basis of biological activity of natural products, but also the discovery of new bioactive scaffolds.     

Coupling Mass Spectral and Genomic Information to Improve Bacterial Natural Product Discovery Workflows

Bacterial natural products possess potent bioactivities and high structural diversity and are typically encoded in biosynthetic gene clusters. Traditional natural product discovery approaches rely on UV- and bioassay-guided fractionation and are limited in terms of dereplication. Recent advances in mass spectrometry, sequencing and bioinformatics have led to large-scale accumulation of genomic and mass spectral data that is increasingly used for signature-based or correlation-based mass spectrometry genome mining approaches that enable rapid linking of metabolomic and genomic information to accelerate and rationalize natural product discovery. In this mini-review, these approaches are presented, and discovery examples provided. Finally, future opportunities and challenges for paired omics-based natural products discovery workflows are discussed.     

Application of Feature-Based Molecular Networking for Comparative Metabolomics and Targeted Isolation of Stereoisomers from Algicolous Fungi

Seaweed endophytic (algicolous) fungi are talented producers of bioactive natural products. We have previously isolated two strains of the endophytic fungus, Pyrenochaetopsis sp. FVE-001 and FVE-087, from the thalli of the brown alga Fucus vesiculosus. Initial chemical studies yielded four new decalinoylspirotetramic acid derivatives with antimelanoma activity, namely pyrenosetins A–C (1–3) from Pyrenochaetopsis sp. strain FVE-001, and pyrenosetin D (4) from strain FVE-087. In this study, we applied a comparative metabolomics study employing HRMS/MS based feature-based molecular networking (FB MN) on both Pyrenochaetopsis strains. A higher chemical capacity in production of decalin derivatives was observed in Pyrenochaetopsis sp. FVE-087. Notably, several decalins showed different retention times despite the same MS data and MS/MS fragmentation pattern with the previously isolated pyrenosetins, indicating they may be their stereoisomers. FB MN-based targeted isolation studies coupled with antimelanoma activity testing on the strain FVE-087 afforded two new stereoisomers, pyrenosetins E (5) and F (6). Extensive NMR spectroscopy including DFT computational studies, HR-ESIMS, and Mosher’s ester method were used in the structure elucidation of compounds 5 and 6. The 3’R,5’R stereochemistry determined for compound 6 was identical to that previously reported for pyrenosetin C (3), whose stereochemistry was revised as 3’S,5’R in this study. Pyrenosetin E (5) inhibited the growth of human malignant melanoma cells (A-375) with an IC₅₀ value of 40.9 μM, while 6 was inactive. This study points out significant variations in the chemical repertoire of two closely related fungal strains and the versatility of FB MN in identification and targeted isolation of stereoisomers. It also confirms that the little-known fungal genus Pyrenochaetopsis is a prolific source of complex decalinoylspirotetramic acid derivatives.     

Bio-Guided Isolation of Antimalarial Metabolites from the Coculture of Two Red Sea Sponge-Derived Actinokineospora and Rhodococcus spp.

Coculture is a productive technique to trigger microbes’ biosynthetic capacity by mimicking the natural habitats’ features principally by competition for food and space and interspecies cross-talks. Mixed cultivation of two Red Sea-derived actinobacteria, Actinokineospora spheciospongiae strain EG49 and Rhodococcus sp. UR59, resulted in the induction of several non-traced metabolites in their axenic cultures, which were detected using LC–HRMS metabolomics analysis. Antimalarial guided isolation of the cocultured fermentation led to the isolation of the angucyclines actinosporins E (1), H (2), G (3), tetragulol (5) and the anthraquinone capillasterquinone B (6), which were not reported under axenic conditions. Interestingly, actinosporins were previously induced when the axenic culture of the Actinokineospora spheciospongiae strain EG49 was treated with signalling molecule N-acetyl-d-glucosamine (GluNAc); this finding confirmed the effectiveness of coculture in the discovery of microbial metabolites yet to be discovered in the axenic fermentation with the potential that could be comparable to adding chemical signalling molecules in the fermentation flask. The isolated angucycline and anthraquinone compounds exhibited in vitro antimalarial activity and good biding affinity against lysyl-tRNA synthetase (PfKRS1), highlighting their potential developability as new antimalarial structural motif.     

