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In this work, we present a novel biostimulant for sustainable crop disease management, PSP1, based on the plant defense-elicitor AsES, an extracellular protease produced by the strawberry fungal pathogen Acremonium strictum. Fungal fermentation conditions and downstream processing were determined to maximize extracellular protein production, product stability and a high plant defense-eliciting activity, as monitored by anthracnose resistance in supernatant-treated strawberry plants subsequently infected with a virulent strain of Colletotrichum acutatum. Fermentation batches were shown to reduce anthracnose development by 30–60% as compared to infected non-treated plants. Product formulation was shown to be stable for 6 months when stored at temperatures up to 45°C and toxicological tests showed that PSP1 was harmless to beneficial organisms and non-toxic to mammalian species at concentrations 50 times higher than those used in plant experiments. Furthermore, disease protection studies using dilutions of PSP1 indicated that there is a minimum threshold protease activity needed to induce pathogen defense in strawberry and that this induction effect is dose-independent. A significant characteristic of PSP1 is its broad-range protection against different diseases in various crop species. In soybean, PSP1 reduced the symptomatology by 70% of Corynespora cassiicola, etiological agent of the target spot. This protection effect was similar to the commercial inducer BION 500 WG based on BTH, and both products were shown to induce an oxidative burst and up-regulated PR1-gene expression in soybean. Furthermore, a double PSP1-treatment on greenhouse-grown sugarcane plants provided protection against bacterial red stripe disease caused by Acidovorax avenae and a double foliar application of PSP1 on field-grown wheat plants significantly increased resistance against Fusarium graminearum, causal agent of head blight disease, manifested mainly in an increased seed germination rate. In summary, these disease protection studies demonstrated an effective control against both bacterial and fungal pathogens in both monocot and dicot crop species, which together with its low production cost, effectiveness at low concentrations, long shelf-life, tolerance to high temperatures, harmlessness to non-target organisms and simple handling and application, make PSP1 a very promising candidate for effective and sustainable disease management in many crop species.

Las enfermedades sistémicas representan uno de los principales factores que afectan la producción de caña de azúcar. El conocimiento del estado fitosanitario del cultivo y la identificación correcta de los fitopatógenos son claves para reducir las pérdidas por enfermedades. En este sentido, es fundamental contar con técnicas de diagnóstico sensibles, rápidas y fáciles de ejecutar, para realizar un diagnóstico preciso y precoz. A partir del año 2005, en la Estación Exper imental Agroindustrial Obispo Colombres se incorporó el diagnóstico molecular basado en la reacción en cadena de la polimerasa, para la detección específica de cuatro enfermedades sistémicas de la caña de azúcar: raquitismo de la caña soca (Leifsonia xyli sp. xyli), escaldadura de la hoja (Xanthomonas albilineans), mosaico de la caña de azúcar (Sugarcane mosaic virus, ScMV) y síndrome de la hoja amarilla (Sugarcane yellow leaf virus, ScYLV). En este trabajo, se presenta la optimización metodológica del diagnóstico molecular y se compara su eficiencia con la de la técnica inmunoquímica ELISA. El método molecular mostró mayor sensibilidad para las enfermedades evaluadas, tanto bacterianas como virales. El establecimiento del diagnóstico molecular constituye un avance tecnológico de gran importancia para la industria azucarera regional, ya que no solo ayudará a disminuir la incidencia de dichas enfermedades, sino que también evitará el ingreso de otras nuevas al introducir germoplasma de caña de azúcar desde otras regiones.Systemic diseases represent one of the main factors affecting sugarcane production. The knowledge of crop sanitary conditions and the correct identification of phytopathogens are key factors to reduce losses caused by them. To diagnose diseases as early as possible is crucial, so techniques that are sensitive, fast, accurate and easy to use are essential. Since 2005, molecular diagnosis based on polymerase chain reaction has been applied at Estación Experimental Agroindustrial Obispo Colombres, for detecting four systemic sugarcane diseases: ratoon stunting (Leifsonia xyli sp xyli), leaf scald (Xanthomonas albilineans), Sugarcane mosaic virus (ScMV) and yellow leaf syndrome (Sugar cane yellow leaf virus, ScYLV). This paper presents the methodological optimization of molecular diagnosis and compares its efficiency with that of ELISA immunochemistry technique. The molecular approach showed greater sensitivity for the detection of both the bacterial and viral diseases evaluated. The implementation of molecular analysis constitutes a technological advance for regional sugar industry, which will not only help to reduce disease incidence, but also avoid the entrance of new pathogens whenever sugarcane germplasm is introduced from other regions.

