Components of seed production and ergot resistance used as criteria for selection of Brachiaria hybrids

Ergot is a fungal disease and causes reductions in seed productivity and quality. We aimed to identify promising genotypes of brachiaria evaluating: (1) seed production potential (number of tillers, racemes per inflorescence, spikelets per raceme, raceme length, weight and number of seeds, and germination) and (2) resistance of seeds to Ergot (infected tillers, infected seeds). Five genotypes of Brachiaria grasses, four non-natural hybrids named BH1619, BH1810, BH1516 and Mavuno Palisadegrass (Urochloa brizantha x Urochloa ruziziensis), and Marandu Palisadegrass (U. brizantha Hoechst cv. Marandu) were evaluated. The BH1619 hybrid, despite the high weight of seeds, produced less viable, pure seeds. The hybrid BH1516 had a lower percentage of flowered tillers (42%) and seeds infected with ergot (8%). Marandu Palisadegrass had a higher percentage of infected tillers (95%) and infected seeds (38%). The hybrid BH1516 was the most resistant to ergot, followed by Mavuno Palisadegras sand BH1619. Among all materials, Mavuno Palisadegrass and BH1516 have a higher potential for seed production due to the higher percentage of flowered tillers and production of pure seeds with high germination capacity. The BH1516 hybrid was resistant to ergot, making it a better choice for use in infected areas used for seed or forage production.(AU)

Genome-wide analysis of Claviceps paspali: insights into the secretome of the main species causing ergot disease in Paspalum spp.

BACKGROUND: The phytopatogen Claviceps paspali is the causal agent of Ergot disease in Paspalum spp., which includes highly productive forage grasses such as P. dilatatum. This disease impacts dairy and beef production by affecting seed quality and producing mycotoxins that can affect performance in feeding animals. The molecular basis of pathogenicity of C. paspali remains unknown, which makes it more difficult to find solutions for this problem. Secreted proteins are related to fungi virulence and can manipulate plant immunity acting on different subcellular localizations. Therefore, identifying and characterizing secreted proteins in phytopathogenic fungi will provide a better understanding of how they overcome host defense and cause disease. The aim of this work is to analyze the whole genome sequences of three C. paspali isolates to obtain a comparative genome characterization based on possible secreted proteins and pathogenicity factors present in their genome. In planta RNA-seq analysis at an early stage of the interaction of C. paspali with P. dilatatum stigmas was also conducted in order to determine possible secreted proteins expressed in the infection process. RESULTS: C. paspali isolates had compact genomes and secretome which accounted for 4.6-4.9% of the predicted proteomes. More than 50% of the predicted secretome had no homology to known proteins. RNA-Seq revealed that three protein-coding genes predicted as secreted have mayor expression changes during 1 dpi vs 4 dpi. Also, three of the first 10 highly expressed genes in both time points were predicted as effector-like. CAZyme-like proteins were found in the predicted secretome and the most abundant family could be associated to pectine degradation. Based on this, pectine could be a main component affected by the cell wall degrading enzymes of C. paspali. CONCLUSIONS: Based on predictions from DNA sequence and RNA-seq, unique probable secreted proteins and probable pathogenicity factors were identified in C. paspali isolates. This information opens new avenues in the study of the biology of this fungus and how it modulates the interaction with its host. Knowledge of the diversity of the secretome and putative pathogenicity genes should facilitate future research in disease management of Claviceps spp.

Diversity of Claviceps paspali reveals unknown lineages and unique alkaloid genotypes.

Claviceps species affecting Paspalum spp. are a serious problem, as they infect forage grasses such as Paspalum dilatatum and P. plicatulum, producing the ergot disease. The ascomycete C. paspali is known to be the pathogen responsible for this disease in both grasses. This fungus produces alkaloids, including ergot alkaloids and indole-diterpenes, that have potent neurotropic activities in mammals. A total of 32 isolates from Uruguay were obtained from infected P. dilatatum and P. plicatulum. Isolates were phylogenetically identified using partial sequences of the genes coding for the second largest subunit of RNA polymerase subunit II (RPB2), translation elongation factor 1-α (TEF1), ß-tubulin (TUB2), and the nuc rDNA 28S subunit (28S). Isolates were also genotyped by randomly amplified polymorphic DNA (RAPD) and presence of genes within the ergot alkaloid (EAS) and indole-diterpene (IDT) biosynthetic gene clusters. This study represents the first genetic characterization of several isolates of C. paspali. The results from this study provide insight into the genetic and genotypic diversity of Claviceps paspali present in P. dilatatum and suggest that isolates from P. plicatulum could be considered an ecological subspecies or specialized variant of C. paspali. Some of these isolates show hypothetical alkaloid genotypes never reported before.