Unveiling the Chemical Diversity of the Deep-Sea Sponge Characella pachastrelloides

Sponges are at the forefront of marine natural product research. In the deep sea, extreme conditions have driven secondary metabolite pathway evolution such that we might expect deep-sea sponges to yield a broad range of unique natural products. Here, we investigate the chemodiversity of a deep-sea tetractinellid sponge, Characella pachastrelloides, collected from ~800 m depth in Irish waters. First, we analyzed the MS/MS data obtained from fractions of this sponge on the GNPS public online platform to guide our exploration of its chemodiversity. Novel glycolipopeptides named characellides were previously isolated from the sponge and herein cyanocobalamin, a manufactured form of vitamin B₁₂, not previously found in nature, was isolated in a large amount. We also identified several poecillastrins from the molecular network, a class of polyketide known to exhibit cytotoxicity. Light sensitivity prevented the isolation and characterization of these polyketides, but their presence was confirmed by characteristic NMR and MS signals. Finally, we isolated the new betaine 6-methylhercynine, which contains a unique methylation at C-2 of the imidazole ring. This compound showed potent cytotoxicity towards against HeLa (cervical cancer) cells.     

Comparative Metabolomics Reveals Fungal Conversion of Co-Existing Bacterial Metabolites within a Synthetic Aspergillus-Streptomyces Community

In nature, secondary metabolites have been proven to be the essential communication media between co-occurring microorganisms and to influence their relationship with each other. In this study, we conducted a metabolomics survey of the secondary metabolites of an artificial co-culture related to a hydrothermal vent fungal–bacterial community comprising Aspergillus sclerotiorum and Streptomyces and their reciprocal relationship. The fungal strain was found to increase the secretion of notoamides and the compound cyclo(Pro-Trp) produced by the actinomycetes strain was discovered to be the responsible molecule. This led to the hypothesis that the fungi transformed cyclo(Pro-Trp) synthesized by the actinomycetes as the biosynthetic precursors of notoamides in the chemical communication. Further analysis showed Streptomyces sp. WU20 was efficient in transforming amino acids into cyclo(Pro-Trp) and adding tryptophan as well as proline into the chemical communication enhanced the induction of the notoamide accumulation. Thus, we propose that the microbial transformation during the synthetic metabolically-mediated chemical communication might be a promising means of speeding up the discovery of novel bioactive molecules. The objective of this research was to clarify the mechanism of microbial transformation for the chemical communication. Besides, this research also highlights the utility of mass spectrometry-based metabolomics as an effective tool in the direct biochemical analysis of community metabolites.     

Drugs from the Sea -Opportunities and Obstacles

The supply problem with regard to drug development and sustainable production lies in the limited amounts of biomass of most marine invertebrates available from wild stocks. Thus, most pharmacologically active marine natural products can only be isolated in minute yields. Total synthesis of pharmacologically active natural products has been successfully established but is in many cases economically not feasible due to the complexity of the molecular structures and the low yields. To solve the pressing supply issue in marine drug discovery, other strategies appear to be more promising. One of these is mariculture which has successfully been established with the bryozoan Bugula neritina (the source of the bryostatins) and the tunicate Ecteinascidia turbinata (the source of ET-743). Another strategy involves partial synthesis from precursors which are biotechnologically available. An example is ET-743 that can be partially synthesized from safracin B which is a metabolite of Pseudomonas fluorescens. There have been many examples of striking structural similarities between natural products obtained from marine invertebrates and those of microbial origin which suggests that microorganisms living in their invertebrate hosts could be the actual producers of these secondary metabolites. With regard to sustainable biotechnological production of pharmacologically important metabolites from marine invertebrates and their “endosymbionts”, a more advanced strategy is to focus on cloning and expression of the respective key biosynthetic gene clusters. This molecular biological approach will open up new avenues for biotechnological production of drugs or drug candidates from the sea.     

