Histopatología, fisiología y calidad postcosecha de frutos de aguacate (Persea americana Mill.) cv. 'Hass' infectados con el avocado sunblotch viroid (ASBVd) y diagnóstico de la enfermedad.

Frutos de aguacate (Persea americana Mill) cv. ‘Hass’ se cosecharon de árboles infectados con el Avocado sunblotch viroid (ASBVd) en Tingambato, Michoacán, considerando tres condiciones fenotípicas: árboles asintomáticos vigorosos (A), árboles de vigor regular (AR) y árboles con síntomas en frutos (S). Se determinó la presencia del ASBVd mediante RT-PCR e hibridación Northern con ribosondas complementarias al viroide. La presencia del ASBVd se detectó en el exocarpio y mesocarpio de frutos de aguacate “Hass” sintomáticos y asintomáticos y en los tejidos de la semilla (cubierta seminal, cotiledones y eje embrionario) de frutos sintomáticos. Es posible que los síntomas macroscópicos causados por el ASBVd en frutos hayan sido el resultado de cambios anatómicos en la conformación y estructura de las células del exocarpio y mesocarpio, caracterizados principalmente por una marcada desorganización celular, acumulación de polifenoles intercelulares y en las paredes celulares, reducción del contenido citoplásmico y desarrollo de colapso y muerte celular. Los tejidos sintomáticos manifestaron una reducción del 27-35 % en el contenido de clorofila (a y b) y aumento del 33-38 % de compuestos fenólicos. Hubo taponamiento de los elementos de vaso del tejido vascular. La desorganización y colapso celular conducen al hundimiento y formación de grietas en frutos sintomáticos; la reducción del contenido de clorofila y aumento de compuestos fenólicos posiblemente expliquen el desarrollo de coloraciones amarillas o rojizas en el exocarpio. Los frutos asintomáticos no mostraron ningún cambio anatómico a pesar de la presencia del ASBVd. También se analizaron la producción de dióxido de carbono, etileno y patrón de maduración, las variables físicas de calidad (firmeza, pérdida de peso, materia seca y tamaño de fruto) y la composición bromatológica (contenido de aceite, proteínas, azucares, fibra cruda, cenizas y contenido energético) de frutos asintomáticos (A y AR) y sintomáticos (S). Los frutos asintomáticos (A y AR) maduraron de manera uniforme, pero los sintomáticos (S) presentaron maduración irregular, asociada a la menor actividad respiratoria y producción de etileno, generándose pérdidas de 46.6 % debido a senescencia y falta de maduración diez días después de la cosecha. No hubo diferencias significativas (p≤0.05) en la velocidad de pérdida de peso y firmeza entre los frutos asintomáticos (A) y sintomáticos (S), pero los últimos fueron 8-10 % de menor tamaño. Los frutos (A, AR y S) no mostraron diferencias significativas (p≤0.05) en el contenido de proteínas, azúcares, fibra cruda ni cenizas; sin embargo, los frutos sintomáticos y asintomáticos cosechados de árboles de vigor regular, presentaron menor contenido de materia seca (23.5-23.9 %) y aceite (12.7-13.5 g/100g) que causó decremento en 28-32 % del aporte calórico, respecto a frutos asintomáticos cosechados de árboles vigorosos con 19.2 g/100g de aceite y 26.1 % de materia seca. Es importante señalar que todos los frutos analizados (A, AR y S) superaron los valores mínimos de materia seca y aceite necesarios para su cosecha y consumo en fresco. Finalmente, se realizaron muestreos en campo de árboles de aguacate Mexicano procedentes del Estado de México y Puebla y árboles cv. ‘Hass’ en Tingambato, Michoacán. Los árboles de raza mexicana mostraron ser libres del ASBVd en las diferentes zonas de colecta. En aguacate ‘Hass’ se determinó una incidencia del 8 % por hibridación y 11 % por RT-PCR que correspondieron a los árboles que manifestaron síntomas de la enfermedad en huerto, pero la RT-PCR mostró mayor sensibilidad al detectar un árbol asintomático infectado con el viroide. Los materiales