Pyricularia oryzae: Lab star and field scourge.

Pyricularia oryzae (syn. Magnaporthe oryzae), is a filamentous ascomycete that causes a major disease called blast on cereal crops, as well as on a wide variety of wild and cultivated grasses. Blast diseases have a tremendous impact worldwide particularly on rice and on wheat, where the disease emerged in South America in the 1980s, before spreading to Asia and Africa. Its economic importance, coupled with its amenability to molecular and genetic manipulation, have inspired extensive research efforts aiming at understanding its biology and evolution. In the past 40 years, this plant-pathogenic fungus has emerged as a major model in molecular plant-microbe interactions. In this review, we focus on the clarification of the taxonomy and genetic structure of the species and its host range determinants. We also discuss recent molecular studies deciphering its lifecycle. TAXONOMY: Kingdom: Fungi, phylum: Ascomycota, sub-phylum: Pezizomycotina, class: Sordariomycetes, order: Magnaporthales, family: Pyriculariaceae, genus: Pyricularia. HOST RANGE: P. oryzae has the ability to infect a wide range of Poaceae. It is structured into different host-specialized lineages that are each associated with a few host plant genera. The fungus is best known to cause tremendous damage to rice crops, but it can also attack other economically important crops such as wheat, maize, barley, and finger millet. DISEASE SYMPTOMS: P. oryzae can cause necrotic lesions or bleaching on all aerial parts of its host plants, including leaf blades, sheaths, and inflorescences (panicles, spikes, and seeds). Characteristic symptoms on leaves are diamond-shaped silver lesions that often have a brown margin and whose appearance is influenced by numerous factors such as the plant genotype and environmental conditions. USEFUL WEBSITES Resources URL Genomic data repositories http://genome.jouy.inra.fr/gemo/ Genomic data repositories http://openriceblast.org/ Genomic data repositories http://openwheatblast.net/ Genome browser for fungi (including P. oryzae) http://fungi.ensembl.org/index.html Comparative genomics database https://mycocosm.jgi.doe.gov/mycocosm/home T-DNA mutant database http://atmt.snu.kr/ T-DNA mutant database http://www.phi-base.org/ SNP and expression data https://fungidb.org/fungidb/app/.

Combined sensory, volatilome and transcriptome analyses identify a limonene terpene synthase as a major contributor to the characteristic aroma of a Coffea arabica L. specialty coffee.

BACKGROUND: The fruity aromatic bouquet of coffee has attracted recent interest to differentiate high value market produce as specialty coffee. Although the volatile compounds present in green and roasted coffee beans have been extensively described, no study has yet linked varietal molecular differences to the greater abundance of specific substances and support the aroma specificity of specialty coffees. RESULTS: This study compared four Arabica genotypes including one, Geisha Especial, suggested to generate specialty coffee. Formal sensory evaluations of coffee beverages stressed the importance of coffee genotype in aroma perception and that Geisha Especial-made coffee stood out by having fine fruity, and floral, aromas and a more balanced acidity. Comparative SPME-GC-MS analyses of green and roasted bean volatile compounds indicated that those of Geisha Especial differed by having greater amounts of limonene and 3-methylbutanoic acid in agreement with the coffee cup aroma perception. A search for gene ontology differences of ripening beans transcriptomes of the four varieties revealed that they differed by metabolic processes linked to terpene biosynthesis due to the greater gene expression of prenyl-pyrophosphate biosynthetic genes and terpene synthases. Only one terpene synthase (CaTPS10-like) had an expression pattern that paralleled limonene loss during the final stage of berry ripening and limonene content in the studied four varieties beans. Its functional expression in tobacco leaves confirmed its functioning as a limonene synthase. CONCLUSIONS: Taken together, these data indicate that coffee variety genotypic specificities may influence ripe berry chemotype and final coffee aroma unicity. For the specialty coffee variety Geisha Especial, greater expression of terpene biosynthetic genes including CaTPS10-like, a limonene synthase, resulted in the greater abundance of limonene in green beans, roasted beans and a unique citrus note of the coffee drink.

The bZIP transcription factor BIP1 of the rice blast fungus is essential for infection and regulates a specific set of appressorium genes.

