Antifungal efficiency and mechanisms of ethyl ferulate against postharvest pathogens.

Postharvest loss caused by a range of pathogens necessitates exploring novel antifungal compounds that are safe and efficient in managing the pathogens. This study evaluated the antifungal activity of ethyl ferulate (EF) and explored its mechanisms of action against Alternaria alternata, Aspergillus niger, Botrytis cinerea, Penicillium expansum, Penicillium digitatum, Geotrichum candidum and evaluated its potential to inhibit postharvest decay. The results demonstrated that EF exerts potent antifungal activity against a wide board of postharvest pathogens. Results also revealed that its antifungal mechanism is multifaceted: EF may be involved in binding to and disturbing the integrity of the fungal plasma membrane, causing leakage of intracellular content and losing normal morphology and ultrastructure. EF also induced oxidative stress in the pathogen, causing membrane lipid peroxidation and malondialdehyde accumulation. EF inhibited the critical gene expression of the pathogen, affecting its metabolic regulation, antioxidant metabolism, and cell wall degrading enzymes. EF exhibited antifungal inhibitory activity when applied directly into peel wounds or after incorporation with chitosan coating. Due to its wide board and efficient antifungal activity, EF has the potential to provide a promising alternative to manage postharvest decay.

Soil, rhizosphere, and root microbiome in kiwifruit vine decline, an emerging multifactorial disease.

Kiwifruit vine decline syndrome (KVDS) is characterized by severe root system impairment, which leads to irreversible wilting of the canopy. Plants usually collapse rapidly from the appearance of the first aboveground symptoms, without recovery even in the following seasons. The syndrome has been negatively impacting kiwifruit yield in different areas of Italy, the main producing European country, since its first outbreak in 2012. To date, a unique, common causal factor has yet to be found, and the syndrome is referred to as multifactorial. In this article, we investigated the whole biotic community (fungi, bacteria, and oomycetes) associated with the development of KVDS in three different belowground matrices/compartments (soil, rhizosphere, and root). Sampling was performed at both healthy and affected sites located in the main kiwifruit-producing area of Northwestern Italy. To address the multifactorial nature of the syndrome and to investigate the potential roles of abiotic factors in shaping these communities, a physicochemical analysis of soils was also performed. This study investigates the associations among taxonomic groups composing the microbiome and also between biotic and abiotic factors. Dysbiosis was considered as a driving event in shaping KVDS microbial communities. The results obtained from this study highlight the role of the oomycete genus Phytopythium, which resulted predominantly in the oomycete community composition of diseased matrices, though it was also present in healthy ones. Both bacterial and fungal communities resulted in a high richness of genera and were highly correlated to the sampling site and matrix, underlining the importance of multiple location sampling both geographically and spatially. The rhizosphere community associated with KVDS was driven by a dysbiotic process. In addition, analysis of the association network in the diseased rhizosphere revealed the presence of potential cross-kingdom competition for plant-derived carbon between saprobes, oomycetes, and bacteria.

Functional analysis of the apple fruit microbiome based on shotgun metagenomic sequencing of conventional and organic orchard samples.

Fruits harbour abundant and diverse microbial communities that protect them from post-harvest pathogens. Identification of functional traits associated with a given microbiota can provide a better understanding of their potential influence. Here, we focused on the epiphytic microbiome of apple fruit. We suggest that shotgun metagenomic data can indicate specific functions carried out by different groups and provide information on their potential impact. Samples were collected from the surface of 'Golden Delicious' apples from four orchards that differ in their geographic location and management practice. Approximately 1 million metagenes were predicted based on a high-quality assembly. Functional profiling of the microbiome of fruits from orchards differing in their management practice revealed a functional shift in the microbiota. The organic orchard microbiome was enriched in pathways involved in plant defence activities; the conventional orchard microbiome was enriched in pathways related to the synthesis of antibiotics. The functional significance of the variations was explored using microbial network modelling algorithms to reveal the metabolic role of specific phylogenetic groups. The analysis identified several associations supported by other published studies. For example, the analysis revealed the nutritional dependencies of the Capnodiales group, including the Alternaria pathogen, on aromatic compounds.

Current and future trends in the biocontrol of postharvest diseases.

