Expression of cry1Ab gene from a novel Bacillus thuringiensis strain SY49-1 active on pest insects

ABSTRACT In this study, the cry1Ab gene of previously characterized and Lepidoptera-, Diptera-, and Coleoptera-active Bacillus thuringiensis SY49-1 strain was cloned, expressed and individually tested on Ephestia kuehniella (Lepidoptera: Pyralidae) and Plodia interpunctella (Lepidoptera: Pyralidae) larvae. pET-cry1Ab plasmids were constructed by ligating the cry1Ab into pET28a (+) expression vector. Constructed plasmids were transferred to an Escherichia coli BL21 (DE3) strain rendered competent with CaCl2. Isopropyl β-D-1-thiogalactopyranoside was used to induce the expression of cry1Ab in E. coli BL21(DE3), and consequently, ∼130 kDa of Cry1Ab was obtained. Bioassay results indicated that recombinant Cry1Ab at a dose of 1000 µg g-1 caused 40% and 64% mortality on P. interpunctella and E. kuehniella larvae, respectively. However, the mortality rates of Bt SY49-1 strains' spore-crystal mixture at the same dose were observed to be 70% on P. interpunctella and 90% on E. kuehniella larvae. The results indicated that cry1Ab may be considered as a good candidate in transgenic crop production and as an alternative biocontrol agent in controlling stored product moths.

Evaluación de ovitrampas para vigilancia y control de Aedes aegypti en dos centros urbanos del Urabá antioqueño / Ovitraps evaluation for surveillance and control of Aedes aegypti in two urban settlements of Urabá, Antioquia

Introducción. Aedes ( Stegomyia ) aegypti es el principal vector del dengue. En el control del mosquito se han usado insecticidas químicos contra los cuales ha desarrollado resistencia. Es necesaria la evaluación de estrategias alternativas que sean eficientes, económicas y de fácil aplicación, como las ovitrampas con Bacillus thuringiesis var. israeliensis. Objetivo. Evaluar el impacto de ovitrampas con B. thuringiesis var. israeliensis sobre los índices aédicos tradicionales, como estrategia para la vigilancia y el control de A. aegypti . Materiales y métodos. Se seleccionaron ocho barrios de los municipios de Apartadó y Carepa, Antioquia, de los cuales se escogieron dos barrios para la intervención y dos para el control en cada municipio. La intervención consistió en la instalación de una ovitrampa con B. thuringiesis var. israeliensis en cada una de las viviendas. Las ovitrampas se visitaron mensualmente entre septiembre de 2009 y marzo de 2010. Se obtuvieron los índices aédicos tradicionales y se hicieron comparaciones entre los barrios. Se calculó el índice de ovitrampas positivas y el de densidad de huevos, y se generaron los mapas de densidad. Resultados. Se colocaron 519 ovitrampas muestreadas mensualmente. Se hicieron 3.114 muestreos, con un porcentaje de registros positivos de 76,4 %. Se recolectaron 501.425 huevos. En Apartadó se observaron diferencias significativas entre el índice de infestación de viviendas, el índice de infestación de depósitos y el índice de Breteau en los barrios intervenidos comparados con los barrios de control. El índice de ovitrampas positivas evidenció un alto riesgo de infestación por A. aegypti y, los índices tradicionales, niveles de riesgo medio y bajo. Conclusiones. Las ovitrampas tuvieron impacto sobre los índices tradicionales y demostraron ser estrategias útiles en la vigilancia de A. aegypti en el Urabá antioqueño.

Introduction: Aedes (Stegomyia) aegypti is the main vector of dengue. Chemical insecticides have been used to control the mosquito and it has developed resistance. It is necessary to evaluate alternative strategies that are efficient, economical and easy to apply, such as ovitraps with Bacillus thuringiesis israeliensis . Objective: To evaluate the impact of ovitraps loaded with B. t. israeliensis on traditional indexes, as strategies for surveillance and control of A. aegypti . Materials and methods: We selected eight neighborhoods from the municipalities of Apartadó and Carepa. Two neighborhoods in each municipality were chosen for intervention and two for control. The intervention consisted in the installation of ovitraps with B. t. israeliensis in every house. The traps were visited each month between September, 2009, and March, 2010. Traditional indexes were obtained and compared among the neighborhoods. Ovitrap positivity index and egg density index were calculated and we generated density maps. Results: Five hundred and nineteen ovitraps were placed monthly; 3,114 samples were obtained, from which 76.4% were positive. We collected 501,425 eggs. In Apartadó, statistically significant differences were observed in the house, container and Breteau indexes. The ovitrap positivity index showed high risk of A. aegypti infestation and traditional indexes showed medium and low risk levels. Conclusions: Ovitraps had an impact on traditional indexes and proved to be useful strategies for surveillance and control of A. aegypti in Urabá, Antioquia.