Quality not Quantity: The Role of Marine Natural Products in Drug Discovery and Reverse Chemical Proteomics

Reverse chemical proteomics combines affinity chromatography with phage display and promises to be a powerful new platform technology for the isolation of natural product receptors, facilitating the drug discovery process by rapidly linking biologically active small molecules to their cellular receptors and the receptors’ genes. In this paper we review chemical proteomics and reverse chemical proteomics and show how these techniques can add value to natural products research. We also report on techniques for the derivatisation of polystyrene microtitre plates with cleavable linkers and marine natural products that can be used in chemical proteomics or reverse chemical proteomics. Specifically, we have derivatised polystyrene with palau’amine and used reverse chemical proteomics to try and isolate the human receptors for this potent anticancer marine drug.     

Bacillimidazoles A−F,Imidazolium-Containing Compounds Isolated from a Marine Bacillus

Chemical investigations of a marine sponge-associated Bacillus revealed six new imidazolium-containing compounds, bacillimidazoles A–F (1–6). Previous reports of related imidazolium-containing natural products are rare. Initially unveiled by timsTOF (trapped ion mobility spectrometry) MS data, extensive HRMS and 1D and 2D NMR analyses enabled the structural elucidation of 1–6. In addition, a plausible biosynthetic pathway to bacillimidazoles is proposed based on isotopic labeling experiments and invokes the highly reactive glycolytic adduct 2,3-butanedione. Combined, the results of structure elucidation efforts, isotopic labeling studies and bioinformatics suggest that 1–6 result from a fascinating intersection of primary and secondary metabolic pathways in Bacillus sp. WMMC1349. Antimicrobial assays revealed that, of 1–6, only compound six displayed discernible antibacterial activity, despite the close structural similarities shared by all six natural products.     

Structure Elucidation of Calyxoside B,a Bipolar Sphingolipid from a Marine Sponge Cladocroce sp. through the Use of Beckmann Rearrangement

Marine sponges are an excellent source of biologically active secondary metabolites. We focus on deep-sea sponges for our discovery study. A marine sponge Cladocroce sp. exhibited cytotoxic activity in the bioactivity screening. From this sponge a previously unreported cytotoxic glycosphingolipid, calyxoside B, was isolated and the structure of this compound was elucidated by analyses of MS and NMR spectra and chemical derivatization. We converted the ketone in the middle of a long aliphatic chain into an oxime to which was applied Beckmann rearrangement to afford two positional isomers of amides. The products were subjected to acidic hydrolysis followed by LC-MS analysis, permitting us to assign unequivocally the position of the ketone. Calyxoside B shows cytotoxicity against HeLa cells with an IC₅₀ value of 31 µM and also weakly stimulated the production of cytokines in mice.     

Genomics- and Metabolomics-Based Investigation of the Deep-Sea Sediment-Derived Yeast,Rhodotorula mucilaginosa 50-3-19/20B

Red yeasts of the genus Rhodotorula are of great interest to the biotechnological industry due to their ability to produce valuable natural products, such as lipids and carotenoids with potential applications as surfactants, food additives, and pharmaceuticals. Herein, we explored the biosynthetic potential of R. mucilaginosa 50-3-19/20B collected from the Mid-Atlantic Ridge using modern genomics and untargeted metabolomics tools. R. mucilaginosa 50-3-19/20B exhibited anticancer activity when grown on PDA medium, while antimicrobial activity was observed when cultured on WSP-30 medium. Applying the bioactive molecular networking approach, the anticancer activity was linked to glycolipids, namely polyol esters of fatty acid (PEFA) derivatives. We purified four PEFAs (1–4) and the known methyl-2-hydroxy-3-(1H-indol-2-yl)propanoate (5). Their structures were deduced from NMR and HR-MS/MS spectra, but 1–5 showed no anticancer activity in their pure form. Illumina-based genome sequencing, de novo assembly and standard biosynthetic gene cluster (BGC) analyses were used to illustrate key components of the PEFA biosynthetic pathway. The fatty acid producing BGC3 was identified to be capable of producing precursors of PEFAs. Some Rhodotorula strains are able to convert inulin into high-yielding PEFA and cell lipid using a native exo-inulinase enzyme. The genomic locus for an exo-inulinase enzyme (g1629.t1), which plays an instrumental role in the PEFA production via the mannitol biosynthesis pathway, was identified. This is the first untargeted metabolomics study on R. mucilaginosa providing new genomic insights into PEFA biosynthesis.     

