Novel Harzia ixtarensis Fungus on Annona cherimola Fruit in Mexico and Its Synergistic Relationship with Colletotrichum fragariae.

Since 2005 in Íxtaro, Michoacán, symptoms of Harzia infection have been observed on immature Annona cherimola fruit with Colletotrichum fragariae-induced anthracnose lesions and mummified fruit. This study aimed to identify the Harzia sp. and evaluate its pathogenicity. Four isolates were obtained from fruit exhibiting symptoms, cultured in four types of agar under various conditions, and characterized based on concatenated internal transcribes spacer (ITS) + large subunit and ITS + small subunit sequences. Additionally, the isolates were compared with two CBS species (two-type strains and two isolates) of Harzia patula and H. tenella under the same conditions as the Harzia isolates, and all known Harzia spp. in culture were included in two phylogenetic analyses. H. ixtarensis sp. nov. was proposed. Compared with H. patula CBS isolate 121524 which was the most closely phylogenetically related species, H. ixtarensis was characterized by slower colony growth (white to salmonish-beige), different percentages of two forms of conidia (elongated and globose; unicellular and hyaline to subhyaline), and smaller conidia. The conidia mainly germinated with two hyaline tubes without an appressorium. In situ inoculations (1 × 106 ml-1 conidia suspension) of fruit showed that fruit with wounds developed larger lesions than those without wounds. Harzia inoculation on anthracnose lesions (induced by prior inoculation with C. fragariae) produced larger anthracnose lesions than C. fragariae alone. When C. fragariae or H. ixtarensis was inoculated alone, the lesion size was 51 and 99% smaller, respectively, indicating synergy between C. fragariae and H. ixtarensis. Thus, H. ixtarensis may have a parasitic-synergistic and necrotrophic lifestyle, and exhibited symptoms on anthracnose lesions.

Effective In Vitro Control of Two Phytopathogens of Agricultural Interest Using Cell-Free Extracts of Pseudomonas fluorescens and Chitosan.

A biofungicide is a natural product that can be derived from various sources such as, among others, microorganisms, higher plants, animal products, phytochemicals, semiochemicals, and antagonist microorganisms. One of the most important approaches for the production of biofungicides is the combination of biocontrol agents. This study showed the inhibition growth of Alternaria alternata and Fusarium solani treated with cell-free extracts of P. fluorescens. Using thin-layer chromatography and plate assays it was also demonstrated that the cell-free extracts of P. fluorescens contained siderophores and derivates of 4-diacetylphloroglucinol and phenazine. Moreover, the combination of cell-free extracts of P. fluorescens and chitosan [50-1.5% (v/v)] had a synergistic effect since they notably inhibited the mycelial growth of A. altenata and F. solani. Various morphological alterations to the mycelia and conidia of the treated fungi as a result of this combination were also observed. The present study could be a starting point to control other fungal phytopathogens using different cell-free extracts and chitosan as biocontrol agents.

Cellular alterations in Pectobacterium carotovorum treated with nanostructured formulations during the incubation time.

Pectobacterium carotovorum was incubated in formulations of chitosan nanoparticles or thyme essential oil-loaded chitosan nanoparticles for a maximum period of 48 h time. The cellular changes and viability were evaluated by transmission electron microscopy (TEM), and two colorimetric assays 3-(4,5 dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) and alamar blue (AMB), respectively. The incubation time and the addition of the secondary metabolite to the formulation were key factors to the cell damage and cell inhibitory effects on P. carotovorum, TEM observations overall demonstrated on the treated bacterium, cell surface alterations such as deforming and disappearance of the cell wall and the plasmatic membrane, with agglomeration of nanoparticles outside and inside of the cells, loss of cell content and lysis. Cell viability was reduced about 80% (MTT) and 100% (AMB) in the applied treatment of chitosan-loaded thyme essential oil nanoparticles after 48 h incubation, in addition, the total cell inhibition was shown from 6 h incubation onwards with the AMB assay. The TEM micrographs and the cell viability assays provided enough evidence of the antimicrobial activity of the nanostructured formulations compared with the control where no damage was observed.

