scholarly journals Evaluación in vitro de 14 medios de cultivo sobre el crecimiento micelial de Moniliophthora perniciosa (Stahel ) Aime & Phillips-Mora

TAYACAJA ◽  
2021 ◽  
Vol 4 (1) ◽  
pp. 168-179
Author(s):  
Jhojan Sven Huaman Pampañaupa ◽  
Fanny-Rosario Márquez Romero ◽  
Sara Cabrera Márquez ◽  
Dilman Glicerio Paricoto Apaza
Keyword(s):  
Moniliophthora Perniciosa ◽  
Santa Ana

El cultivo de cacao en el Perú se va incrementando por la ampliación de áreas productivas y mejora de los rendimientos, sin embargo, la presencia de las tres principales enfermedades (mazorca parda, escoba de bruja y moniliasis) afectan significativamente los rendimientos en cacaotales con bajo nivel de mantenimiento, teniendo como objetivo determinar los medios de cultivo que permitan mayores crecimientos radiales de Moniliophthora perniciosa, por lo que se hace necesario contar con estrategias viables de manejo y control. Las pruebas in vitro de control químico y biológico requieren que los aislados presentan crecimientos miceliales apropiados, por lo que se planteó la comparación de 14 medios de cultivo sólidos no convencionales con 6 repeticiones. Los tratamientos fueron maltacervezneg, frejolcaraota, achiote, cascmazorca, arroz, maizamarillo, frugos, mbamilaceo, papa, melazacacao, vegetales, masabourd, escobaverde y gelatina, sobre los cuales se sembró basidiocarpos de M. perniciosa proveniente de Cacaopampa-Santa Ana, pegados en el centro de la tapa de las placas petri de 90 mm y se incubaron por 12 días a 25°C de temperatura. Los medios de cultivo que permitieron mayores crecimientos radiales de M. perniciosa fueron medio malta proveniente de cerveza negra, frejol caraota, achiote y cáscara de cacao con 90.00, 87.08, 82.58 y 66.75 mm respectivamente y el medio papa (PDA) presentó un desempeño 50% menor que el medio Maltacerveneg en el crecimiento radial de cultivo monospórico de M. perniciosa.

Genetic characterization of Moniliophthora perniciosa from Ecuador and in vitro sensitivity to compost tea

2019 ◽  
Vol 154 (4) ◽  
pp. 943-959
Author(s):  
María Gabriela Maridueña-Zavala ◽  
Andrea Freire-Peñaherrera ◽  
Rodrigo Fernando Espinoza-Lozano ◽  
Mirian Villavicencio-Vasquez ◽  
Maria Jimenez-Feijoo ◽  
...  
Keyword(s):  
Genetic Characterization ◽  
Compost Tea ◽  
Moniliophthora Perniciosa ◽  
In Vitro Sensitivity

scholarly journals Moniliophthora perniciosa, the Causal Agent of Cacao Witches’ Broom Disease Is Killed in vitro by Saccharomyces cerevisiae and Wickerhamomyces anomalus Yeasts

2021 ◽  
Vol 12 ◽  
Author(s):  
Pedro Ferraz ◽  
Rogelio Lopes Brandão ◽  
Fernanda Cássio ◽  
Cândida Lucas
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Feeding Strategy ◽  
Fungal Growth ◽  
Initial Step ◽  
Yeast Cells ◽  
Moniliophthora Perniciosa ◽  
Wickerhamomyces Anomalus ◽  
Yeast Cultures ◽  
Broom Disease