Sobre el mal de los ardientes o del fuego de San Antonio / Saint Anthony's fire illness

Resumen El ergotismo es el resultado de la intoxicación con alcaloides derivados del ergot. Durante la edad media y hasta el siglo XIX fue el responsable de grandes epidemias en Europa por el consumo de granos contaminados con el cornezuelo del centeno provocando manifestaciones isquémicas características y afección del sistema nervioso. El ergotismo como se conoció en el Medioevo ha desaparecido, pero aún hoy en día se reportan casos en el marco de uso terapéutico de ergotamínicos. Se pretenden describir algunos de sus aspectos históricos y clínicos distintivos.

Abstract Ergotism is the result of intoxication with alkaloids derived from ergot. During the middle ages and until the nineteenth century it was responsible for major epidemics in Europe for the consumption of grains contaminated with ergot, causing characteristic ischemic manifestations and nervous system involvement. Ergotism as it was known in the Middle Ages has disappeared, but even today cases are reported in the therapeutic use of ergotamine. Some of its distinctive historical and clinical aspects are intended to be described.

Biological activity of Claviceps gigantea in juvenile New Zealand rabbits.

The Ascomycete fungus Claviceps gigantea infects maize kernels and synthetizes several alkaloids, mostly dihydrolysergamides. There is limited information on the damage these toxins cause in mammals, despite reports from infested areas with 90% presence of the fungus sclerotia. With this background, it was decided to determine the biological activity of chemical compounds present in sclerotia of C. gigantea in rabbits 38 days after weaning. Sclerotia of C. gigantea were collected in fields with high incidence of the disease, ground and analysed for nutrients. Experimental diets were prepared with four treatments, where sclerotial powder was added, substituting for alfalfa flour in increasing proportions [C. gigantea/alfalfa flour (0:100, 5:95, 15:85 and 25:75)]. Total ergot alkaloid content was analysed by high-performance liquid chromatography. Male juvenile rabbits were utilised and distributed in completely randomised design with four replications. Initial weight was recorded in each animal, and experimental diet was offered. In this study, weight of animals, feed consumption and feed conversion were evaluated in individual animals. Blood samples were taken for haemograms, and finally euthanasia was practiced. The consumption of C. gigantea had a negative effect on body weight and feed consumption. The necropsies showed anomalies proportional to the consumption of feed contaminated with the fungus.

Evolutionary history of ergot with a new infrageneric classification (Hypocreales: Clavicipitaceae: Claviceps).

The ergot, genus Claviceps, comprises approximately 60 species of specialised ovarial grass parasites famous for the production of food toxins and pharmaceutics. Although the ergot has been known for centuries, its evolution have not been resolved yet. Our approach combining multilocus phylogeny, molecular dating and the study of ecological, morphological and metabolic features shows that Claviceps originated in South America in the Palaeocene on a common ancestor of BEP (subfamilies Bambusoideae, Ehrhartoideae, Pooideae) and PACMAD (subfamilies Panicoideae, Aristidoideae, Chloridoideae, Micrairoideae, Arundinoideae, Danthonioideae) grasses. Four clades described here as sections diverged during the Paleocene and Eocene. Since Claviceps are parasitic fungi with a close relationship with their host plants, their evolution is influenced by interactions with the new hosts, either by the spread to a new continent or the radiation of the host plants. Three of the sections possess very narrow host ranges and biogeographical distributions and have relatively low toxicity. On the contrary, the section Claviceps, comprising the rye ergot, C. purpurea, is unique in all aspects. Fungi in this section of North American origin have spread all over the world and infect grasses in all subfamilies as well as sedges, and it is the only section synthesising toxic ergopeptines and secalonic acids. The evolutionary success of the Claviceps section members can be explained by high toxin presence, serving as feeding deterrents and playing a role in their protective mutualism with host plants. Closely related taxa Neoclaviceps monostipa and Cepsiclava phalaridis were combined into the genus Aciculosporium.