Functional properties of flours and protein concentrates of 3 strains of the edible mushroom Pleurotus ostreatus.

Pleurotus ostreatus is an edible mushroom with significant nutritional properties and highly valuable protein concentrates can be obtained from its fruit bodies. Functional properties of flours and protein concentrates derived from 3 Pleurotus ostreatus strains (PCM, POS and hybrid PCM × POS) were evaluated in this investigation. Fruit bodies were produced on wheat straw substrate, flours were obtained from dried and grinded fruit bodies and protein concentrates were extracted from flours by alkaline solubilization. For all 3 strains, pale yellow flours were obtained and protein concentrates showed a grayish brown color. Flour bulk densities ranged from 0.52 to 0.64 g/mL, a higher value than those for protein concentrates, i.e. 0.30-0.35 g/mL. The highest water absorption capacities (WAC) were observed for flours (300-418.8%) while protein concentrates presented higher oil absorption capacity (OAC) (173.3-214.1%). Flours and protein concentrates presented a minimal gelation concentration of 2%. Protein concentrates showed a higher foam capacity formation (FC) at pH 8. Likewise, flours and protein concentrates presented higher foam stability (FS) at alkaline pH (8 and 10). Emulsion activity index (EAI) for flours ranged from 3.96 to 26.68 m2 g-1 whereas for protein concentrates ranged from 1.55 to 10.28 m2 g-1. These results indicate that flours and protein concentrates from P. ostreatus have remarkable functional properties, valuable in food industry where foaming and emulsifying properties are required.

Utilización de marcadores ITS e ISSR para la caracterización molecular de cepas híbridas de Pleurotus djamor / Use of ITS and ISSR markers in the molecular characterisation of Pleurotus djamor hybrid strains

Antecedentes. La caracterización molecular de cepas silvestres de Pleurotus es importante para la conservación del germoplasma y su posterior uso en la mejora genética. No se han realizado estudios moleculares con los monocariontes utilizados para la producción de cepas híbridas, ni de las cepas reconstituidas obtenidas al aparear tales monocariontes. Por consiguiente, la caracterización molecular de cepas dicarióticas parentales, híbridas y reconstituidas, así como de cepas monocarióticas, es de suma importancia. Objetivos. Caracterizar molecularmente cepas dicarióticas silvestres e híbridas, así como cepas monocarióticas de Pleurotus djamor. Métodos. Se recolectaron cinco cepas silvestres de P. djamor en diferentes estados de México y se identificaron molecularmente mediante la secuenciación de la región ITS1-5.8-ITS2 con los oligonucleótidos universales ITS1 e ITS4. Se seleccionaron dos cepas silvestres y se generaron cuatro cepas híbridas por apareamiento de neohaplontes compatibles. Para la caracterización molecular de las cepas monocarióticas y dicarióticas seleccionadas y producidas se utilizaron seis marcadores ISSR. Resultados. Con los marcadores ITS se obtuvo un producto de amplificación de 700 pb en las cinco cepas silvestres con una similitud del 99-100% con la especie P. djamor. Con los marcadores ISSR se obtuvieron un total de 95 fragmentos con un 99% de polimorfismo. Conclusiones. Las cepas silvestres se identificaron como P. djamor. Los marcadores ISSR generaron bandas polimórficas en las cepas monocarióticas y dicarióticas, y separaron ambos tipos de cepas. El alto grado de polimorfismo indica la diversidad genética de P. djamor, una ventaja para la producción de hongos y para la mejora de la especie (AU)