Cacao plantations from South America have been afflicted with the severe fungal disease known as Witches’ Broom Disease (WBD), caused by the basidiomycete Moniliophthora perniciosa. Yeasts are increasingly recognized as good fungal biocides, although their application is still mostly restricted to the postharvest control of plant and fruit decay. Their possible utilization in the field, in a preharvest phase, is nevertheless promising, particularly if the strains are locally adapted and evolved and if they belong to species considered safe for man and the environment. In this work, a group of yeast strains originating from sugarcane-based fermentative processes in Brazil, the cacao-producing country where the disease is most severe, were tested for their ability to antagonize M. perniciosa in vitro. Wickerhamomyces anomalus LBCM1105 and Saccharomyces cerevisiae strains LBCM1112 from spontaneous fermentations used to produce cachaça, and PE2 widely used in Brazil in the industrial production of bioethanol, efficiently antagonized six strains of M. perniciosa, originating from several South American countries. The two fastest growing fungal strains, both originating from Brazil, were further used to assess the mechanisms underlying the yeasts’ antagonism. Yeasts were able to inhibit fungal growth and kill the fungus at three different temperatures, under starvation, at different culture stages, or using an inoculum from old yeast cultures. Moreover, SEM analysis revealed that W. anomalus and S. cerevisiae PE2 cluster and adhere to the hyphae, push their surface, and fuse to them, ultimately draining the cells. This behavior concurs with that classified as necrotrophic parasitism/mycoparasitism. In particular, W. anomalus within the adhered clusters appear to be ligated to each other through roundish groups of fimbriae-like structures filled with bundles of microtubule-sized formations, which appear to close after cells detach, leaving a scar. SEM also revealed the formation of tube-like structures apparently connecting yeast to hypha. This evidence suggests W. anomalus cells form a network of yeast cells connecting with each other and with hyphae, supporting a possible cooperative collective killing and feeding strategy. The present results provide an initial step toward the formulation of a new eco-friendly and effective alternative for controlling cacao WBD using live yeast biocides.

76 SURVIVAL AND IN VITRO FUNCTIONALITY OF DOMESTIC CAT EPIDIDYMAL SPERMATOZOA FOLLOWING CRYOPRESERVATION IN EXTENDERS WITH OR WITHOUT EGG YOLK

2009 ◽  
Vol 21 (1) ◽  
pp. 138
Author(s):  
J. R. Saenz ◽  
C. Dumas ◽  
B. L. Dresser ◽  
M. C. Gómez ◽  
R. A. Godke ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Egg Yolk ◽  
Human Sperm ◽  
Domestic Cat ◽  
Blastocyst Development ◽  
Santa Ana ◽  
Sperm Suspension ◽  
Epididymal Spermatozoa ◽  
One Way Anova

Our purpose was to compare in vitro survivability and functionality of cat epididymal spermatozoa cryopreserved in TEST egg-yolk buffered extender (TYB) with that obtained by use of clear Tris-citrate and HEPES-buffered extenders containing BSA. Testes were transported to the lab in HEPES saline; epididymides were dissected in HEPES-199 medium (HE-199) and repeatedly sliced. The sperm suspension was filtered (40 μm), layered onto a density gradient column (Isolater, Irving Scientific, Santa Ana, CA), and centrifuged at 600g for 20 min. Aliquots of the sperm pellet were extended in TYB, Human Sperm Preservation Medium (HSPM), or Tris-citrate + 10% BSA (TCBSA). After cooling to 4°C, samples were diluted 1:1 with extender + 12% glycerol in 4 steps as modified from Gao DY et al. 1995 Hum. Reprod. 10, 1109–1122. Then, samples were loaded into 0.25-mL straws, sealed, and frozen on a dry ice block (–80°C) for 20 min before storage in LN2. Straws were thawed by exposure to air (22°C) for 5 s and immersion in a 60°C water bath for 5 s. Samples were diluted by addition of HE-199 in 7 steps as modified from Gao DY et al. 1995 Hum. Reprod. 10, 1109–1122, centrifuged at 200g for 10 min and pellets resuspended in HE-199. Motility (MOT, phase contrast, 37°C), membrane integrity (MI, SYBR 14–PI), and acrosomal status (AS, FITC–PNA) were evaluated at 0 h, after gradual cooling to 4°C, and after freezing at 0 h and 3 h post-thaw (37°C). Cumulus oocyte complexes (COC) were placed in modified TCM-199 and cultured for 24 h in 5% O2, 5% CO2, and 90% N2 at 38°C (IVM). For IVF, COC were co-incubated with spermatozoa frozen in either TYB or HSPM in droplets (1 million sperm mL–1) of IVF medium under 5% CO2 in air at 38°C. After 18 h, oocytes were rinsed and cultured using a 3-step system (Pope CE et al. 2006 Theriogenology 66, 59–71) until blastocyst development was evaluated (Day 8). There were no treatment differences at any time/temperature point for the 3 sperm parameters evaluated (one-way ANOVA; P > 0.05). As shown in Table 1, sperm motility in TCBSA and HSPM decreased by 20% after cooling to 4°C and another 20% after freezing, whereas motility in TYB was maintained after cooling and decreased <30% after freezing. Membrane integrity and acrosomal status values were 12 to 15% greater at collection, at 4°C and at 0 h post-thaw, and 25% greater at 3 h post-thaw than were the motility values. Cleavage frequency and blastocyst development rate of 203 IVM oocytes after IVF using sperm frozen in TYB and HSPM was 36 v. 33% and 50 v. 44%, respectively. In summary, we have shown that cat epididymal spermatozoa can be frozen successfully in cryoprotectant solutions that do not contain egg yolk. Table 1.Motility, membrane integrity and acrosomal status of cat epididymal sperm after cryo-storage