Influencia de factores bióticos sobre la eficacia de Bacillus thuringiensis var. israelensis contra Aedes aegypti (Diptera: Culicidae) / Influence of biotic factors on the efficacy of Bacillus thuringiensis var. Israelensis against Aedes aegypti (Diptera: Culicidae)

Introducción: Bacillus thuringiensis H-14 ha mostrado alta eficacia sobre las larvas de Aedes aegypti, pero su efectividad puede ser influida por numerosos factores. El conocimiento de estos es de gran importancia porque permite optimizar los parámetros de aplicación en condiciones reales, para garantizar la eficiencia de los tratamientos y reducir el impacto ambiental. Objetivo: evaluar la influencia de los factores bióticos sobre la eficacia de Bacillus thuringiensis H-14 contra las larvas de Aedes aegypti. Métodos: se realizó un estudio experimental mediante bioensayos de laboratorio, siguiendo la metodología de la Organización Mundial de la Salud. Se evaluó la influencia de la disponibilidad de alimento, densidad y estadio larvario sobre la eficacia de Bacillus thuringiensis H-14 para Aedes aegypti. Se utilizó un biolarvicida comercial (Bactivec, Labiofam) y una cepa de referencia de Aedes aegypti (Rockefeller). Resultados: la eficacia de Bacillus thuringiensis H-14 fue menor en la medida en que avanzó la densidad y el estadio de desarrollo larvario, lo que puede estar en relación con la conducta de alimentación y la disponibilidad del principio activo. La presencia abundante de alimento también limita la eficacia por un efecto de competencia. Conclusiones: la eficacia de Bacillus thuringiensis H-14 se afectó por la disponibilidad de alimento, el estadio de desarrollo y la densidad larvaria, por lo que debe tenerse en cuenta esto para las aplicaciones del biolarvicida en el terreno.

Introduction: Bacillus thuringiensis H-14 has shown high efficacy on Aedes aegypti larvae. But a number of factors could affect its effectiveness. Knowing these factors is of vital importance for improving the application parameters in real conditions in order to guarantee the treatment's efficiency and to reduce environmental impact. Objective: to evaluate the influence of some biotic factors on Bacillus thuringiensis H-14 efficacy against Aedes aegypti (Rockefeller). Methods: an experimental study was conducted following the WHO guidelines. The influence of the food availability, the larval density and the larval staging was evaluated on laboratory bioassays. A commercial Bacillus thuringiensis H-14 formulation against a reference Aedes aegypti strain was used. Results: the efficacy of Bacillus thuringiensis H-14 decreased in a linear manner with increasing larval density and development stage. This could be related to food intake and availability of the active principle. Competition in food intake resulted in lower efficacy of Bacillus thuringiensis H-14 applications. Conclusions: the Bacillus thuringiensis H-14 efficacy was affected for the availability of food, the larval development stage and the larval density, so all this should be borne in mind when applying the biolarvicide in situ.

Interação entre transgênicos (Bt) e inseticidas no controle de pragas-chave em híbridos de milho-safrinha / Interaction between transgenics and insecticides in the control of key pests on off-season maize hybrids