The rice blast fungus Magnaporthe oryzae differentiates specialized cells called appressoria that are required for fungal penetration into host leaves. In this study, we identified the novel basic leucine zipper (bZIP) transcription factor BIP1 (B-ZIP Involved in Pathogenesis-1) that is essential for pathogenicity. BIP1 is required for the infection of plant leaves, even if they are wounded, but not for appressorium-mediated penetration of artificial cellophane membranes. This phenotype suggests that BIP1 is not implicated in the differentiation of the penetration peg but is necessary for the initial establishment of the fungus within plant cells. BIP1 expression was restricted to the appressorium by both transcriptional and post-transcriptional control. Genome-wide transcriptome analysis showed that 40 genes were down regulated in a BIP1 deletion mutant. Most of these genes were specifically expressed in the appressorium. They encode proteins with pathogenesis-related functions such as enzymes involved in secondary metabolism including those encoded by the ACE1 gene cluster, small secreted proteins such as SLP2, BAS2, BAS3, and AVR-Pi9 effectors, as well as plant cuticle and cell wall degrading enzymes. Interestingly, this BIP1 network is different from other known infection-related regulatory networks, highlighting the complexity of gene expression control during plant-fungal interactions. Promoters of BIP1-regulated genes shared a GCN4/bZIP-binding DNA motif (TGACTC) binding in vitro to BIP1. Mutation of this motif in the promoter of MGG_08381.7 from the ACE1 gene cluster abolished its appressorium-specific expression, showing that BIP1 behaves as a transcriptional activator. In summary, our findings demonstrate that BIP1 is critical for the expression of early invasion-related genes in appressoria. These genes are likely needed for biotrophic invasion of the first infected host cell, but not for the penetration process itself. Through these mechanisms, the blast fungus strategically anticipates the host plant environment and responses during appressorium-mediated penetration.

Whole-genome sequences of Bipolaris bicolor, Curvularia hawaiiensis, Curvularia spicifera, and Exserohilum rostratum isolated from rice in Burkina Faso, France, Mali, and Pakistan.

Different fungal species of the Pleosporaceae family infect rice, causing similar symptoms. Reference genomic sequences are useful tools to study the evolution of these species and to develop accurate molecular diagnostic tools. Here, we report the complete genome sequences of Bipolaris bicolor, Curvularia hawaiiensis, Curvularia spicifera, and Exserohilum rostratum.

The Combination of Both Heat and Water Stresses May Worsen Botryosphaeria Dieback Symptoms in Grapevine.

(1) Background: Grapevine trunk diseases (GTDs) have become a global threat to vineyards worldwide. These diseases share three main common features. First, they are caused by multiple pathogenic micro-organisms. Second, these pathogens often maintain a long latent phase, which makes any research in pathology and symptomatology challenging. Third, a consensus is raising to pinpoint combined abiotic stresses as a key factor contributing to disease symptom expression. (2) Methods: We analyzed the impact of combined abiotic stresses in grapevine cuttings artificially infected by two fungi involved in Botryosphaeria dieback (one of the major GTDs), Neofusicoccum parvum and Diplodia seriata. Fungal-infected and control plants were subjected to single or combined abiotic stresses (heat stress, drought stress or both). Disease intensity was monitored thanks to the measurement of necrosis area size. (3) Results and conclusions: Overall, our results suggest that combined stresses might have a stronger impact on disease intensity upon infection by the less virulent pathogen Diplodia seriata. This conclusion is discussed through the impact on plant physiology using metabolomic and transcriptomic analyses of leaves sampled for the different conditions.

A thousand-genome panel retraces the global spread and adaptation of a major fungal crop pathogen.

Human activity impacts the evolutionary trajectories of many species worldwide. Global trade of agricultural goods contributes to the dispersal of pathogens reshaping their genetic makeup and providing opportunities for virulence gains. Understanding how pathogens surmount control strategies and cope with new climates is crucial to predicting the future impact of crop pathogens. Here, we address this by assembling a global thousand-genome panel of Zymoseptoria tritici, a major fungal pathogen of wheat reported in all production areas worldwide. We identify the global invasion routes and ongoing genetic exchange of the pathogen among wheat-growing regions. We find that the global expansion was accompanied by increased activity of transposable elements and weakened genomic defenses. Finally, we find significant standing variation for adaptation to new climates encountered during the global spread. Our work shows how large population genomic panels enable deep insights into the evolutionary trajectory of a major crop pathogen.