El cultivo in vitro de tejidos vegetales puede producir variación somaclonal, fenómeno que consiste en modificaciones genéticas en las células y tejidos cultivados. Esto puede limitar la aplicación de dicha técnica para la micropopagación masiva, especialmente si la variación provoca un cambio fenotípico de importancia agronómica. En este trabajo se optimizó una metodología basada en la comparación de perfiles de marcadores moleculares AFLP (del inglés "Amplified Fragment Length Polymorphism"), para la detección de la variación somaclonal en vitroplantas de caña de azúcar. Para la optimización de la técnica de AFLP en caña de azúcar, se utilizaron plantas de seis genotipos propagados convencionalmente y dos tipos de muestras: hojas tiernas y meristemas. La variación somaclonal fue evaluada en líneas de vitroplantas de los mismos genotipos al final del cultivo in vitro, luego de seis meses de micropropagación. Con las 19 combinaciones de cebadores utilizadas, se diferenciaron los perfiles moleculares de los seis genotipos. En los plantines micropropagados se detectaron perfiles diferenciales en las variedades LCP85-384 y TUCCP77-42 con 3 de las 19 combinaciones de cebadores utilizadas. Este resultado muestra la validez de la técnica para detectar variantes somaclonales, y deja en evidencia la diferencia de susceptibilidad de los genotipos al cultivo in vitro. Esto permitió ajustar la metodología de micropropagación para cada genotipo multiplicado y asegurar la pureza genética de cada vitroplanta.In vitro culture of plant tissue can produce somaclonal variation, which consists of genetic modifications in cultured cells and tissues. This may constrain the use of this technique in massive micropopagation, especially if such change causes an agronomically relevant phenotypical modification. In this work, a methodology based on the comparison of AFLP (Amplified Fragment Length Polymorphism) molecular marker profiles was developed for detecting somaclonal variation in in vitro propagated sugarcane plants. To optimize AFLP technique application to sugarcane plants, six conventionally propagated genotypes and two types of samples (tender leaves and meristems) were used. Somaclonal variation was determined in micropropagated lines of these genotypes after six months of micropropagation. Molecular profile differentiation of the selected genotypes was achieved with 19 primer combinations. Differential profiles were detected in LCP85-384 and TUCCP77-42 micropropagated lines with 3 of the 19 primer combinations. This result demonstrated that the technique can be used to detect somaclonal variants and that there are different susceptibility levels to in vitro culture among genotypes. Therefore, micropropagation methodology was adjusted to each multiplied genotype so as to ensure genetic purity of in vitro propagated plants.

El mosaico de la caña de azúcar es una de las enfermedades sistémicas de mayor importancia de este cultivo. El virus del mosaico de la caña de azúcar (SCMV) y el virus del mosaico del sorgo (SrMV) son los principales agentes causales de esta enfermedad. Mediante RT-PCR-RFLP (siglas del "reverse transcriptase-polymerase chain reaction"-"restriction fragment length polymorphism") y secuenciación del gen de la proteína de la cápside, se analizaron muestras de caña de azúcar con síntomas de mosaico para evaluar la presencia y estimar la diversidad genética de ambos virus en el área cañera de Tucumán. Noventa y tres por ciento de las muestras resultaron positivas para SCMV al utilizar los cebadores específicos SCMVF4/R3 y 33% de ellas exhibió el perfil RFLP de la raza E. El resto de las muestras presentó nueve perfiles diferentes que no se correspondieron con los de las razas descritas. El análisis de la secuencia indicó que 20% de las muestras de perfil RFLP desconocido presentó una alta identidad con la raza D, mientras que las muestras restantes resultaron divergentes entre sí. Mediante microscopía electrónica de transmisión, se confirmó la presencia de los típicos viriones flexuosos de los potyvirus. Asimismo, se detectó la presencia de SrMV en 90% de las muestras con los cebadores específicos y la coexistencia de ambos virus en 85% de las mismas. Mediante RFLP, se identificaron las razas M e I del virus del sorgo en 68% y 14% de las muestras, respectivamente, y en 18% de los casos se detectó coexistencia entre las razas M y H de esta virosis. Solo una de las muestras no amplificó para ninguno de los dos virus, lo que sugeriría la existencia de otro agente responsable de una sintomatología similar.Sugarcane mosaic is one of the most important systemic diseases of sugarcane. Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV) are the causal agents of the disease. Sugarcane leaves with mosaic symptoms were analysed by RT-PCR-RFLP (reverse transcriptase-polymerase chain reaction- restriction fragment length polymorphism) and the nucleotide sequences of the coat protein (CP) genes amplified to assess the presence and genetic diversity of both viruses in the sugarcane producing area in Tucumán. Using the primers SCMVR3/F4, 93% of samples were SCMV positive and 33% of them had the E strain RFLP profile, while the rest produced nine different profiles that did not match those of any known strains. Sequence analyses showed that 20% of the samples with the unknown profiles were highly identical to the SCMV D strain, while the rest differed significantly from each other. The presence of the flexuous virions typical of potyviruses was confirmed by transmission electron microscopy. Also, using the specific primers, the presence of SrMV was detected in 90% of the samples, and co-existence of both viruses was found in 85% of the samples. RFLP analysis determined the presence of SrMV strains M and I in 68% and 14% of the samples, respectively, while in approximately 18% of the cases, both M and H strains were present. No RT-PCR product was produced by either SCMV or SrMV primer pairs in one symptomatic sample, suggesting the presence of another pathogen producing similar symptomatology.