Postharvest diseases of fruits and vegetables cause significant economic losses to producers and marketing firms. Many of these diseases are caused by necrotrophic fungal pathogens that require wounded or injured tissues to establish an infection. Biocontrol of postharvest diseases is an evolving science that has moved from the traditional paradigm of one organism controlling another organism to viewing biocontrol as a system involving the biocontrol agent, the pathogen, the host, the physical environment, and most recently the resident microflora. Thus, the paradigm has shifted from one of simplicity to complexity. The present review provides an overview of how the field of postharvest biocontrol has evolved over the past 40 years, a brief review of the biology of necrotrophic pathogens, the discovery of BCAs, their commercialization, and mechanisms of action. Most importantly, current research on the use of marker-assisted-selection, the fruit microbiome and its relationship to the pathobiome, and the use of double-stranded RNA as a biocontrol strategy is discussed. These latter subjects represent evolving trends in postharvest biocontrol research and suggestions for future research are presented.

Assembly and dynamics of the apple carposphere microbiome during fruit development and storage.

Microbial communities associated with fruit can contribute to quality and pathogen resistance, but little is known about their assembly and dynamics during fruit development and storage. Three apple cultivars growing under the same environmental conditions were utilized to examine the apple carposphere microbiome composition and structure at different developmental stages and storage. There was a significant effect (Adonis, p ≤ 0.001) of fruit genotype and its developmental stages and storage times on the fruit surface microbial assemblage and a strong temporal microbial community succession was detected (Mantel test: R ≤ 0.5, p = 0.001) in both bacterial and fungal communities. A set of 15 bacterial and 35 fungal core successional taxa and members exhibiting differential abundances at different fruit stages were identified. For the first time, we show the existence of underlying universal dynamics in the assembly of fruit-associated microbiomes. We also provide evidence of strong microbial cross-domain associations and uncover potential microbe-microbe correlations in the apple carposphere. Together our findings shed light on how the fruit carposphere assemble and change over time, and provide new insights into fruit microbial ecology.

Contrasting effects of genotype and root size on the fungal and bacterial communities associated with apple rootstocks.

The endophytic microbiome of plants is believed to have a significant impact on its physiology and disease resistance, however, the role of host genotype in determining the composition of the endophytic microbiome of apple root systems remains an open question that has important implications for defining breeding objectives. In the current study, the bacterial and fungal microbiota associated with four different apple rootstocks planted in April, 2018 in the same soil environment and harvested in May, 2019 were evaluated to determine the role of genotype on the composition of both the bacterial and fungal communities. Results demonstrated a clear impact of genotype and root size on microbial composition and diversity. The fungal community was more affected by plant genotype whereas the bacterial community was shaped by root size. Fungal and bacterial abundance was equal between different-sized roots however, significantly higher microbial counts were detected in rhizosphere samples compared to root endosphere samples. This study provides information that can be used to develop a comprehensive and readily applicable understanding of the impact of genotype and environmental factors on the establishment of plant microbiome, as well as its potential function and impact on host physiology.

Evidence for host-microbiome co-evolution in apple.

Plants evolved in association with a diverse community of microorganisms. The effect of plant phylogeny and domestication on host-microbiome co-evolutionary dynamics are poorly understood. Here we examined the effect of domestication and plant lineage on the composition of the endophytic microbiome of 11 Malus species, representing three major groups: domesticated apple (M. domestica), wild apple progenitors, and wild Malus species. The endophytic community of M. domestica and its wild progenitors showed higher microbial diversity and abundance than wild Malus species. Heirloom and modern cultivars harbored a distinct community composition, though the difference was not significant. A community-wide Bayesian model revealed that the endophytic microbiome of domesticated apple is an admixture of its wild progenitors, with clear evidence for microbiome introgression, especially for the bacterial community. We observed a significant correlation between the evolutionary distance of Malus species and their microbiome. This study supports co-evolution between Malus species and their microbiome during domestication. This finding has major implications for future breeding programs and our understanding of the evolution of plants and their microbiomes.

A Sustainable Alternative for Postharvest Disease Management and Phytopathogens Biocontrol in Fruit: Antagonistic Yeasts.