Marine Microorganism-Invertebrate Assemblages: Perspectives to Solve the “Supply Problem” in the Initial Steps of Drug Discovery

The chemical diversity associated with marine natural products (MNP) is unanimously acknowledged as the “blue gold” in the urgent quest for new drugs. Consequently, a significant increase in the discovery of MNP published in the literature has been observed in the past decades, particularly from marine invertebrates. However, it remains unclear whether target metabolites originate from the marine invertebrates themselves or from their microbial symbionts. This issue underlines critical challenges associated with the lack of biomass required to supply the early stages of the drug discovery pipeline. The present review discusses potential solutions for such challenges, with particular emphasis on innovative approaches to culture invertebrate holobionts (microorganism-invertebrate assemblages) through in toto aquaculture, together with methods for the discovery and initial production of bioactive compounds from these microbial symbionts.     

Efficacy of medicinal plant extracts against Formosan subterranean termite, Coptotermes formosanus

The Formosan subterranean termite, Coptotermes formosanus Shiraki, is among the most devastating termite pests. Natural products derived from plant extracts were tested in a discovery programme for effective, environment friendly termite control agents. Screening for anti-termitic activity of plant extracts with some known medicinal attributes could lead to the discovery of new agents for termite control. The aim of this study was to investigate the anti-termitic activity of crude leaf hexane, ethyl acetate, acetone and methanol extracts of Andrographis lineata Wallich ex Nees. (Acanthaceae), Andrographis paniculata (Burm.f.) Wall. ex Nees. (Acanthaceae), Argemone mexicana L. (Papaveraceae), Aristolochia bracteolata Lam. (Aristolochiaceae), Datura metel L. (Solanaceae), Eclipta prostrata L. (Asteraceae), Sesbania grandiflora (L.) Pers. (Fibaceae) and Tagetes erecta L. (Compositae) against C. formosanus. An impregnated filter paper no-choice bioassay method was followed. All the crude extracts showed anti-termitic activity in a dose-dependent manner and exhibited a significant activity after 24 h and 48 h of exposure; the highest termite mortality was found in leaf hexane extract of A. bracteolata, ethyl acetate extract of A. paniculata, D. metel, E. prostrata, methanol extract of A. lineata and D. metel after 24 h (LD₅₀ = 363, 371, 298, 292, 358 and 317 ppm; LD₉₀ = 1433, 1659, 1308, 1538, 1703 and 1469 ppm), respectively. The hexane extract of T. erecta, acetone extract of A. mexicana, methanol extract of S. grandiflora and T. erecta showed activity after 48 h (LD₅₀ = 245, 253, 289, 409 ppm; LD₉₀ = 1378, 1511, 1508 and 2425 ppm), respectively. Among the natural products tested, may provide a renewable source of safe natural wood preservatives. These findings corroborate traditional insecticidal application of selected plants and the results can be extended for the control of termites. The primary objective of the present study was to identify novel, natural chemotypes from biologically active crude plant extracts that may be useful as part of termite treatment regimens in their natural form or as synthons for structure-activity studies in the future. The results reported here open the possibility of further investigations of efficacy on their anti-termitic properties of natural product extracts.     

Untargeted Metabolomics Approach for the Discovery of Environment-Related Pyran-2-Ones Chemodiversity in a Marine-Sourced Penicillium restrictum