CusProSe: a customizable protein annotation software with an application to the prediction of fungal secondary metabolism genes.

We report here a new application, CustomProteinSearch (CusProSe), whose purpose is to help users to search for proteins of interest based on their domain composition. The application is customizable. It consists of two independent tools, IterHMMBuild and ProSeCDA. IterHMMBuild allows the iterative construction of Hidden Markov Model (HMM) profiles for conserved domains of selected protein sequences, while ProSeCDA scans a proteome of interest against an HMM profile database, and annotates identified proteins using user-defined rules. CusProSe was successfully used to identify, in fungal genomes, genes encoding key enzyme families involved in secondary metabolism, such as polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS), hybrid PKS-NRPS and dimethylallyl tryptophan synthases (DMATS), as well as to characterize distinct terpene synthases (TS) sub-families. The highly configurable characteristics of this application makes it a generic tool, which allows the user to refine the function of predicted proteins, to extend detection to new enzymes families, and may also be applied to biological systems other than fungi and to other proteins than those involved in secondary metabolism.

Genetic diversity and structure of Bipolaris oryzae and Exserohilum rostratum populations causing brown spot of rice in Burkina Faso based on genotyping-by-sequencing.

In recent years, Brown spot disease of rice (BSR) has been observed on leaves and seeds of rice in all rice-growing areas of Burkina Faso. Bipolaris oryzae and Exserohilum rostratum are the main fungal species isolated from BSR infected tissues and they are frequently observed in the same field. However, we are lacking information on the genetic diversity and population structure of these fungi in Burkina Faso. The mode of reproduction is also unknown. The genetic diversity of isolates of B. oryzae (n=61) and E. rostratum (n=151), collected from major rice-growing areas of Burkina Faso, was estimated using genotyping-by-sequencing (GBS). The mean values for nucleotide diversity (π) were 1.9 x10-4 for B. oryzae and 4.8 x10-4 for E. rostratum. There is no genetic differentiation between the geographical populations of each species. The analysis of molecular variance revealed that 89% and 94% of the genetic variances were within the populations of B. oryzae and E. rostratum, respectively. For each species, four genetic clusters were identified by two clustering methods (DAPC and sNMF). The distribution of these genetic groups was independent of the geographical origin of the isolates. Evidence of recombination was detected in the populations of B. oryzae and E. rostratum. For B. oryzae balanced mating type ratios were supporting sexual reproduction. For E. rostratum overrepresentation of MAT1-2 isolates (79%) suggested a predominant asexual reproduction. This study provides important information on the biology and genetics of the two major fungi causing brown spot disease of rice in Burkina Faso.

Effect of spontaneous fermentation location on the fingerprint of volatile compound precursors of cocoa and the sensory perceptions of the end-chocolate.

Cocoa pod-opening delay and bean fermentation promote the organoleptic quality of chocolate. The present research investigated the changes in the volatile fingerprint of cocoa harvested at a traditional plantation. Cocoa beans extracted from 2-days pod-opening delay were simultaneously fermented for 5 days using container and then sun-dried to 7-8% moisture content at five different locations: Akoupé, San Pedro, Soubré, Djekanou and Daloa. The aromatic analysis were done on cocoa using the HS-SPME-GC/MS technique. Professional panelists evaluated the sensory perceptions of the chocolate. The results shows that cocoa fermented in both Daloa and Soubré regions were differentiated by 2,3-butanediol while those processed in other regions presented highest acetoin content. However, fermented cocoa from Soubré region exhibited most amount of 2,3-butanediol, diacetate A whereas 2,3,5,6-tetramethylpyrazine differentiated those from Daloa region. Sensory properties of chocolate were not linked to the aromatic compound precursors profile of beans. The fermentation performed in San Pédro region promote both the generation of more desirable aromatic compounds of cocoa and sensory attributes of the finished chocolate. The fermentation location generates a greater differentiation of the volatile fingerprint of cocoa and the sensory perceptions of the finished chocolate.

Diversity and determinants of bitterness, astringency, and fat content in cultivated Nacional and native Amazonian cocoa accessions from Ecuador.