112 EFFECT OF EGG YOLK CONCENTRATION IN TEST BUFFERED EXTENDER ON SURVIVAL AND IN VITRO FUNCTIONALITY OF DOMESTIC CAT EPIDIDYMAL SPERMATOZOA FOLLOWING CRYOPRESERVATION

2010 ◽  
Vol 22 (1) ◽  
pp. 214
Author(s):  
J. R. Saenz ◽  
C. Dumas ◽  
B. L. Dresser ◽  
M. C. Gómez ◽  
R. A. Godke ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Egg Yolk ◽  
Domestic Cat ◽  
Blastocyst Development ◽  
Santa Ana ◽  
Sperm Suspension ◽  
Epididymal Spermatozoa ◽  
One Way Anova ◽  
Cryoprotectant Solution

Our purpose was to examine the effect of egg yolk concentration (EY; 2, 5, or 10%) on in vitro survivability and functionality of cat epididymal spermatozoa cryopreserved in TEST-buffered extender (TYB). Testes were transported in HEPES saline; epididymes were dissected in HEPES 199 medium (He199) and repeatedly sliced. The sperm suspension was filtered (40 μ), layered onto a density gradient column (Isolate®, Irving Scientific, Santa Ana, CA, USA), and centrifuged at 650 g for 20 min. Aliquots of the sperm pellet were extended in TYB containing 2, 5, or 10% EY. After cooling to 4°C, samples were diluted 1:1 with TYB containing 2, 5, or 10% EY + 12% glycerol in 4 steps as modified from Gao DY et al. Hum. Reprod. 1995 10, 1109-1122. Then, samples were loaded into 0.25-mL straws, sealed, and frozen on a dry ice block (-80°C) for 20 min before storage in LN2. Straws were thawed by exposure to air (˜22°C) for 5 s and immersion in a 60°C water bath for 5 s. Samples were diluted by addition of He199 in 7 steps as modified from Gao DY et al. Hum. Reprod. 1995 10, 1109-1122 centrifuged at 200g for 10 min, and pellets resuspended in He199. Motility (Mot, phase contrast, 37°C), membrane integrity (M.I., SYBR 14-PI), and acrosomal status (A.S., FITC-PNA) were evaluated at 0 h, after gradual cooling to 4°C and after freezing at 0 and 3 h post-thaw (37°C). Ten replicates were done. Cumulus oocyte complexes (COC) were placed in modified TCM-199 and cultured for 24 h in 5% O2, 5% CO2, and 90% N2 at 38°C (IVM). For IVF, COC were co-incubated with spermatozoa frozen in TYB + 2% egg yolk or HSPM (no egg yolk) in droplets (1 million sperm/mL) of IVF medium under 5% CO2 in air at 38°C. After 18 h, oocytes were rinsed and cultured until blastocyst development was evaluated (Day 8). There were no treatment differences at any time or temperature point for the 3 sperm characteristics evaluated (one-way ANOVA; P > 0.05). As shown in the Table 1, at 0 h post-thawing, sperm in each group retained ˜70% of their initial pre-freeze motility. After 3 h of post-thaw incubation, motility decreased to ˜50% of the pre-freeze value. Cooling to 4°C did not affect membrane integrity or acrosomal status, but post-thaw values were reduced by 30-35% as compared with pre-freeze. Cleavage frequency and blastocyst development of 284 IVM oocytes after IVF using sperm frozen in TYB + 2% EY and HSPM were 53 v. 52% and 42 v. 38%, respectively (P > 0.05). In summary, we have shown that cat epididymal spermatozoa can be frozen successfully in a cryoprotectant solution containing minimal egg yolk (2%). Table 1.Motility, membrane integrity, and acrosomal status of cat epididymal sperm after cryo-storage

scholarly journals Enzymatic and Antagonist Activity of Endophytic Fungi from Sapindus saponaria L. (Sapindaceae)