Very little is known about chemical interactions between fungi and their mollusc host within marine environments. Here, we investigated the metabolome of a Penicillium restrictum MMS417 strain isolated from the blue mussel Mytilus edulis collected on the Loire estuary, France. Following the OSMAC approach with the use of 14 culture media, the effect of salinity and of a mussel-derived medium on the metabolic expression were analysed using HPLC-UV/DAD-HRMS/MS. An untargeted metabolomics study was performed using principal component analysis (PCA), orthogonal projection to latent structure discriminant analysis (O-PLSDA) and molecular networking (MN). It highlighted some compounds belonging to sterols, macrolides and pyran-2-ones, which were specifically induced in marine conditions. In particular, a high chemical diversity of pyran-2-ones was found to be related to the presence of mussel extract in the culture medium. Mass spectrometry (MS)- and UV-guided purification resulted in the isolation of five new natural fungal pyran-2-one derivatives—5,6-dihydro-6S-hydroxymethyl-4-methoxy-2H-pyran-2-one (1), (6S, 1’R, 2’S)-LL-P880β (3), 5,6-dihydro-4-methoxy-6S-(1’S, 2’S-dihydroxy pent-3’(E)-enyl)-2H-pyran-2-one (4), 4-methoxy-6-(1’R, 2’S-dihydroxy pent-3’(E)-enyl)-2H-pyran-2-one (6) and 4-methoxy-2H-pyran-2-one (7)—together with the known (6S, 1’S, 2’S)-LL-P880β (2), (1’R, 2’S)-LL-P880γ (5), 5,6-dihydro-4-methoxy-2H-pyran-2-one (8), (6S, 1’S, 2’R)-LL-P880β (9), (6S, 1’S)-pestalotin (10), 1’R-dehydropestalotin (11) and 6-pentyl-4-methoxy-2H-pyran-2-one (12) from the mussel-derived culture medium extract. The structures of 1-12 were determined by 1D- and 2D-MMR experiments as well as high-resolution tandem MS, ECD and DP4 calculations. Some of these compounds were evaluated for their cytotoxic, antibacterial, antileishmanial and in-silico PTP1B inhibitory activities. These results illustrate the utility in using host-derived media for the discovery of new natural products.     

Application of LC–MS–MS to identify niacin in aleurone layers of yellow corn,barley and wheat kernels

Previous studies identified type II inclusions in-situ of aleurone layers as niacin reserves and quantified niacin using colorimetric methods. Such identification and quantification was based on the König reaction which lacks specificity because cyanogen bromide reacts with all pyridine derivatives. The aim of this investigation was to define the structure of niacin in aleurone layers and aleurone cell contents of yellow corn, wheat and barley using LC–MS/MS. Aleurone layers were manually separated and cell contents released by ultrasonic processing and the residue and pellets were examined microscopically. Niacin was extracted from the samples by autoclaving in alkali. The extracts were analysed using HPLC and the structural identity of niacin was confirmed using quadrupole time of flight mass spectrometry in positive mode. Niacin concentrations were highest in wheat aleurone and lowest in corn aleurone. The MS/MS spectra of pure niacin and the purported niacin peak in sample extracts showed similar fragmentation patterns. The major ion product occurred at m/z 80 representing loss of CO₂ and parent ion at m/z 124 (M + H)⁺. These findings define some of the structural characteristics of niacin in aleurone layer of cereal grains, and demonstrate the possibility of using an ultrasonic processor to release cell contents.     

Mining Indonesian Microbial Biodiversity for Novel Natural Compounds by a Combined Genome Mining and Molecular Networking Approach

Indonesia is one of the most biodiverse countries in the world and a promising resource for novel natural compound producers. Actinomycetes produce about two thirds of all clinically used antibiotics. Thus, exploiting Indonesia’s microbial diversity for actinomycetes may lead to the discovery of novel antibiotics. A total of 422 actinomycete strains were isolated from three different unique areas in Indonesia and tested for their antimicrobial activity. Nine potent bioactive strains were prioritized for further drug screening approaches. The nine strains were cultivated in different solid and liquid media, and a combination of genome mining analysis and mass spectrometry (MS)-based molecular networking was employed to identify potential novel compounds. By correlating secondary metabolite gene cluster data with MS-based molecular networking results, we identified several gene cluster-encoded biosynthetic products from the nine strains, including naphthyridinomycin, amicetin, echinomycin, tirandamycin, antimycin, and desferrioxamine B. Moreover, 16 putative ion clusters and numerous gene clusters were detected that could not be associated with any known compound, indicating that the strains can produce novel secondary metabolites. Our results demonstrate that sampling of actinomycetes from unique and biodiversity-rich habitats, such as Indonesia, along with a combination of gene cluster networking and molecular networking approaches, accelerates natural product identification.     