A cultura do milho é atacada por diversos lepidópteros-praga. Recentemente, foi liberada no Brasil a comercialização de híbridos de milho Bt, integrando em seu genoma o gene Cry1Ab, proveniente de Bacillus thuringiensis subsp. kurstaki, que codifica a proteína com efeito tóxico sobre insetos da ordem Lepidooptera. Dessa forma, o objetivo deste trabalho foi avaliar, em condições de campo, a eficácia de híbridos de milho convencionais e transgênicos para o controle de pragas, submetidos ou não à pulverização com inseticidas. O ensaio foi realizado em área experimental do Polo Apta Centro Norte, em Pindorama, Estado de São Paulo. Foram avaliados cinco híbridos de milho convencionais em comparação com os respectivos híbridos isogênicos Bt. A avaliação dos danos visuais de Spodoptera frugiperda (Smith) foi realizada a cada 10 dias, em 20 plantas das duas linhas centrais da parcela, e para Helicoverpa zea (Bod.) e Diatraea saccharalis (Fab.) na pré-colheita. As interações entre os híbridos e a transgenia ocorreram em todas as avaliações a partir dos 40 dia após emergência (DAE). Os híbridos transgênicos (Bt) avaliados apresentaram as menores notas de danos visuais de S. frugiperda em todas as avaliações. A transgenia também proporcionou menor percentagem de espigas danificadas por H. zea e S. frugiperda e de colmos atacados por D. saccharalis. Conclui-se que a transgenia proporciona redução nos danos visuais ocasionados pelas pragas, embora apresente desempenho diferente entre os híbridos.

Diverse lepidopteran pests attack the maize crop. The commercialization of hybrids of transgenic Bt maize, integrating in its genome the gene Cry1Ab, from Bacillus thuringiensis subsp. kurstaki, which codifies the protein with toxic effect on insects of the Lepidoptera order, has been recently introduced in Brazil. The objective of this work was to evaluate, under field conditions, the effectiveness of conventional and transgenic maize hybrids for the control of pests, sprayed or not with insecticides. The assay was carried out in an experimental area of the Polo Apta Centro Norte, in Pindorama, São Paulo State, Brazil. Five conventional maize hybrids were evaluated in comparison with the respective isogenic Bt hybrids. The evaluation was carried out every 10 days, in 20 plants of the 2 central lines of the parcel for Spodoptera frugiperda (Smith) damage. For damages of Helicoverpa zea (Bod.) and Diatraea saccharalis (Fab.) the evaluations were carried out in the pre-harvest. The interactions between the hybrids and the transgeny occurred in all evaluations starting at the 40th day after emergence (DAE). The Bt hybrids evaluated presented the lowest scores for visual S. frugiperda damages in all the evaluations. The transgenic maize also provided the lowest percentage of cobs damaged by H. zea and S. frugiperda, and of stalks attacked by D. saccharalis. In conclusion, the transgenic maize provided reduction in visual damages caused by the pests, however, it presented different performance among the hybrids.

Preferential protection of domains II and III of bacillus thuringiensis cry1Aa toxin by brush border membrane vesicles / Protección preferencial de los dominios II y III de la toxina cry1Aa de bacillus thuringiensis en vesículas de membrana de borde de cepillo

The surface exposed Leucine 371 on loop 2 of domain II, in Cry1Aa toxin, was mutated to Lysine to generate the trypsin-sensitive mutant, L371K. Upon trypsin digestion L371K is cleaved into approximately 37 and 26 kDa fragments. These are separable on SDS-PAGE, but remain as a single molecule of 65 kDa upon purification by liquid chromatography. The larger fragment is domain I and a portion of domain II (amino acid residues 1 to 371). The smaller 26-kDa polypeptide is the remainder of domain II and domain III (amino acids 372 to 609). When the mutant toxin was treated with high dose of M. sexta gut juice both fragments were degraded. However, when incubated with M. sexta BBMV, the 26 kDa fragment (domains II and III) was preferentially protected from gut juice proteases. As previously reported, wild type Cry1Aa toxin was also protected against degradation by gut juice proteases when incubated with M. sexta BBMV. On the contrary, when mouse BBMV was added to the reaction mixture neither Cry1Aa nor L371K toxins showed resistance to M. sexta gut juice proteases and were degraded. Since the whole Cry1Aa toxin and most of the domain II and domain III of L371K are protected from proteases in the presence of BBMV of the target insect, we suggest that the insertion of the toxin into the membrane is complex and involves all three domains.