Cocoa (Theobroma cacao L.) is the only tree that can produce cocoa. Cocoa beans are highly sought after by chocolate makers to produce chocolate. Cocoa can be fine aromatic, characterized by floral and fruity notes, or it can be described as standard cocoa with a more pronounced cocoa aroma and bitterness. In this study, the genetic and biochemical determinants of sensorial notes and nonvolatile compounds related to bitterness, astringency, fat content, and protein content will be investigated in two populations: a cultivated modern Nacional population and a population of cocoa accessions collected recently in the Ecuadorian South Amazonia area of origin of the Nacional ancestral variety. For this purpose, a genome-wide association study (GWAS) was carried out on both populations, with results of biochemical compounds evaluated by near-infrared spectroscopy (NIRS) assays and with sensory evaluations. Twenty areas of associations were detected for sensorial data especially bitterness and astringency. Fifty-three areas of associations were detected linked to nonvolatile compounds. A total of 81 candidate genes could be identified in the areas of the association.

Blocked at the Stomatal Gate, a Key Step of Wheat Stb16q-Mediated Resistance to Zymoseptoria tritici.

Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici, is among the most threatening wheat diseases in Europe. Genetic resistance remains one of the main environmentally sustainable strategies to efficiently control STB. However, the molecular and physiological mechanisms underlying resistance are still unknown, limiting the implementation of knowledge-driven management strategies. Among the 22 known major resistance genes (Stb), the recently cloned Stb16q gene encodes a cysteine-rich receptor-like kinase conferring a full broad-spectrum resistance against Z. tritici. Here, we showed that an avirulent Z. tritici inoculated on Stb16q quasi near isogenic lines (NILs) either by infiltration into leaf tissues or by brush inoculation of wounded tissues partially bypasses Stb16q-mediated resistance. To understand this bypass, we monitored the infection of GFP-labeled avirulent and virulent isolates on Stb16q NILs, from germination to pycnidia formation. This quantitative cytological analysis revealed that 95% of the penetration attempts were unsuccessful in the Stb16q incompatible interaction, while almost all succeeded in compatible interactions. Infectious hyphae resulting from the few successful penetration events in the Stb16q incompatible interaction were arrested in the sub-stomatal cavity of the primary-infected stomata. These results indicate that Stb16q-mediated resistance mainly blocks the avirulent isolate during its stomatal penetration into wheat tissue. Analyses of stomatal aperture of the Stb16q NILs during infection revealed that Stb16q triggers a temporary stomatal closure in response to an avirulent isolate. Finally, we showed that infiltrating avirulent isolates into leaves of the Stb6 and Stb9 NILs also partially bypasses resistances, suggesting that arrest during stomatal penetration might be a common major mechanism for Stb-mediated resistances.

Patterns of Volatile Diversity Yield Insights Into the Genetics and Biochemistry of the Date Palm Fruit Volatilome.

Volatile organic compounds are key components of the fruit metabolome that contribute to traits such as aroma and taste. Here we report on the diversity of 90 flavor-related fruit traits in date palms (Phoenix dactylifera L.) including 80 volatile organic compounds, which collectively represent the fruit volatilome, as well as 6 organic acids, and 4 sugars in tree-ripened fruits. We characterize these traits in 148 date palms representing 135 varieties using headspace solid-phase microextraction gas chromatography. We discovered new volatile compounds unknown in date palm including 2-methoxy-4-vinylphenol, an attractant of the red palm weevil (Rhynchophorus ferrugineus Olivier), a key pest that threatens the date palm crop. Associations between volatile composition and sugar and moisture content suggest that differences among fruits in these traits may be characterized by system-wide differences in fruit metabolism. Correlations between volatiles indicate medium chain and long chain fatty acid ester volatiles are regulated independently, possibly reflecting differences in the biochemistry of fatty acid precursors. Finally, we took advantage of date palm clones in our analysis to estimate broad-sense heritabilities of volatiles and demonstrate that at least some of volatile diversity has a genetic basis.

Impact of crossflow microfiltration on aroma and sensory profiles of a potential functional citrus-based food.