Postharvest diseases of fruits caused by phytopathogens cause losses up to 50% of global production. Phytopathogens control is performed with synthetic fungicides, but the application causes environmental contamination problems and human and animal health in addition to generating resistance. Yeasts are antagonist microorganisms that have been used in the last years as biocontrol agents and in sustainable postharvest disease management in fruits. Yeast application for biocontrol of phytopathogens has been an effective action worldwide. This review explores the sustainable use of yeasts in each continent, the main antagonistic mechanisms towards phytopathogens, their relationship with OMIC sciences, and patents at the world level that involve yeast-based-products for their biocontrol.

Analysis of Stored Wheat Grain-Associated Microbiota Reveals Biocontrol Activity among Microorganisms against Mycotoxigenic Fungi.

Wheat grains are colonized by complex microbial communities that have the potential to affect seed quality and susceptibility to disease. Some of the beneficial microbes in these communities have been shown to protect plants against pathogens through antagonism. We evaluated the role of the microbiome in seed health: in particular, against mycotoxin-producing fungi. Amplicon sequencing was used to characterize the seed microbiome and determine if epiphytes and endophytes differ in their fungal and bacterial diversity and community composition. We then isolated culturable fungal and bacterial species and evaluated their antagonistic activity against mycotoxigenic fungi. The most prevalent taxa were found to be shared between the epiphytic and endophytic microbiota of stored wheat seeds. Among the isolated bacteria, Bacillus strains exhibited strong antagonistic properties against fungal pathogens with noteworthy fungal load reduction in wheat grain samples of up to a 3.59 log10 CFU/g compared to untreated controls. We also found that a strain of the yeast, Rhodotorula glutinis, isolated from wheat grains, degrades and/or metabolizes aflatoxin B1, one of the most dangerous mycotoxins that negatively affects physiological processes in animals and humans. The mycotoxin level in grain samples was significantly reduced up to 65% in the presence of the yeast strain, compared to the untreated control. Our study demonstrates that stored wheat grains are a rich source of bacterial and yeast antagonists with strong inhibitory and biodegradation potential against mycotoxigenic fungi and the mycotoxins they produce, respectively. Utilization of these antagonistic microorganisms may help reduce fungal and mycotoxin contamination, and potentially replace traditionally used synthetic chemicals.

Microbial Biosurfactant: A New Frontier for Sustainable Agriculture and Pharmaceutical Industries.

In the current scenario of changing climatic conditions and the rising global population, there is an urgent need to explore novel, efficient, and economical natural products for the benefit of humankind. Biosurfactants are one of the latest explored microbial synthesized biomolecules that have been used in numerous fields, including agriculture, pharmaceuticals, cosmetics, food processing, and environment-cleaning industries, as a source of raw materials, for the lubrication, wetting, foaming, emulsions formulations, and as stabilizing dispersions. The amphiphilic nature of biosurfactants have shown to be a great advantage, distributing themselves into two immiscible surfaces by reducing the interfacial surface tension and increasing the solubility of hydrophobic compounds. Furthermore, their eco-friendly nature, low or even no toxic nature, durability at higher temperatures, and ability to withstand a wide range of pH fluctuations make microbial surfactants preferable compared to their chemical counterparts. Additionally, biosurfactants can obviate the oxidation flow by eliciting antioxidant properties, antimicrobial and anticancer activities, and drug delivery systems, further broadening their applicability in the food and pharmaceutical industries. Nowadays, biosurfactants have been broadly utilized to improve the soil quality by improving the concentration of trace elements and have either been mixed with pesticides or applied singly on the plant surfaces for plant disease management. In the present review, we summarize the latest research on microbial synthesized biosurfactant compounds, the limiting factors of biosurfactant production, their application in improving soil quality and plant disease management, and their use as antioxidant or antimicrobial compounds in the pharmaceutical industries.

The Potential Application of Endophytes in Management of Stress from Drought and Salinity in Crop Plants.

Endophytic microorganisms present inside the host plant play an essential role in host fitness, nutrient supply and stress tolerance. Endophytes are often used in sustainable agriculture as biofertilizers, biopesticides and as inoculants to mitigate abiotic stresses including salinity, drought, cold and pH variation in the soil. In changing climatic conditions, abiotic stresses create global challenges to achieve optimum crop yields in agricultural production. Plants experience stress conditions that involve endogenous boosting of their immune system or the overexpression of their defensive redox regulatory systems with increased reactive oxygen species (ROS). However, rising stress factors overwhelm the natural redox protection systems of plants, which leads to massive internal oxidative damage and death. Endophytes are an integral internal partner of hosts and have been shown to mitigate abiotic stresses via modulating local or systemic mechanisms and producing antioxidants to counteract ROS in plants. Advancements in omics and other technologies have been made, but potential application of endophytes remains largely unrealized. In this review article, we will discuss the diversity, population and interaction of endophytes with crop plants as well as potential applications in abiotic stress management.