La superficie de la toxina Cry1Aa, en el asa 2 del dominio II contiene expuesta la leucina 371, la cual fue modificada a lisina produciendo una mutante sensible a la tripsina, L371K. Esta mutante produce dos fragmentos de 37 y 26 kDa por acción de la tripsina que son separables por SDS-PAGE, pero que a la purificación por cromatografía líquida se mantienen como una sola molécula de 65 kDa. El fragmento grande contiene al dominio I y una parte del dominio II (aminoácidos 1 al 371). El polipéptido de 26 kDa contiene la parte restante del dominio II y dominio III (aminoácidos 372 al 609). Cuando la toxina mutante fue tratada con dosis altas de jugo intestinal de Manduca sexta, ambos fragmentos fueron degradados. Sin embargo, cuando fueron incubados en VMBC de M. sexta, el fragmento de 26 kDa fue protegido preferencialmente de las proteasas intestinales. Como se ha reportado, la toxina silvestre Cry1Aa también es protegida de la degradación de las proteasas cuando es incubada en VMBC de M. sexta. Sin embargo, cuando se adicionó VMBC de ratón a la mezcla de reacción, ni la toxina Cry1Aa ni la mutante L371K mostraron resistencia a las proteasas y fueron degradadas. Dado que la toxina completa de Cry1Aa y casi todo de los dominios II y III de L371K están protegidos de proteasas en presencia de VMBC del insecto, este estudio sugiere que la inserción de la toxina en la membrana involucra los tres dominios.

Estandarización de un bioensayo y evaluación preliminar de tres formulaciones comerciales de bacillus thuringiensis sobre tuta absoluta (meyrick) (lepidoptera: gelechiidae) / Standardising a bioassay for the preliminary evaluation of threecommercial bacillus thuringiensis formulations against tutaabsoluta (meyrick) (lepidoptera: gelechiidae)

La polilla del tomate (Tuta absoluta Meyrick; Lepidoptera: Gelechiidae) es una de las plagas más devastadorasdel tomate en Colombia y países suramericanos, produciendo pérdidas de hasta el 100% en cultivos sin protección.En 2009, T. absoluta se detectó en España, Portugal y países del mediterráneo, además de Inglaterra,Bulgaria y Alemania. Para su control se utilizan insecticidas químicos que generan resistencia e impactoambiental y de salud. La alternativa de utilizar biopesticidas contra esta plaga es de importancia creciente. Eneste estudio se evaluaron cinco métodos de bioensayo para medir adecuadamente la toxicidad sobre larvasde T. absoluta de tres productos comerciales: Dipel®, XenTary® y Turilav®, formulaciones a base de Bacillusthuringiensis (Bt). El método de Inmersión del folíolo, con el producto Dipel®, causó el 100% de mortalidadde larvas y 96% de supervivencia del testigo; este método presentó diferencias significativas al segundo(F=0,025, p>0,05) y cuarto (F=0,0018, p>0,05) día después de la aplicación (DDA). El método de Aspersiónfoliar por aerógrafo produjo 100% de mortalidad de larvas con Dipel® al segundo DDA (F=7,94x10-10,p> 0,05), y produjo diferencias significativas también al cuarto DDA (F=3,45x10-6, p>0,05). Los métodosFoliolos sumergidos y Medio de cultivo provocaron una alta mortalidad en el control por lo que fueron rechazados.El uso de Dipel®, XenTari® y Turilav® en concentración de 1,25 g/L causó entre 80-100% demortalidad entre el segundo y octavo DDA en tres métodos evaluados válidos (1, 2, 3), además corrobora laactividad biológica de B. thuringiensis sobre este insecto plaga.

The tomato moth (Tuta absoluta Meyrick; Lepidoptera: Gelechiidae) is one of the most devastating tomatopests in Colombia and South-American countries, producing losses of up to 100% in unprotected crops. T.absoluta was detected in Spain, Portugal and Mediterranean countries in 2009, as well as England, Bulgaria andGermany. Chemical insecticides are used for controlling it; however, they produce resistance and an environmentaland human health impact. Finding an alternative to using biopesticides against this pest is becomingincreasingly important. This study evaluated five bioassay methods measuring three commercial products’toxicity on T. absoluta larvae: Dipel, XenTary and Turilav Bacillus thuringiensis (Bt) -based formulations. The leafdipping bioassay method caused 100% larvae mortality with Dipel, the control group having 95% survivalrate. The other products showed significant differences on the 2nd (F=0.025, p>0.05) and 4th (F=0.0018,p>0.05) days after application (DAA). The leaf spray airbrush method produced 100% larvae mortality withDipel on the 2nd DAA, having significant differences from the other products tested on 2nd (DAA F=7.94 x10-10, p>0.05 ), 4th (F=3.45x10-6, p>0.05 ) and 8th (F=1.07x10-5, p>0.05 ) DAA. Submerged leaflet and culturemedium methods caused high mortality in controls and were thus rejected. A variation of the leaflet immersionmethod was standardised. The three commercial products produced high mortality in Lab conditionsregarding T. absolute larvae control at 1.25 g/L concentration, thereby corroborating the biological activity ofB. thuringiensis against this insect pest.