BACKGROUND: Citrus juices can be cold-concentrated by crossflow microfiltration (CMF) in order to obtain functional foods enriched in carotenoids, flavonoids and pectins. The work aimed to characterize the organoleptic quality of this type of micronutrient-dense foods through their aroma profile and sensory analysis. Two citrus concentrates with and without a diafiltration step were compared. RESULTS: Both citrus products were very different, linked to aroma compound, sugar and organic acid contents. Due to its sugar/acidity balance and its better aromatic profile responsible for the citrus-floral flavour, the concentrate without diafiltration was preferred by the sensory panel. Thanks to a simple transfer model, we showed that retention of volatiles clearly varied from one aroma compound to another. The terpene hydrocarbons were the most retained by the membrane during CMF, probably because they were strongly associated with insoluble solids by adsorption. CONCLUSION: Even though the process modified their organoleptic profiles, both citrus-based products were well rated and can be consumed directly as pleasant functional drinks. © 2022 Society of Chemical Industry.

Diversity of Neofusicoccum parvum for the Production of the Phytotoxic Metabolites (-)-Terremutin and (R)-Mellein.

Two Neofusicoccumparvum isolates and a UV mutant were characterized for their phytotoxin production in vitro, their pathogenicity on grapevine, and their genome sequenced. The isolate Np-Bt67 produced high level of (-)-terremutin, but almost no (R)-mellein, and it was the most aggressive on grapevine, triggering apoplexy. Similar symptoms were not induced by purified (-)-terremutin. The isolate Bourgogne S-116 (Np-B) produced 3-fold less (-)-terremutin and high amounts of (R)-mellein, but it was less aggressive on grapevine than Np-Bt67. The UV9 mutant obtained from Np-B (NpB-UV9) no longer produced (-)-terremutin but overproduced (R)-mellein by 2.5-fold, and it was as pathogenic as its parent. NpB-UV9 differed from its parent by simple mutations in two genes (transcription factor UCR-NP2_6692, regulatory protein UCR-NP2_9007), not located neither near (R)-mellein, nor (-)-terremutin biosynthetic genes, but likely involved in the control of (-)-terremutin biosynthesis. Grapevine immunity was disturbed upon challenge with these pathogens or purified phytotoxins, leading to an upregulation of SA-dependent defenses, while (-)-terremutin interfered with host JA/ET-dependent defenses. Our results suggest that neither (-)-terremutin nor (R)-mellein alone is essential for the pathogenicity of N. parvum on grapevine, since isolate/mutant non-producing these toxins in vitro is pathogenic. However, these phytotoxins could play a quantitative role in the infection process.

Impacts of Sodium Arsenite on Wood Microbiota of Esca-Diseased Grapevines.

Although sodium arsenite was widely used in Europe until its ban in 2003, its effects on microorganisms is not clearly understood. To improve our understanding of sodium arsenite curative effect on GTDs, grapevines displaying esca-foliar symptoms from different French regions (Alsace, Champagne, Languedoc) were treated or not with sodium arsenite, and analyzed for their wood microbiota. Using metabarcoding, we identified the fungal and bacterial taxa composition of microbiota colonizing woody trunk tissues. Large differences in fungal microbiota composition between treated and untreated grapevines were observed while no major impacts were observed on bacteria microbiota. The main fungal species detected in untreated necrotic woody tissues was Fomitiporia mediterranea (63-94%), a fungal pathogen associated with esca. The relative abundance of this fungal species significantly decreased after sodium arsenite treatment in the three vineyards, in particular in white-rot necrotic tissues and their borders (-90%). F. mediterranea was the most sensitive to sodium arsenite among fungi from grapevine woody tissues. These results strongly suggest that the effect of sodium arsenite on GTDs is due to its ability to efficiently and almost specifically eliminate F. mediterranea from white-rot necrotic tissues, allowing saprobic fungi to colonize the tissues previously occupied by this pathogenic fungus.

Multi-block classification of chocolate and cocoa samples into sensory poles.

The present study aims at developing an analytical methodology which allows correlating sensory poles of chocolate to their chemical characteristics and, eventually, to those of the cocoa beans used for its preparation. Trained panelists investigated several samples of chocolate, and they divided them into four sensorial poles (characterized by 36 different descriptors) attributable to chocolate flavor. The same samples were analyzed by six different techniques: Proton Transfer Reaction-Time of Flight-Mass Spectrometry (PTR-ToF-MS), Solid Phase Micro Extraction-Gas Chromatography-Mass Spectroscopy (SPME-GC-MS), High-Performance Liquid Chromatography (HPLC) (for the quantification of eight organic acids), Ultra High Performance Liquid Chromatography coupled to triple-quadrupole Mass Spectrometry (UHPLC-QqQ-MS) for polyphenol quantification, 3D front face fluorescence Spectroscopy and Near Infrared Spectroscopy (NIRS). A multi-block classification approach (Sequential and Orthogonalized-Partial Least Squares - SO-PLS) has been used, in order to exploit the chemical information to predict the sensorial poles of samples. Among thirty-one test samples, only two were misclassified.