Unravelling the fruit microbiome: The key for developing effective biological control strategies for postharvest diseases.

Fruit-based diets are recognized for their benefits to human health. The safety of fruit is a global concern for scientists. Fruit microbiome represents the whole microorganisms that are associated with a fruit. These microbes are either found on the surfaces (epiphytes) or in the tissues of the fruit (endophytes). The recent knowledge gained from these microbial communities is considered relevant to the field of biological control in prevention of postharvest fruit pathology. In this study, the importance of the microbiome of certain fruits and how it holds promise for solving the problems inherent in biocontrol and postharvest crop protection are summarized. Research needs on the fruit microbiome are highlighted. Data from DNA sequencing and "meta-omics" technologies very recently applied to the study of microbial communities of fruits in the postharvest context are also discussed. Various fruit parameters, management practices, and environmental conditions are the main determinants of the microbiome. Microbial communities can be classified according to their structure and function in fruit tissues. A critical mechanism of microbial biological control agents is to reshape and interact with the microbiome of the fruit. The ability to control the microbiome of any fruit is a great potential in postharvest management of fruits. Research on the fruit microbiome offers important opportunities to develop postharvest biocontrol strategies and products, as well as the health profile of the fruit.

Global analysis of the apple fruit microbiome: are all apples the same?

We present the first worldwide study on the apple (Malus × domestica) fruit microbiome that examines questions regarding the composition and the assembly of microbial communities on and in apple fruit. Results revealed that the composition and structure of the fungal and bacterial communities associated with apple fruit vary and are highly dependent on geographical location. The study also confirmed that the spatial variation in the fungal and bacterial composition of different fruit tissues exists at a global level. Fungal diversity varied significantly in fruit harvested in different geographical locations and suggests a potential link between location and the type and rate of postharvest diseases that develop in each country. The global core microbiome of apple fruit was represented by several beneficial microbial taxa and accounted for a large fraction of the fruit microbial community. The study provides foundational information about the apple fruit microbiome that can be utilized for the development of novel approaches for the management of fruit quality and safety, as well as for reducing losses due to the establishment and proliferation of postharvest pathogens. It also lays the groundwork for studying the complex microbial interactions that occur on apple fruit surfaces.

Metagenomics Approaches for the Detection and Surveillance of Emerging and Recurrent Plant Pathogens.

Globalization has a dramatic effect on the trade and movement of seeds, fruits and vegetables, with a corresponding increase in economic losses caused by the introduction of transboundary plant pathogens. Current diagnostic techniques provide a useful and precise tool to enact surveillance protocols regarding specific organisms, but this approach is strictly targeted, while metabarcoding and shotgun metagenomics could be used to simultaneously detect all known pathogens and potentially new ones. This review aims to present the current status of high-throughput sequencing (HTS) diagnostics of fungal and bacterial plant pathogens, discuss the challenges that need to be addressed, and provide direction for the development of methods for the detection of a restricted number of related taxa (specific surveillance) or all of the microorganisms present in a sample (general surveillance). HTS techniques, particularly metabarcoding, could be useful for the surveillance of soilborne, seedborne and airborne pathogens, as well as for identifying new pathogens and determining the origin of outbreaks. Metabarcoding and shotgun metagenomics still suffer from low precision, but this issue can be limited by carefully choosing primers and bioinformatic algorithms. Advances in bioinformatics will greatly accelerate the use of metagenomics to address critical aspects related to the detection and surveillance of plant pathogens in plant material and foodstuffs.

Multiple transcriptomic analyses and characterization of pathogen-related core effectors and LysM family members reveal their differential roles in fungal growth and pathogenicity in Penicillium expansum.