Synchronicity of pollination and inoculation with Claviceps africana and its effects on pollen-pistil compatibility and seed production in sorghum.

Sorghum ergot (caused by Claviceps africana) is a disease that affects sorghum seed development and yield. The interaction between pollen tube growth and hyphal development determines whether ovaries will be fertilized or colonized. Thus their respective deposition times on the stigma are critical. The effect of the time interval between pollination and inoculation on stigma receptivity and seed production was measured under field conditions in the male-sterile line A9 at Montecillo, State of México (2240m altitude). Pollination and inoculation treatments, from simultaneous application to 2 and 4h difference, were imposed when all stigmas on the panicle had emerged. Control panicles were either only pollinated or only inoculated. Eighteen hours later, pollen grains that adhered to, and germinated within the stigma, pollen tubes in the style and ovary, and fertilized pistils were counted. Pistils showing some disease expression (germinated spores, mycelium growth, or tissue necrosis) at 18, 48, and 72h were recorded. The number of diseased florets was registered at the dough growth stage, while number of seeds, grain yield and 100-seeds weight was measured at the physiological maturity. The pathogen applied in a water suspension of macro and secondary conidia caused a decrease in stigma receptivity; the greatest decrease (40-60%) occurred when the pollen and the inoculum were deposited almost simultaneously, regardless of which was deposited first. The route of the pollen tube was also the route for fungal infection. On average, treatments first inoculated had 60% more diseased florets and 36% less grain yield, 30% fewer seeds and seed size decreased 8%, than those first pollinated.

Metabolic pathway of oligosaccharides in sorghum honeydew caused by Claviceps africana / Rota metabólica de oligossacarídeos em exsudato da "doença açucarada" do sorgo causado por Claviceps africana

The occurrence of the alditol oligosaccharides in the Claviceps afriana honeydew is partly as a rational expression of the pathogen's selective nutritive metabolism of the sucrose supplied by the host plant. The experiments were carried out in laboratory and when 14C-D-sucrose, 14C-D-fructose or 14C-D-mannitol radiolabelled saccharides were incorporated into: a) sorghum plant infected by C. africana, b) whole and macereted micelia tissue and c) cell-free honeydew of C. africana, it was observed that the glucose moiety of sucrose was not involved in oligosaccharides formation. Glucose was used by the pathogen as nutritional source. Part of the unused fructose moiety was reduced to mannitol by the pathogen's enzymes which was also excreted into honeydew where reductase activity accepted 14C mannitol. The mannitol was linked with fructose in a 2-position synthesizing the disaccharide 1-beta-D-fructofuranosyl-D-mannitol and then the process was repeated by the mannitol moiety of the disaccharide to yield the trisaccharide 1,6-di-beta-D-fructofuranosyl-D-mannitol, which became dominant. The direct formation of alditol saccharides from monosaccharides in this way seems to be unique to C. africana, contrasting with the fructosyl transfer from sucrose to sucrose which is usual in others ergot parasites.(AU)

A ocorrência de oligossacarídeos contendo alditol em exsudações da doença açucarada produzidos por Claviceps africana é em parte uma expressão racional do metabolismo seletivo de nutrição do patógeno na tansformação da sacarose fornecida pela planta. Quando açúcares radioativos marcados com 14C (D-sucrose, D-fructose e D-mannitol) foram incorporados em: a) planta do sorgo infectada por C. africana, b) tecido micelial inteiro e macerado isolado de C. africana e c) exsudato livre de células de C. africana, foi observado que a glicose da sacarose não estava envolvida na formação de oligossacarídeos, sendo usada pelo patógeno como fonte nutritiva. Parte da frutose não utilizada da sacarose foi reduzida a mannitol por enzimas do patógeno. As enzimas envolvidas também atuaram no exsudato da "doença açucarada", ativando a ação da redutase pela incorporação do 14C manitol. O manitol foi ligado na posição 2 da fructose, formando o dissacarídeo 1-beta-D-fructofuranosyl-D-mannitol e, posteriormente, adicionada mais uma fructose ao mannitol do dissacarídeo, formando o trisacarídeo 1,6-di-beta-D-fructofuranosyl-D-manitol, que se tornou predominate. A formação direta de oligossacarídeos contendo alditol a partir de monossacarídeos parece ser exclusiva em C. africana, contrastando com a ação enzimática da frutose transferase a partir de sucrose para sucrose, a qual é muito comum em outros patógenos tipo "ergot".(AU)

Urgencias tocoginecológicas: la fitoterapia ¿una alternativa o un peligro? / Tocoginecologic urgencies: fitotherapy, an alternative or a risk?