negativos al ASBVd pueden ser utilizados como fuente de semilla para la obtención de portainjertos o como fuente de vareta durante la propagación de plantas. _______________ ABSTRACT: Fruits of avocado (Persea Americana Mill) cv. ‘Hass’ were harvested in Tingambato, Michoacán from trees infected with the Avocado sunblotch viroid (ASBVd) taking into account three phenotypic conditions: asymptomatic vigorous trees (A), trees of regular vigor (AR) and viroid symptoms in fruits (S). Symptomatic (S) and asymptomatic fruits (A, AR) were analyzed by RT-PCR and dot-blot hybridization with complementary riboprobes for ASBVd detection. The presence of the ASBVd was detected in the exocarp and mesocarp of symptomatic and asymptomatic “Hass” avocado fruit sand in seed tissues (seed coat, cotyledon and embryo) from symptomatic fruits. It is possible than the macroscopic symptoms caused by the ASBVd in fruits were the result of anatomical changes in the composition and structure of the exocarp and mesocarp cells, which are typified by a marked cellular disorganization, accumulation of phenolic compounds in the cytoplasm and cell walls, reduction of cytoplasmic content and development of cell collapse and death. Symptomatic tissues showed a reduction of 27-35 % of chlorophyll content (a and b) and an increment of 33-38% in phenolic compounds. Conducting tissues showed obstruction of xylem elements. Disorganization and cell collapse leads to grooves formation in symptomatic fruits and the reduction of chlorophyll content and increase of phenolic compounds probably leads to the development of yellow or red symptoms on rind. Asymptomatic fruits did not show any anatomical change despite the presence of the ASBVd. Ethylene and carbon dioxide production rates, physical quality variables [pulp firmness, weight loss (%), dry matter and fruit size] and bromatological composition (oil, proteins, sugars, crude fiber, ashes and energetic contents) were also evaluated. The asymptomatic fruits (A y AR) showed normal maturation, but it was irregular in the symptomatic (S) ones due to reduction in the rate of ethylene and carbon dioxide production, which caused fruit losses of 46.6 % due to senescence and lack of maturity in fruits 10 days after harvest. There were no differences (p≤0.05) in the rate of weight loss (%) and pulp firmness among the asymptomatic (A) and symptomatic (S) fruits, but the symptomatic ones showed 8-10 % less fruit size. Fruits (A, AR y S) did not show differences (p≤0.05) in proteins, sugars, crude fiber nor ash content, but symptomatic and asymptomatic fruits harvested from trees of regular vigor presented less dry matter (23.5-23.9 %) and oil (12.7-13.5 g/100g) content, which decreased the caloric contribution in 28-32 %, compared to asymptomatic fruits harvested from vigorous trees, which presented 26.1 % of dry matter and 19.2 g/100g of oil. It is important to point it out that all fruits analyzed (A, AR y S) reached the minimum dry matter and oil content demanded for harvest and to develop good quality for consumption in fresh. Finally, samples of Mexican avocado trees were taken in the field from orchard from the State of Mexico and Puebla and trees cv. 'Hass' from Tingambato, Michoacán. The trees of Mexican races showed to be free of ASBVd from the different areas of collection. The incidence determined in 'Hass' avocado by hybridization was 8 % and 11 % by RT-PCR, which corresponded to trees expressing symptoms of the disease in the orchard, but the RT-PCR showed greater sensitivity by detecting an asymptomatic tree infected with the viroid. Negative materials to the ASBVd can be used as source of seed for obtaining rootstocks or scion, during propagation of plants.Tesis (Doctorado en Ciencias, especialista en Fitopatología).- Colegio de Postgraduados, 2011.Consejo Nacional de Ciencia y Tecnología (CONACyT)