Penicillium expansum is a destructive phytopathogen causing postharvest decay on many stored fruits. To develop effective and safe management strategies, it is important to investigate its pathogenicity-related mechanisms. In this study, a bioinformatic pipeline was constructed and 50 core effector genes were identified in P. expansum using multiple RNA-seq data sets and their putative functions were implicated by comparatively homologous analyses using pathogen-host interaction database. To functionally characterize P. expansum LysM domain proteins during infection, null mutants for the 15 uncharacterized putative LysM effectors were constructed and the fungal growth rate on either PDA or Cazpek medium or lesion expansion rate on the infected apple fruits was evaluated. The results showed the growth rate of knockout mutants from PeLysM5, PeLysM12 and PeLysM15 was retarded on PDA medium. No significant difference in growth rate was observed between wild type and all mutants on solid Cazpek medium. Nevertheless, the hypha of wild type displayed deeper yellow on the back of Cazpek medium than those of knockout mutants. On the infecting apples fruits, the knockout mutants from PeLysM5, PeLysM7, PeLysM8, PeLysM9, PeLysM10, PeLysM11, PeLysM14, PeLysM15, PeLysM16, PeLysM18 and PeLysM19 showed enhanced fungal virulence, with faster decaying on infected fruits than those from wild type. By contrast, the knockout mutation at PeLysM12 locus led to reduced lesion expansion rate on the infected apple fruits. In addition, P. expansum-apple interaction RNA-seq experiment was performed using apple fruit tissues infected by the wild type and knockout mutant ΔPeLysM15, respectively. Transcriptome analyses indicated that deletion of PeLysM15 could activate expression of several core effector genes, such as PEX2_055830, PEX2_036960 and PEX2_108150, and a chitin-binding protein, PEX2_064520. These results suggest PeLysM15 may play pivotal roles in fungal growth and development and involve pathogen-host interaction by modulating other effector genes' expression. Our results could provide solid data reference and good candidates for further pathogen-related studies in P. expansum.

Effect of Washing, Waxing and Low-Temperature Storage on the Postharvest Microbiome of Apple.

There is growing recognition of the role that the microbiome plays in the health and physiology of many plant species. However, considerably less research has been conducted on the postharvest microbiome of produce and the impact that postharvest processing may have on its composition. Here, amplicon sequencing was used to study the effect of washing, waxing, and low-temperature storage at 2 °C for six months on the bacterial and fungal communities of apple calyx-end, stem-end, and peel tissues. The results of the present work reveal that tissue-type is the main factor defining fungal and bacterial diversity and community composition on apple fruit. Both postharvest treatments and low temperature storage had a strong impact on the fungal and bacterial diversity and community composition of these tissue types. Distinct spatial and temporal changes in the composition and diversity of the microbiota were observed in response to various postharvest management practices. The greatest impact was attributed to sanitation practices with major differences among unwashed, washed and washed-waxed apples. The magnitude of the differences, however, was tissue-specific, with the greatest impact occurring on peel tissues. Temporally, the largest shift occurred during the first two months of low-temperature storage, although fungi were more affected by storage time than bacteria. In general, fungi and bacteria were impacted equally by sanitation practices, especially the epiphytic microflora of peel tissues. This research provides a foundation for understanding the impact of postharvest management practices on the microbiome of apple and its potential subsequent effects on postharvest disease management and food safety.

Characterizing the Fungal Microbiome in Date (Phoenix dactylifera) Fruit Pulp and Peel from Early Development to Harvest.

Date palm (Phoenix dactylifera) is considered to be a highly important food crop in several African and Middle Eastern countries due to its nutritional value and health-promoting properties. Microbial contamination of dates has been of concern to consumers, but very few works have analyzed in detail the microbial load of the different parts of date fruit. In the present work, we characterized the fungal communities of date fruit using a metagenomic approach, analyzing the data for differences between microbial populations residing in the pulp and peel of "Medjool" dates at the different stages of fruit development. The results revealed that Penicillium, Cladosporium, Aspergillus, and Alternaria were the most abundant genera in both parts of the fruit, however, the distribution of taxa among the time points and tissue types (peel vs. pulp) was very diverse. Penicillium was more abundant in the pulp at the green developmental stage (Kimri), while Aspergillus was more frequent in the peel at the brown developmental stage (Tamer). The highest abundance of Alternaria was detected at the earliest sampled stage of fruit development (Hababauk stage). Cladosporium had a high level of abundance in peel tissues at the Hababauk and yellow (Khalal) stages. Regarding the yeast community, the abundance of Candida remained stable up until the Khalal stage, but exhibited a dramatic increase in abundance at the Tamer stage in peel tissues, while the level of Metschnikowia, a genus containing several species with postharvest biocontrol activity, exhibited no significant differences between the two tissue types or stages of fruit development. This work constitutes a comprehensive metagenomic analysis of the fungal microbiome of date fruits, and has identified changes in the composition of the fungal microbiome in peel and pulp tissues at the different stages of fruit development. Notably, this study has also characterized the endophytic fungal microbiome present in pulp tissues of dates.