Se debe evitar el uso de plantas con fines terapéuticos sin un adecuado control médico, sobre todo en el período gestacional. Se distinguen las plantas adecuadas para ser utilizadas durante el embarazo, y las que son peligrosas, especialmente el cornezuelo de centeno y sus derivados

Urgencias tocoginecológicas: la fitoterapia ¿una alternativa o un peligro? / Tocoginecologic urgencies: fitotherapy, an alternative or a risk?

Se debe evitar el uso de plantas con fines terapéuticos sin un adecuado control médico, sobre todo en el período gestacional. Se distinguen las plantas adecuadas para ser utilizadas durante el embarazo, y las que son peligrosas, especialmente el cornezuelo de centeno y sus derivados(AU)

Urgencias tocoginecológicas: la fitoterapia ¿una alternativa o un peligro? / Tocoginecologic urgencies: fitotherapy, an alternative or a risk?

Se debe evitar el uso de plantas con fines terapéuticos sin un adecuado control médico, sobre todo en el período gestacional. Se distinguen las plantas adecuadas para ser utilizadas durante el embarazo, y las que son peligrosas, especialmente el cornezuelo de centeno y sus derivados(AU)

Hyperthermic syndrome in dairy cattle associated with consumption of ergots of Claviceps purpurea in southern Brazil.

A hyperthermic syndrome (HS) associated with consumption of ration contaminated with Claviceps purpurea is described in 4 dairy cattle herds from different counties in the state of Rio Grande do Sul, southern Brazil. Outbreaks of this mycotoxicosis occurred during the summer of 1999-2000 and autumn of 2002 when 25% to 70% of cattle from those herds were affected. The disease occurred after the introduction of a new batch of feed contaminated with sclerotia of C. purpurea. First clinical signs were observed up to 3 mo after the ration was introduced and consisted of pyrexia, intense salivation and drooling, open-mouth, difficult breathing, in some cases with the tongue protruded, decrease in milk yield, decrease in food intake and weight loss. Affected cattle compulsively sought shade or water ponds. Signs were more pronounced during daytime. The severity of the clinical picture varied according to changes in environmental temperature during the day. There was complete recovery approximately 60 d after feeding with the contaminated ration was discontinued. Abortions occurred during the outbreak and soon after the remission of the clinical signs associated with HS. In one of the herds, reproductive performance and fertility were seriously impaired even after clinical signs of HS completely disappeared. Diagnosis was based on epidemiology, clinical signs and pathology. The pathogenetic mechanisms involved in the occurrence of HS are briefly discussed.

Agalactia in mares fed with grain contaminated with Claviceps purpurea.

This article reports an outbreak of intoxication of female horses with Claviceps purpurea in southern Brazil. The outbreak affected twelve pregnant mares which were fed with black oat (Avena strigosa) during the pre-delivery period. Underdevelopment of the mammary gland in the pre-delivery period resulting in post-delivery agalactia was the most pronounced finding. These mares delivered weak and unviable foals, which showed no suckling reflex and died within a few hours of birth. Laboratory analysis of oat samples fed to the animals resulted in the identification of Claviceps purpurea sclerotia. The fungus was identified in 0.22% of the examined seeds.

Síndrome distérmica (hipertermia) em bovinos associada à intoxicaçäo por Claviceps purpurea / Dysthermic syndrome (hyperthermia) in cattle associated with poisoning by Claviceps purpurea