Major changes in grapevine wood microbiota are associated with the onset of esca, a devastating trunk disease.

Esca, a major grapevine trunk disease in old grapevines, is associated with the colonization of woody tissues by a broad range of plant pathogenic fungi. To identify which fungal and bacterial species are involved in the onset of this disease, we analysed the microbiota from woody tissues of young (10-year-old) grapevines at an early stage of esca. Using meta-barcoding, 515 fungal and 403 bacterial operational taxonomic units (OTUs) were identified in woody tissues. In situ hybridization showed that these fungi and bacteria co-inhabited in grapevine woody tissues. In non-necrotic woody tissues, fungal and bacterial microbiota varied according to organs and seasons but not diseased plant status. Phaeomoniella chlamydospora, involved in the Grapevine trunk disease, was the most abundant species in non-necrotic tissues from healthy plants, suggesting a possible non-pathogenic endophytic behaviour. Most diseased plants (70%) displayed cordons, with their central white-rot necrosis colonized essentially by two plant pathogenic fungi (Fomitiporia mediterranea: 60%-90% and P. chlamydospora: 5%-15%) and by a few bacterial taxa (Sphingomonas spp. and Mycobacterium spp.). The occurrence of a specific association of fungal and bacterial species in cordons from young grapevines expressing esca-foliar symptoms strongly suggests that that microbiota is involved in the onset of this complex disease.

Infection cushions of Fusarium graminearum are fungal arsenals for wheat infection.

Fusarium graminearum is one of the most destructive plant pathogens worldwide, causing fusarium head blight (FHB) on cereals. F. graminearum colonizes wheat plant surfaces with specialized unbranched hyphae called runner hyphae (RH), which develop multicelled complex appressoria called infection cushions (IC). IC generate multiple penetration sites, allowing the fungus to enter the plant cuticle. Complex infection structures are typical for several economically important plant pathogens, yet with unknown molecular basis. In this study, RH and IC formed on the surface of wheat paleae were isolated by laser capture microdissection. RNA-Seq-based transcriptomic analyses were performed on RH and IC and compared to mycelium grown in complete medium (MY). Both RH and IC displayed a high number of infection up-regulated genes (982), encoding, among others, carbohydrate-active enzymes (CAZymes: 140), putative effectors (PE: 88), or secondary metabolism gene clusters (SMC: 12 of 67 clusters). RH specifically up-regulated one SMC corresponding to aurofusarin biosynthesis, a broad activity antibiotic. IC specifically up-regulated 248 genes encoding mostly putative virulence factors such as 7 SMC, including the mycotoxin deoxynivalenol and the newly identified fusaoctaxin A, 33 PE, and 42 CAZymes. Furthermore, we studied selected candidate virulence factors using cellular biology and reverse genetics. Hence, our results demonstrate that IC accumulate an arsenal of proven and putative virulence factors to facilitate the invasion of epidermal cells.

Genome-wide association mapping of date palm fruit traits.

Date palms (Phoenix dactylifera) are an important fruit crop of arid regions of the Middle East and North Africa. Despite its importance, few genomic resources exist for date palms, hampering evolutionary genomic studies of this perennial species. Here we report an improved long-read genome assembly for P. dactylifera that is 772.3 Mb in length, with contig N50 of 897.2 Kb, and use this to perform genome-wide association studies (GWAS) of the sex determining region and 21 fruit traits. We find a fruit color GWAS at the R2R3-MYB transcription factor VIRESCENS gene and identify functional alleles that include a retrotransposon insertion and start codon mutation. We also find a GWAS peak for sugar composition spanning deletion polymorphisms in multiple linked invertase genes. MYB transcription factors and invertase are implicated in fruit color and sugar composition in other crops, demonstrating the importance of parallel evolution in the evolutionary diversification of domesticated species.