Toxicity of organic-coffee-approved products to the southern red mite Oligonychus ilicis and to its predator Iphiseiodes zuluagai

Organic coffee growers in Brazil often spray a wide range of organic-farming compatible products to control pests including the southern red mite, Oligonychus ilicis. However, most of these products have not yet been proven to be effective for mite control and there is no information about their side effects on natural enemies. We evaluated the lethal and sublethal effects of three organic compatible products, the biofertilizer Supermagro, a fresh bovine manure supplemented with micronutrients, bone meal, unrefined sugar, and milk to stimulate fermentation, an enriched Bordeaux mixture (Viça Café Plus^®) and lime sulfur to O. ilicis and to its predatory mite Iphiseiodes zuluagai. Additionally, we evaluated the efficacy of these products against O. ilicis under greenhouse conditions. Only lime sulfur showed acute lethal effects at concentrations lower than the field recommended rates (20–40 ml/l). The predatory mite I. zuluagai was more tolerant than its prey to the three products based on acute lethal concentration bioassays. The instantaneous rate of increase (r_i) of O. ilicis when exposed to lime sulfur at field recommended rates was negative but r_i of the predatory mite, I. zuluagai was not. The r_i of the predatory mite was lower than that of its prey under Supermagro and Viça Café exposure, including at their field recommended rates (200 ml/l and 10 g/l, respectively). In a greenhouse experiment, the control efficacy against O. ilicis 14 days after spraying reached levels higher than 80% for all three products. Lime sulfur has the potential to control O. ilicis in organic coffee plantations. Although, Supermagro and Viça Café exhibited potential to control O. ilicis in the greenhouse, the product concentrations used were very high and may lead to selectivity problems. Therefore, spraying suitable concentrations of such products taking into account their effectiveness against O. ilicis and their selectivity to natural enemies are important for ecological pest management in organic coffee besides benefiting coffee plant nutrition.     

Indigenous legume fallows (indifallows) as an alternative soil fertility resource in smallholder maize cropping systems

Widening the range of organic nutrient resources, especially N sources, is a major challenge for improving crop productivity of smallholder farms in southern Africa. A study was conducted over three seasons to evaluate different species of indigenous legumes for their biomass productivity, N₂-fixation and residual effects on subsequent maize crops on nutrient-depleted fields belonging to smallholder farmers under contrasting rainfall zones in Zimbabwe. Under high rainfall (>800 mm yr⁻¹), 1-year indigenous legume fallows (indifallows), comprising mostly species of the genera Crotalaria, Indigofera and Tephrosia, yielded 8.6 t ha⁻¹ of biomass within 6 months, out-performing sunnhemp (Crotalaria juncea L.) green manure and grass (natural) fallows by 41% and 74%, respectively. A similar trend was observed under medium (650–750 mm yr⁻¹) rainfall in Chinyika, where the indifallow attained a biomass yield of 6.6 t ha⁻¹ compared with 2.2 t ha⁻¹ for natural fallows. Cumulatively, over two growing seasons, the indifallow treatment under high rainfall at Domboshawa produced biomass as high as 28 t ha⁻¹ compared with ∼7 t ha⁻¹ under natural fallow. The mean total N₂ fixed under indifallows ranged from 125 kg ha⁻¹ under soils exhibiting severe nutrient depletion in Chikwaka, to 205 kg ha⁻¹ at Domboshawa. Indifallow biomass accumulated up to 210 kg N ha⁻¹, eleven-fold higher than the N contained in corresponding natural fallow biomass at time of incorporation. Application of P to indifallows significantly increased both biomass productivity and N₂-fixation, translating into positive yield responses by subsequent maize. Differences in maize biomass productivity between indifallow and natural fallow treatments were already apparent at 2 weeks after maize emergence, with the former yielding significantly (P < 0.05) more maize biomass than the latter. The first maize crop following termination of 1-year indifallows yielded grain averaging 2.3 t ha⁻¹, significantly out-yielding 1-year natural fallows by >1 t ha⁻¹. In the second season, maize yields were consistently better under indifallows compared with natural fallows in terms of both grain and total biomass. The first maize crop following 2-year indifallows yielded ∼3 t ha⁻¹ of grain, significantly higher than the second maize crop after 1-year indifallows and natural fallows. The study demonstrated that indigenous legumes can generate N-rich biomass in sufficient quantities to make a significant influence on maize productivity for more than a single season. Maize yield gains under indifallow systems on low fertility sandy soils exceeded the yields attained with either mineral fertilizer alone or traditional green manure crop of sunnhemp.