2019 ◽  
Vol 24 (2) ◽  
pp. 322-330 ◽  
Author(s):  
Caroline Menicoze dos Santos ◽  
Amanda Da Silva Ribeiro ◽  
Adriana Garcia ◽  
Andressa Domingos Polli ◽  
Julio Cesar Polonio ◽  
...  
Keyword(s):  
Endophytic Fungi ◽  
Molecular Taxonomy ◽  
Phytopathogenic Fungi ◽  
Enzyme Assays ◽  
Antagonist Activity ◽  
Moniliophthora Perniciosa ◽  
Biotechnological Potential ◽  
Sapindus Saponaria ◽  
Medicinal Properties

Endophyte microorganisms have great biotechnological interest, with features applicable to different areas and are potentially useful in agriculture. The current study determines the biotechnological potential of endophytic fungi, isolated from leaves of Sapindus saponaria, to control phytopathogenic fungi and evaluate their enzyme production. Molecular taxonomy was performed by sequencing of the ITS1-5.8S-ITS2 ribosomal DNA region, identifying the genera Phomopsis, Sordariomycetes, Diaporthe, and Colletotrichum. In vitro antagonism against phytopathogens showed better results against Fusarium solani and provided inhibition indices between 41.8 % and 67.5 %. The endophytic strain SS81 (Diaporthe citri) presented the highest antagonism index against the pathogen. Against Glomerella sp. and Moniliophthora perniciosa, inhibition rates ranged between 18.7 % and 57.4 % and between 38.3 % and 64.8 %, respectively. Enzyme assays revealed that strain SS65 (Diaporthe sp.) produced 1.16 UI µmol/min of amylase; strain SS77 (Diaporthe sp.) produced 2.74 UI µmol/min of pectinase, and strain SS08 (Diaporthe sp.) produced 1.51 UI µmol/min of cellulase. Thus, the current study shows evidence the importance of isolated endophytes with phytoprotective properties of plants with medicinal properties as alternatives for biological control and natural sources of products with biotechnological interest.

scholarly journals Evaluation of Trichoderma spp for the biocontrol of Moniliophthora perniciosa Subgroup 1441

2011 ◽  
Vol 3 (1) ◽  
Author(s):  
Maria Lucia Garcia Simoes ◽  
Samia Maria Tauk-Tornisielo ◽  
Givaldo Rocha Niella ◽  
Daniel Mario Tapia Tapia
Keyword(s):  
Trichoderma Harzianum ◽  
Spore Production ◽  
Potential Growth ◽  
Trichoderma Spp ◽  
Moniliophthora Perniciosa ◽  
Trichoderma Species ◽  
Biological Control Potential ◽  
Biocontrol Potential ◽  
The Individual

Trichoderma species , isolated from different producer regions of cocoa (Bahia, Brazil), were evaluated as for their capacity of usage in the biocontrol of the basidiomycete Moniliophthora perniciosa subgroup 1441, which causes the witches’ broom in cocoa. The isolates of Trichoderma were evaluated through individual indices so called %AP (Antagonistic Potential to Moniliophthora perniciosa subgroup 1441), %PG (Potential Growth in vitro) and %PSPr (Potential of Spore Production on rice) These indices were evaluated together, also they were used for the determination of Biological Control Potential (%BCP) of each antagonistic specie to the evaluated pathogen. Afterwards, the ability of the antagonistic to colonize and to produce spores on sterilized dry brooms was also evaluated. Some of the isolates Trichoderma spp showed a high %AP to the pathogen and high %PG, but did not present a significant %PSPr, turning impossible the spore production for biocontrol at commercial level. Significant differences were found within the individual indices among the species and isolates of the same species of Trichoderma spp, pointing out a great genetic variability among them. Trichoderma harzianum 911 showed to have the best biocontrol potential to the pathogen when compared to the other isolates, presenting a %BCP de 91.86% (mainly by the high %AP of 97,76%) a %PSPr of 99.53%, also producing 22.67 spores x 109. mL-1 by dry broom segment. Trichoderma harzianum 911 showed to be as promising isolate for future researches on biocontrol of cocoa witches’ broom.