Genome Sequence, Assembly, and Characterization of the Antagonistic Yeast Candida oleophila Used as a Biocontrol Agent Against Post-harvest Diseases.

Candida oleophila is an effective biocontrol agent used to control post-harvest diseases of fruits and vegetables. C. oleophila I-182 was the active agent used in the first-generation yeast-based commercial product, Aspire®, for post-harvest disease management. Several action modes, like competition for nutrients and space, induction of pathogenesis-related genes in host tissues, and production of extracellular lytic enzymes, have been demonstrated for the biological control activity exhibited by C. oleophila through which it inhibits post-harvest pathogens. In the present study, the whole genome of C. oleophila I-182 was sequenced using PacBio and Illumina shotgun sequencing technologies, yielding an estimated genome size of 14.73 Mb. The genome size is similar in length to that of the model yeast strain Saccharomyces cerevisiae S288c. Based on the assembled genome, protein-coding sequences were identified and annotated. The predicted genes were further assigned with gene ontology terms and clustered in special functional groups. A comparative analysis of C. oleophila proteome with the proteomes of 11 representative yeasts revealed 2 unique and 124 expanded families of proteins in C. oleophila. Availability of the genome sequence will facilitate a better understanding the properties of biocontrol yeasts at the molecular level.

Yeasts and Bacterial Consortia from Kefir Grains Are Effective Biocontrol Agents of Postharvest Diseases of Fruits.

Fungal pathogens in fruits and vegetables cause significant losses during handling, transportation, and storage. Biological control with microbial antagonists replacing the use of chemical fungicides is a major approach in postharvest disease control, and several products based on single antagonists have been developed but have limitations related to reduced and inconsistent performance under commercial conditions. One possible approach to enhance the biocontrol efficacy is to broaden the spectrum of the antagonistic action by employing compatible microbial consortia. Here, we explore commercial kefir grains, a natural probiotic microbial consortium, by culture-dependent and metagenomic approaches and observed a rich diversity of co-existing yeasts and bacterial population. We report effective inhibition of the postharvest pathogen Penicillium expansum on apple by using the grains in its fresh commercial and milk-activated forms. We observed few candidate bacteria and yeasts from the kefir grains that grew together over successive enrichment cycles, and these mixed fermentation cultures showed enhanced biocontrol activities as compared to the fresh commercial or milk-activated grains. We also report several individual species of bacteria and yeasts with biocontrol activities against Penicillium rots on apple and grapefruit. These species with antagonistic properties could be further exploited to develop a synthetic consortium to achieve enhanced antagonistic effects against a wide range of postharvest pathogens.

Biocontrol of Aspergillus flavus in Ensiled Sorghum by Water Kefir Microorganisms.

The capacity of microorganisms from water kefir (WK) to control Aspergillus flavus growth during the aerobic phase of ensiled sorghum grains was determined. Sorghum inoculated with A. flavus was treated with filter-sterilized and non-sterilized water kefir, ensiled, and incubated 7 days at 25 °C. A. flavus growth was quantified by qPCR after incubation. Mold growth was inhibited in the presence of water kefir while no inhibition was observed when filter-sterilized water kefir was applied, demonstrating the relevant role of the microorganisms in the kefir water in the biocontrol process. Fungal and bacterial diversity in treated sorghum mini-silos was analyzed by high-throughput sequencing. Firmicutes was the predominant bacterial phyla and Lactobacillus represented the most abundant genus, while Ascomycota was the predominant fungal phyla with Saccharomyces and Pichia as the major genera. Bacterial and yeast counts before and after incubation indicated that the microbial community obtained from WK was able to grow in the sorghum mini-silos in the presence of A. flavus. Results of the present work indicate that the use of a mixed inoculum of microorganisms present in WK may represent an alternative management practice to avoid the growth of A. flavus in ensiled sorghum grains and the concomitant contamination with aflatoxins.