Bacterial wilt and canker of tomato: fundamentals of a complex biological system

"Tomato (Solanum lycopersicum) is well-known as a model for study of plant–pathogen interactions, since it is a crop of global relevance and susceptible to multiple bacterial, fungal, viral and nematode pathogens. Among bacterial phytopathogens, the actinomycete Clavibacter michiganensis subsp. michiganensis (Cmm) is the causal agent of bacterial wilt and canker of tomato, considered a quarantine disease at international level. The tomato–Cmm interaction has been studied to decipher the pathogenicity mechanisms in Cmm, susceptibility mechanisms in tomato, molecular basis of resistance to Cmm in wild species relative to domesticated tomato, and the level of genetic variability in Cmm. The objective of this review is to discuss recent advances in tomato–Cmm compatible interaction, which can be integrated for application in early diagnosis and biological control of bacterial wilt and canker of tomato. Further study of plant–microorganism interactions is a promising field for improvements in tomato pathogen resistance.

Raman Spectroscopy and Machine-Learning for Early Detection of Bacterial Canker of Tomato: The Asymptomatic Disease Condition

Bacterial canker of tomato is caused by Clavibacter michiganensis subsp. michiganensis (Cmm). The disease is highly destructive, because it produces latent asymptomatic infections that favor contagion rates. The present research aims consisted on the implementation of Raman spectroscopy (RS) and machine-learning spectral analysis as a method for the early disease detection. Raman spectra were obtained from infected asymptomatic tomato plants (BCTo) and healthy controls (HTo) with 785 nm excitation laser micro-Raman spectrometer. Spectral data were normalized and processed by principal component analysis (PCA), then the classifiers algorithms multilayer perceptron (PCA + MLP) and linear discriminant analysis (PCA + LDA) were implemented. Bacterial isolation and identification (16S rRNA gene sequencing) were realized of each plant studied. The Raman spectra obtained from tomato leaf samples of HTo and BCTo exhibited peaks associated to cellular components, and the most prominent vibrational bands were assigned to carbohydrates, carotenoids, chlorophyll, and phenolic compounds. Biochemical changes were also detectable in the Raman spectral patterns. Raman bands associated with triterpenoids and flavonoids compounds can be considered as indicators of Cmm infection during the asymptomatic stage. RS is an efficient, fast and reliable technology to differentiate the tomato health condition (BCTo or HTo). The analytical method showed high performance values of sensitivity, specificity and accuracy, among others

Detection of Clavibacter michiganensis subsp. michiganensis Assisted by Micro-Raman Spectroscopy under Laboratory Conditions

Clavibacter michiganensis subsp. michiganesis (Cmm) is a quarantine-worthy pest in México. The implementation and validation of new technologies is necessary to reduce the time for bacterial detection in laboratory conditions and Raman spectroscopy is an ambitious technology that has all of the features needed to characterize and identify bacteria. Under controlled conditions a contagion process was induced with Cmm, the disease epidemiology was monitored. Micro-Raman spectroscopy (532 nm λ laser) technique was evaluated its performance at assisting on Cmm detection through its characteristic Raman spectrum fingerprint. Our experiment was conducted with tomato plants in a completely randomized block experimental design (13 plants × 4 rows). The Cmm infection was confirmed by 16S rDNA and plants showed symptoms from 48 to 72 h after inoculation, the evolution of the incidence and severity on plant population varied over time and it kept an aggregated spatial pattern. The contagion process reached 79% just 24 days after the epidemic was induced. Micro-Raman spectroscopy proved its speed, efficiency and usefulness as a non-destructive method for the preliminary detection of Cmm. Carotenoid specific bands with wavelengths at 1146 and 1510 cm⁻¹ were the distinguishable markers. Chemometric analyses showed the best performance by the implementation of PCA-LDA supervised classification algorithms applied over Raman spectrum data with 100% of performance in metrics of classifiers (sensitivity, specificity, accuracy, negative and positive predictive value) that allowed us to differentiate Cmm from other endophytic bacteria (Bacillus and Pantoea). The unsupervised KMeans algorithm showed good performance (100, 96, 98, 91 y 100%, respectively)

Compilación de Proyectos de Investigacion de 1984-2002

Instituto Politecnico Nacional. UPIICS

Bacterial wilt and canker of tomato: fundamentals of a complex biological system

"Tomato (Solanum lycopersicum) is well-known as a model for study of plant–pathogen interactions, since it is a crop of global relevance and susceptible to multiple bacterial, fungal, viral and nematode pathogens. Among bacterial phytopathogens, the actinomycete Clavibacter michiganensis subsp. michiganensis (Cmm) is the causal agent of bacterial wilt and canker of tomato, considered a quarantine disease at international level. The tomato–Cmm interaction has been studied to decipher the pathogenicity mechanisms in Cmm, susceptibility mechanisms in tomato, molecular basis of resistance to Cmm in wild species relative to domesticated tomato, and the level of genetic variability in Cmm. The objective of this review is to discuss recent advances in tomato–Cmm compatible interaction, which can be integrated for application in early diagnosis and biological control of bacterial wilt and canker of tomato. Further study of plant–microorganism interactions is a promising field for improvements in tomato pathogen resistance.