La mayoría de las bacterias fitopatógenas viven de forma epifítica sobre las plantas antes de la colonización y sobreviven en la superficie del tejido del hospedante, mediante la formación de biopelículas. La capacidad para producir exopolisacáridos es crítica para la formación de las mismas en diversas bacterias. En este trabajo, se estudió la formación de biopelículas de X. citri subsp. citri, bacteria responsable de la cancrosis de los cítricos, y su relación con la supervivencia epifítica y el desarrollo del cancro sobre hojas de plantas de limonero. Mediante tinción con cristal violeta y análisis de microscopía confocal de barrido láser, se observó que X. citri subsp. citri forma estructuras de biopelículas sobre hojas de limonero. En contraste, una cepa mutante defectiva en la biosíntesis del exopolisacárido xantano, denominada X. citri subsp. citri gumB, fue incapaz de formar dichas estructuras y su supervivencia epifítica y capacidad de desarrollar la enfermedad en la planta se vieron comprometidas. Estos resultados sugieren que la formación de biopelículas tiene una importancia clave en la fase epifítica de X. citri subsp. citri antes y durante el desarrollo del cancro.

In sugarcane (Saccharum spp.) breeding programs, parents used in crosses are classified as male or female based on the relative amounts of viable pollen produced. High pollen production favored by environmental conditions reduces female inflorescence availability and restricts the possibility of cross combinations. However, male parents could be employed as female parents when an efficient emasculation treatment is used. An ideal approach for hybridity testing is using molecular markers, especially microsatellites (SSR). To determine the effectiveness of an emasculation treatment (immersion of the panicle in water at 50ºC for five minutes) employed in the Sugarcane Breeding Program of Estación Experimental Agroindustrial Obispo Colombres (EEAOC), Tucumán, Argentina, six cross combinations (selfings and reciprocals) between two varieties commonly used as males, LCP 85-384 and RA 87-3, were evaluated by using SSRs. Samples were amplified with one primer pair that produced seven polymorphic and three monomorphic bands between the two progenitors. While Mendelian segregation may be difficult to observe in the progeny of a complex polyploid like sugarcane, the analysis showed that each marker segregated in a Mendelian fashion (as evaluated by ?2 tests, P=0.05) for each cross combination. Results indicated that the emasculation treatment was successful and that SSRs made it possible to identify true hybrid progeny routinely in sugarcane breeding.

Better knowledge of sugarcane genetic diversity will provide useful information concerning genotypic value for breeding programs and should help to improve the use and conservation of genetic resources and the protection of sugarcane varieties by intellectual property rights. Morphological descriptors are traditional tools to characterise varieties; however, they vary phenotypically because of environmental effects. Therefore, molecular markers have become increasingly important for identifying genotypes and estimating diversity, as they are accurate, readily available, and are not affected by the environment. The aim of this research was to evaluate genotypes used as parental materials in the Sugarcane Breeding Program of Estación Experimental Agroindustrial Obispo Colombres (EEAOC), Argentina, by using molecular markers (AFLP and SSR) and morphological traits, and by comparing the data obtained with two statistical software programs (NTSys and InfoStat). All cultivars grouped in one main cluster of the dendrogram when using both programs and at least 150 data points. Local Argentine genotypes grouped together with US-varieties and no clear genetic differentiation was found, probably due to regular germplasm exchange. Although morphological traits reflected external resemblance only, the topology of the dendrogram was not modified when combining both molecular and morphological data. These results suggest that both characterisation methods should be used to estimate genetic diversity. Molecular markers should be included internationally for sugarcane variety protection.