Histopatologia da interação de Bacillus thuringiensis e extratos vegetais no intestino médio de Spodoptera frugiperda (Lepidoptera: Noctuidae) / Histopathology of the interaction of Bacillus thuringiensis and plant extracts in the midgut of Spodoptera frugiperda (Lepidoptera: Noctuidae)

Plantas são fontes naturais de substâncias inseticidas, já que podem ser produzidas pelo vegetal em resposta a ataques de insetos podendo representar uma alternativa no manejo de insetos-praga, como Spodoptera frugiperda. Dessa forma, o presente trabalho objetivou a análise histopatológica do intestino médio de lagartas de S. frugiperda, após a ingestão dos extratos obtidos por maceração e infusão de Petiveria alliacea. Zingiber officinale. Cymbopogon citratus. Malva silvestris. Baccharis genistelloides e Ruta graveolens, assim como a associação desses extratos com Bacillus thuringiensis aizawai. As lagartas foram tratadas in vivo com cada extrato e a bactéria, mais a associação desses extratos com B. thuringiensis e após, uma reação de cinética entre 3 e 27 horas, foram fixadas para o preparo de cortes histológicos, os quais foram corados e analisados comparativamente às testemunhas em microscopia óptica. Os resultados mostraram mudanças na histologia do intestino médio das lagartas de S. frugiperda, 3 horas após a aplicação dos tratamentos à base de alliacea. Z. officinale. C. citratus e M. silvestris, enquanto que para B. genistelloides e R. graveolens só foram observados alterações após 6h. Na interação dos extratos com B. thuringiensis observou-se alterações nas microvilosidades, desorganização do intestino médio e a hipertrofia das células epiteliais que projetaram-se para o lúmen. Os resultados desse trabalho mostram que o efeito histopatológico de Z. officinale. M. silvestris. R. graveolens e B. genistelloides, foram mais ativos quando comparados aos extratos de P. alliacea e C. citratus, os quais apresentaram uma interação positiva com B. thuringiensis.

The natural insecticidal substances produced by plants in response to insect attack may represent an alternative for the management of pest insects such as Spodoptera frugiperda. This study was therefore aimed to histopathologically analyze the midgut of larvae of S. frugiperda, after ingestion of extracts obtained by maceration and infusion of Petiveria alliacea. Zingiber officinale. Cymbopogon citratus. Malva silvestris. Baccharis genistelloides and Ruta graveolens, and the association of these extracts with Bacillus thuringiensis aizawai. The larvae were treated in vivo with each extract, B. thuringiensis, and the combination of these extracts with the bacteria, and after a reaction kinetics of between 3 to 27 hours they were fixed for the preparation of histological sections, which were stained and analyzed in comparison to the controls with light microscopy. The results showed changes in the histology of the midgut of the larvae of S. frugiperda, 3 hours after application of treatments based on P. alliacea. Z. officinale. C. citratus and M. silvestris, while for B. genistelloides and R. graveolens changes were observed only after 6h. In the interaction of extracts with B. thuringiensis the analysis revealed changes in microvilli, disorganization of the midgut and hypertrophy of epithelial cells that projected into the lumen. The findings show that the histopathological effects of Z. officinale. M. silvestris. R. graveolens and B. genistelloides were more active when compared to extracts of P. alliacea and C. citratus, which showed a positive interaction with B. thuringiensis.