Background. Molecular characterisation of wild type Pleurotus species is important for germplasm conservation and its further use for genetic improvement. No molecular studies have been performed with monokaryons used for producing hybrid strains, either with the reconstituted strains obtained by pairing those monokaryons. The molecular characterisation of parental dikaryons, hybrid, and reconstituted strains as well as monokaryotic strains, is therefore of utmost importance. Aims. To carry out the molecular identification of Pleurotus djamor strains, i.e. dikaryotic wild type strains, hybrid strains, and the monokaryotic strains used for the hybrid formation. Methods. Five wild type strains of P. djamor from different states in Mexico were collected and molecularly identified by sequencing the ITS1-5.8-ITS2 region using ITS1 and ITS4 universal oligonucleotides. Four hybrid strains were obtained by pairing neohaplonts of two wild type strains selected. Six ISSR markers were used for the molecular characterisation of monokaryotic and dikaryotic strains. Results. Using the ITS markers, an amplified product of 700bp was obtained in five wild type strains, with a 99-100% similarity with P. djamor. A total of 95 fragments were obtained using the ISSR markers, with 99% of polymorphism. Conclusions. Wild type strains were identified as P. djamor, and were clearly grouped with Mexican strains from other states of Mexico. ISSR markers allowed the generation of polymorphic bands in monokaryotic and dikaryotic strains, splitting both types of strains. The high degree of polymorphism indicates the genetic diversity of P. djamor, an advantage in mushroom production and in the improving of the species (AU)

[Use of ITS and ISSR markers in the molecular characterisation of Pleurotus djamor hybrid strains]. / Utilización de marcadores ITS e ISSR para la caracterización molecular de cepas híbridas de Pleurotus djamor.

BACKGROUND: Molecular characterisation of wild type Pleurotus species is important for germplasm conservation and its further use for genetic improvement. No molecular studies have been performed with monokaryons used for producing hybrid strains, either with the reconstituted strains obtained by pairing those monokaryons. The molecular characterisation of parental dikaryons, hybrid, and reconstituted strains as well as monokaryotic strains, is therefore of utmost importance. AIMS: To carry out the molecular identification of Pleurotus djamor strains, i.e. dikaryotic wild type strains, hybrid strains, and the monokaryotic strains used for the hybrid formation. METHODS: Five wild type strains of P. djamor from different states in Mexico were collected and molecularly identified by sequencing the ITS1-5.8-ITS2 region using ITS1 and ITS4 universal oligonucleotides. Four hybrid strains were obtained by pairing neohaplonts of two wild type strains selected. Six ISSR markers were used for the molecular characterisation of monokaryotic and dikaryotic strains. RESULTS: Using the ITS markers, an amplified product of 700bp was obtained in five wild type strains, with a 99-100% similarity with P. djamor. A total of 95 fragments were obtained using the ISSR markers, with 99% of polymorphism. CONCLUSIONS: Wild type strains were identified as P. djamor, and were clearly grouped with Mexican strains from other states of Mexico. ISSR markers allowed the generation of polymorphic bands in monokaryotic and dikaryotic strains, splitting both types of strains. The high degree of polymorphism indicates the genetic diversity of P. djamor, an advantage in mushroom production and in the improving of the species.

Ripening in papaya fruit is altered by ACC oxidase cosuppression.

Papaya (Carica papaya) is a very important crop in many tropical countries but it is highly susceptible to parasitic diseases, physiological disorders, mechanical damage and fruit overripening. Here we report a study on ACC oxidase cosuppression and its effects on papaya fruit ripening. Papaya ACC oxidase was isolated using PCR and embriogenic cells transformed by biolistic using the CaMV 35S promoter to drive the expression of the PCR fragment in sense orientation. Fifty transgenic lines were recovered and 20 of those were grown under field conditions. Southern analysis showed incorporation of the transgene in different copy numbers in the papaya genome. Fruits were evaluated in terms of texture (firmness), colour development, respiration and ethylene production. A sharp reduction in ethylene and CO2 production was detected, whereas softening and colour development of the peel were also altered. Overall, transgenic fruits showed a delay in ripening rate. A reduction in mRNA level for ACC oxidase in transgenic fruit was clearly detectable by northern blot. More studies are necessary before this technology can be used to extend the shelf life of papaya fruit.