Descrevem-se três surtos de síndrome distérmica (hipertermia) associada à intoxicaçäo por Claviceps purpurea, em bovinos de leite durante o veräo de 1999-2000, em três estabelecimentos do Rio Grande do Sul. De um total de 66 bovinos que ingeriram a raçäo contaminada com o fungo, 37 (56 por cento) adoeceram até 3 meses após a introduçäo da raçäo contaminada. Os principais sinais clínicos foram temperatura retal elevada, pêlos compridos, longos e sem brilho, salivaçäo intensa, respiraçäo ofegante, com a boca aberta e, em alguns casos, com a língua para fora da cavidade oral. Os animais acometidos procuravam sombra ou permaneciam dentro d'água. Houve diminuiçäo de 10 a 30 por cento no consumo de alimentos e perda de peso. A reduçäo na produçäo de leite foi de 30 a 50 por cento. Os sinais clínicos se intensificavam durante o dia e eram diretamente proporcionais à elevaçäo da temperatura ambiental. Os achados de necropsia em um bovino que foi eutanasiado, incluíram leve enfisema pulmonar, principalmente na regiäo dorsal dos lobos pulmonares diafragmáticos. Histologicamente havia moderada hipertrofia da musculatura lisa dos bronquíolos e ruptura de septos alveolares formando cotos alveolares em clava. Nos três estabelecimentos onde ocorreram os surtos, escleródios de C. purpurea foram observados nas amostras de raçäo fornecida aos bovinos. Os animais afetados recuperaram-se após aproximadamente 60 dias da retirada da raçäo contaminada. O diagnóstico baseou-se em dados epidemiológicos, sinais clínicos, na presença de escleródios de C. purpurea na raçäo fornecida aos animais, nos achados de necropsia e na histopatologia. A patogenia e o quadro clínico-patológico observados sao discutidos e comparados com outros relatos dessa enfermidade

Síndrome distérmica (hipertermia) em bovinos associada à intoxicação por Claviceps purpurea / Dysthermic syndrome (hyperthermia) in cattle associated with poisoning by Claviceps purpurea

Descrevem-se três surtos de síndrome distérmica (hipertermia) associada à intoxicação por Claviceps purpurea, em bovinos de leite durante o verão de 1999-2000, em três estabelecimentos do Rio Grande do Sul. De um total de 66 bovinos que ingeriram a ração contaminada com o fungo, 37 (56 por cento) adoeceram até 3 meses após a introdução da ração contaminada. Os principais sinais clínicos foram temperatura retal elevada, pêlos compridos, longos e sem brilho, salivação intensa, respiração ofegante, com a boca aberta e, em alguns casos, com a língua para fora da cavidade oral. Os animais acometidos procuravam sombra ou permaneciam dentro d'água. Houve diminuição de 10 a 30 por cento no consumo de alimentos e perda de peso. A redução na produção de leite foi de 30 a 50 por cento. Os sinais clínicos se intensificavam durante o dia e eram diretamente proporcionais à elevação da temperatura ambiental. Os achados de necropsia em um bovino que foi eutanasiado, incluíram leve enfisema pulmonar, principalmente na região dorsal dos lobos pulmonares diafragmáticos. Histologicamente havia moderada hipertrofia da musculatura lisa dos bronquíolos e ruptura de septos alveolares formando cotos alveolares em clava. Nos três estabelecimentos onde ocorreram os surtos, escleródios de C. purpurea foram observados nas amostras de ração fornecida aos bovinos. Os animais afetados recuperaram-se após aproximadamente 60 dias da retirada da ração contaminada. O diagnóstico baseou-se em dados epidemiológicos, sinais clínicos, na presença de escleródios de C. purpurea na ração fornecida aos animais, nos achados de necropsia e na histopatologia. A patogenia e o quadro clínico-patológico observados sao discutidos e comparados com outros relatos dessa enfermidade (AU)

Características biológicas de claviceps paspali stev & hall / Biological characteristics of claviceps paspali stev & hall

Se estudia el ciclo biológico de claviceps paspali, un ascomycete parásito, altamente específico de gramíneas del género paspalum (pasto miel). Desarrolla un ciclo asexual (sphacelia deliquescens) en el interior de las flores del pasto miel y uno sexual, en la superficie del suelo. El primero, comienza con la llegada de las ascosporas a la flor, que germina desarrollando un micelio y una secreción melosa muy rica en conidios, con el tiempo ésta se transforma en una masa esclerotial. El segundo se inicia con la germinación del esclerocio, dando 2 a 4 cabezuelas estromáticas, en cuyo interior se encuetran peritecios que contienen ascos con 8 ascosporas, las cuales son expulsadas activamente y los insectos las transportan nuevamente a la flor, cerrándo así el ciclo. La fase asexual es la encargada de la difusión masiva del hongo y la sexual de mantener el hongo viable hasta el verano siguiente