Changes in the haemocytes of bombyx mori larvae [Lepidoptera: bombycidae] in relation to Escherichia coli and bacillus thuringensis infection

Injection of Bombyx mori fifth instar larvae with non- pathogenic bacteria Escherichia coli and entomopathogenic bacteria Bacillus thuringeinsis affected in the total haemocyte count and changes in the different haemocyte population during bacterial infection. Cellular immunity responses of B. mori against bacteria and the hormonal regulation of immunity were also studied

Biological activity of Bacillus thuringiensis strains against larvae of the Blowfly Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae)

Different strains of Bacillus thuringiensis Berliner were proved to be a powerful biologic insecticide against larvae of several insect orders. Due to the epidemiological importance of blowflies of the Chrysomya Robineau-Desvoidy genus in the production of secondary cutaneous myiasis and mechanic transmission of pathogenic agents, the performance of two strains of B. thuringiensis (LFB-FIOCRUZ 907 and LFB-FIOCRUZ 856) was tested against larvae of Chrysomya putoria (Wiedemann). The LFB-FIOCRUZ 907 strain was tested in four different concentrations, added to the diet; the LFB-FIOCRUZ 856 strain was tested in three concentrations. C. putoria larvae showed sensibility to the treatment with the LFB-FIOCRUZ 907 strain at the tested concentrations. The higher concentration presented the best efficiency, causing higher mortality and reducing larval weight and adult emergence more intensely. The LFB-FIOCRUZ 856 strain showed low toxicity, sliglity reducing emergence time of adults at 326 mg/ 25 g concentration and larval weight at 326 mg/ 25 g and 86 mg/ 25 g concentrations.

Diferentes linhagens de Bacillus thuringiensis Berliner têm provado ser poderoso inseticida biológico contra larvas de várias ordens de insetos. Dada a importância epidemiológica das moscas do gênero Chrysomya Robineau-Desvoidy na produção de miíases cutâneas secundárias e transmissão mecânica de agentes patogênicos, avaliou-se a atividade de duas linhagens de B. thuringiensis (LFB-FIOCRUZ 907 e LFB-FIOCRUZ 856) sobre larvas de Chrysomya putoria (Wiedemann). A linhagem LFB-FIOCRUZ 907 foi testada em quatro diferentes concentrações misturadas à dieta e a linhagem LFB-FIOCRUZ 856 em três concentrações. As larvas de C. putoria apresentaram sensibilidade ao tratamento com a linhagem LFB-FIOCRUZ 907, nas concentrações testadas, sendo a concentração mais alta a de maior eficiência, causando maior mortalidade e reduzindo mais intensamente o peso larval e a taxa de emergência dos adultos. A linhagem LFB-FIOCRUZ 856 apresentou toxicidade muito baixa, redizindo ligeiramente a emergência dos adultos na concentração de 326 mg/ 25 g e o peso larval nas concentrações de 326 mg/ 25 g e 86 mg/ 25 g.

Development of a self floating slow release formulation of Bacillus thuringiensis var. israelensis and its larvicidal activity.

Alginate encapsulated B. thuringiensis var. israelensis (B. t. i.) self floating type formulations were prepared. Its spore release rate, floating efficacy and larvicidal activity against Culex quinquefasiatus were tested in the laboratory. The larval mortality of 91-100% was induced by the floating formulation with a mean spore release of 3.04 x 10(4)/ml/day from 6th day to 27th day. From day 28 to 33 the mean number of spores released were 1.16 x 10(4)/ml/day which caused 72.2-88.2% mortality. From 34th day to 40th day the mean number of spores released were 4.97 x 10(3)/ml/day which caused 42.2-67.2% mortality. However, the self floating alginate encapsulated beads were intact and found to float upto 40 days.

Influencia de algunos iones en la esporulación y formación de delta-endotoxina en cultivos de Bacillus thuringiensis / Influence of several ions on growth in sporulation and delta-endotoxin formation Bacillus thurigiensis

El estudio del efecto de algunos iones en medios de cultivo para Bacillus thuringiensis en los que se emplean fuentes nitrogenadas complejas, revela la necesidad de suplementación de estos medios con sales de Ca+**2, Mg+**2 y Mn+**2 en concentraciones adecuadas, puesto que el aporte de dichos iones por la fuente nitrogenada compleja, resulta insuficiente para lograr óptimos rendimientos en delta endotoxina. La constancia de la relación proteína/UFC pone en evidencia que la ausencia de uno o más iones afecta por igual el fenómeno de esporulación y formación del cristal proteico. En medios deficitarios en Mn+**2 se observa una sensible disminución de las UFC, manifestándose la importancia de dicho ión en el mecanismo de esporulación. La ausencia de Ca+**2 si bien no cumple una función esencial en la formación del cristal, tiene implicancia a nivel industrial, en el manejo de las pastas esporo-cristal