scholarly journals Differential Gene Expression Between the Biotrophic-Like and Saprotrophic Mycelia of the Witches' Broom Pathogen Moniliophthora perniciosa

2008 ◽  
Vol 21 (7) ◽  
pp. 891-908 ◽  
Author(s):  
Johana Rincones ◽  
Leandra M. Scarpari ◽  
Marcelo F. Carazzolle ◽  
Jorge M. C. Mondego ◽  
Eduardo F. Formighieri ◽  
...  
Keyword(s):  
Gene Expression ◽  
Oxidative Phosphorylation ◽  
Differential Gene Expression ◽  
Molecular Mechanisms ◽  
Expressed Sequence Tag ◽  
Lower Growth ◽  
Moniliophthora Perniciosa ◽  
Differential Gene ◽  
Combined Strategy

Moniliophthora perniciosa is a hemibiotrophic fungus that causes witches' broom disease (WBD) in cacao. Marked dimorphism characterizes this fungus, showing a monokaryotic or biotrophic phase that causes disease symptoms and a later dikaryotic or saprotrophic phase. A combined strategy of DNA microarray, expressed sequence tag, and real-time reverse-transcriptase polymerase chain reaction analyses was employed to analyze differences between these two fungal stages in vitro. In all, 1,131 putative genes were hybridized with cDNA from different phases, resulting in 189 differentially expressed genes, and 4,595 reads were clusterized, producing 1,534 unigenes. The analysis of these genes, which represent approximately 21% of the total genes, indicates that the biotrophic-like phase undergoes carbon and nitrogen catabolite repression that correlates to the expression of phytopathogenicity genes. Moreover, downregulation of mitochondrial oxidative phosphorylation and the presence of a putative ngr1 of Saccharomyces cerevisiae could help explain its lower growth rate. In contrast, the saprotrophic mycelium expresses genes related to the metabolism of hexoses, ammonia, and oxidative phosphorylation, which could explain its faster growth. Antifungal toxins were upregulated and could prevent the colonization by competing fungi. This work significantly contributes to our understanding of the molecular mechanisms of WBD and, to our knowledge, is the first to analyze differential gene expression of the different phases of a hemibiotrophic fungus.

scholarly journals CLONAGEM E EXPRESSÃO DA PORÇÃO ENZIMÁTICA DA QUITINA SINTASE DE Moniliophthora perniciosa (STAHEL) AIME & PHILLIPS-MORA A PARTIR DE CÉLULAS DE Escherichia coli

2019 ◽  
Author(s):  
Afonso Menezes do Nascimento Neto
Keyword(s):  
Escherichia Coli ◽  
Theobroma Cacao ◽  
Moniliophthora Perniciosa

A quitina é um homopolissacarídeo linear com uma função estrutural de extrema importância na composição das membranas celulares dos fungos. Sendo um desses o Moniliophtora perniciosa, causador da fitopatologia da cultura do Theobroma cacao, a Vassoura-de-bruxa, que infecta as regiões meristemáticas da planta, especialmente frutos novos, lançamentos e almofadas florais, causando queda acentuada na produção, dano nas almofadas florais e causando enfraquecimento da planta (Brasil, 2013). Com os conhecimentos adquiridos a partir da descoberta da sequência do genoma do Moniliophtora perniciosa por Souza (2012), foram criadas alternativas para o controle do fungo nas plantações de cacau, onde a inibição ou desregulação das principais enzimas são objetivos importantes para o desenvolvimento de fungicidas (Merzendorfer, 2003). No qual, este presente trabalho se baseia no pressuposto da elaboração de um inibidor para enzimas da rota de síntese da quitina, como a Quitina Sintase, dispondo-se a fazer a clonagem da porção enzimática da Quitina Sintase que proporcionará testes in vitro de possíveis inibidores para controle do Moniliophtora perniciosa.

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