Ergotismo por automedicación / Ergotism by automedication

Los autores informan sobre 7 casos clínicos de ergotismo gangrenoso (6 mujeres y 1 hombre) provocado por la ingestión inmoderada de ergotamina ministrada para aliviar las crisis de migraña. En todos los enfermos estaban presentes síntomas y signos clínicos, ecográficos y angiográficos de constricción arterial severa y ninguno sufrió alucinaciones. El ergotismo fue tratado con la suspensión de la ergotamina, la ministración de vasodilatadores y la simpatectomía. Después del tratamiento, la mejoría de los 7 pacientes fue evidente por la desaparición de los síntomas y signos de isquemia distal y aumento significativo (P < 0.05) del índice pletismográfico de perfusión sanguínea, medido por medio de la ecografía Doppler. Estos cambios fueron observados aún en un paciente que perdió 2 dedos del pie derecho, secundariamente al ergotismo gangrenoso. Si bien, ninguno de los enfermos manifestó alucinaciones, los autores hacen referencia al primer uso del cornezuelo de centeno, reconocido históricamente, en las ceremonias mágico religiosas de Eleusis de la Grecia clásica; así, como al caso de brujería de Salem, Nueva Inglaterra, en 1692. La migraña no puede ser considerada banal; frecuentemente, causa desesperación del enfermo, que se intoxica con la ergotamina, cuando es sólo este medicamento el recomendado por su médico tratante. En consecuencia, el médico especialista, familiar, general o rural, debe conocer perfectamente la terapia profiláctica de las crisis de migraña con medicamentos vasodilatadores y así, reducir la posibilidad de ergotismo.

Ergotismo: ¿iatrogenia o idiosincrasia? / Ergotism: Iatrogenic or idiosyncrasy?

El ergotismo continúa siendo una causa rara de insuficiencia arterial crónica y aguda en pacientes en edad productiva, que si no es detectada y tratada a tiempo puede causar daño neurológico permanente o pérdida de la extremidad y como consecuencia invalidez del paciente. En un lapso de dos años se recibieron en el servicio cuatro pacientes con diagnóstico de ergotismo, tres mujeres y un hombre, se manejaron con interrupción de la ergotamina, vasodilatadores y dos de ellos con simpatectomía lumbar; dos de los pacientes presentaron secuelas y determinar si el ergotismo es una iatrogenia causada por el uso prolongado de este medicamento

Ergotism and photosensitization in swine produced by the combined ingestion of Claviceps purpurea sclerotia and Ammi majus seeds.

Poisoning of domestic animals happens frequently in the southeast of Buenos Aires Province (Argentina). Intoxications are produced mainly by the ingestion of plants and mycotoxins, but animals are rarely affected simultaneously by both types of agents. One herd of pigs suffered simultaneous intoxications by ergot alkaloids from Claviceps purpurea sclerotia and furocoumarins from Ammi majus seeds. Pigs were fed a diet composed of wheat (poor quality) or corn and protein and vitamin supplements. This diet was completed with forage sorghum. Nervous signs were first observed 5-7 days after the initiation of feeding the suspect ration. These signs were followed by cutaneous irritation. Snout ulcers, eyelid edema, and conjunctivitis were observed in several piglets. Ten days after the start of feeding the incriminated ration, 8 abortions were observed. Many of the sows that were nursing piglets developed udder edema and teat cracking. Dermal lesions were observed in most of the animals with unpigmented areas in the skin but not in a Duroc-Jersey boar. Removal of the incriminated diet and feeding of another diet prepared with good-quality wheat allowed all the animals to recover in 15 days. The herd experienced normal pregnancies and parturitions, litter sizes, and piglet weights when fed a cleaned portion of the poor-quality wheat. No photosensitization lesions were observed. Examination of impurities in the suspected wheat indicated the presence of 2.2% of A. majus seeds and 0.14% of C. purpurea sclerotia. The quantitative analysis indicated the presence of 3.2 g xanthotoxin and 0.65 g bergaptene/100 g A. majus seeds and 0.73 g ergot alkaloids (expressed as ergonovine) per 100 g of C. purpurea sclerotia. Qualitative analysis demonstrated the presence of ergotamine, ergocristine, and ergonovine. These results indicate that clinical signs and lesions were caused by the ingestion of large quantities of these biologically active compounds.