Astolfo Zoina

Polymorphisms in nuclear rDNA and mtDNA reveal the polyphyletic nature of isolates of Phomopsis pathogenic to sunflower and a tight monophyletic clade of defined geographic origin

The molecular diversity of Diaporthe helianthi (anamorph Phomopsis helianthi), the causal agent of sunflower stem canker, was studied in 16 isolates of different geographic origin using nuclear and mitochondrial markers. PCR products corresponding to the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal RNA gene, and to the mitochondrial atp6 gene were sequenced. The ITS1 and ITS2 sequences were compared with those of Phomopsis spp. and Diaporthe spp. obtained from databases. The diversity in the region surrounding the atp6 gene was also studied by restriction analysis using four enzymes. The analyses revealed a marked diversity within the sunflower-isolated strains, which appear to belong to phylogenetically unrelated groups. Noticeably, all the isolates collected in France and in the former Yugoslavia, where severe epiphytotics of sunflower stem canker are frequently reported, showed high similarity to each other forming a clade which clearly differentiated from all other ones within the genus Phomopsis. Conversely, all the isolates collected in Italy, where, despite favourable environmental conditions, the incidence of the disease is low, were only distantly related to the former group and showed sequence similarity with other previously established phylogenetic clades within the Phomopsis/Diaporthe complex.

RESISTANCE TO BLACKLEG AND TUBER SOFT ROT IN SEXUAL AND SOMATIC INTERSPECIFIC HYBRIDS WITH DIFFERENT GENETIC BACKGROUND

Diploid interspecific hybrids from crosses betweenSolanum tuberosum haploids andS. canasense, S. multidissectum, andS. tarijense, along with tetraploid and hexaploid somatic hybrids S.tuberosum (+)S. commersonii, were screened for resistance to blackleg and tuber soft rot byErwinia carotovora subsp.atroseptica andE. carotovora subsp.carotovora. Among the diploid hybrids, those involving clone mlt la of S.multidissectum and tar 2b ofS. tarijense yielded the highest number of genotypes resistant to both blackleg and tuber soft rot. By contrast, all the hybrids involving clone tar 1lb ofS. tarijense were susceptible to both bacterial diseases. As far as resistance of somatic hybrids is concerned, the most interesting genotype was the tetraploid one which showed resistance to both blackleg and tuber soft rot Significant correlations were found betweenE. carotovora subsp.atroseptica andE. carotovora subsp.carotovora both in resistance to blackleg and in resistance to tuber soft rot, suggesting that resistance to the two subspecies is controlled by the same gene/s or that the relevant genes are linked. On the other hand, correlations between blackleg and tuber soft rot were never significant, indicating that different mechanisms may control resistance in tuber and stems. Alternatively, the resistance could be controlled by the same gene/s under a different spatial/temporal expression pattern. Twelve diploid sexual hybrids and one tetraploid somatic hybrid were selected for resistance to blackleg and/or tuber soft rot and for tuber characteristics.CompendioHíbridos diploides interespecíficos producto de cruzamientos entre haploides deSolanum tuberosum y S. canasense, S. multidissectum y S. tarijense, conjuntamente con híbridos somáticos tetraploides y hexaploides deS. tuberosum (+) S. commersonii, fueron evaluados por su resistencia a la pierna negra del tallo y la pudrición blanda del tubérculo causadas porErwinia carotovora subsp.atroseptica y E. carotovora subsp. carotovora. Entre los híbridos diploides, aquéllos que involucran al don mlt la de S.multidissectum y al clon tar 2b deS. tarijense dieron como resultado el número más alto de genotipos resistentes a la pierna negra y a la pudrición blanda del tubérculo En contraste, todos los híbridos involucrando al clon tar 11b deS. tarijense fueron susceptibles a ambas emfermedades bacterianas. En cuanto a la resistencia de los híbridos somáticos, el genotipo más interesante fue el tetraploide que mostró resistencia a la pierna negra y a la pudrición blanda del tubérculo. Se encontraron correlaciones significativas entreE. carotovora subsp.atroseptica E. carotavora subsp.carotovora en la resistencia tanto a la pierna negra como a la pudrición blanda del tubérculo, sugiriendo que la resistencia a ambas subespecies es controlada por el mismo gen o los mismos genes o que los genes pertinentes están ligados. Por otro lado, las correlaciones entre la pierna negra y la pudrición blanda del tubérculo nunca fueron significativas, indicando que diferentes mecanismos podrían controlar la resistencia en tubérculos y tallos. Alternativamente, la resistencia podría ser controlada por el mismo gen o genes bajo un modelo diferente de expresión espacial o temporal. Doce híbridos sexuales diploides y un híbrido somático tetraploide fueron seleccionados por su resistencia a la pierna negra o pudrición blanda del tubérculo y por sus características de tubérculo.

Induction of novel organelle DNA variation and transfer of resistance to frost and Verticillium wilt in Solanum tuberosum through somatic hybridization with 1EBN S. commersonii

Somatic hybridization can be used to induce genetic variability in plastidial and mitochondrial genomes, and transfer multiple uncloned genes across sexual barriers. Somatic hybrids were produced between a dihaploid clone of the common potato, S. tuberosum subsp. tuberosum, and the wild sexually incongruent diploid species S. commersonii. Fusion products were selected on the basis of callus growth and regeneration in vitro. Genome composition of putative somatic hybrids was determined by flow cytometric analysis of nuclear DNA content, RAPD analysis, and Southern analysis with probes specific to organellar DNA. All regenerated fusion products proved to be hybrids based on RAPD analysis. Seventy per cent of somatic hybrids were (near) tetraploids, 22% (near) hexaploids and 8%(near) octoploids. A high correlation was found between the nuclear DNA content determined by flow cytometry and the number of chloroplasts in stomata guard cell pairs. Somatic hybrids inherited the parental plastids in a random manner. On the contrary, they preferentially inherited the mitochondrial DNA fragments of S. tuberosum. The majority of them had a rearranged mitochondrial genome with fragments from both parents. Hybrids were highly vigorous and morphologically more similar to the cultivated than to the wild parent, produced tubers on long stolons under long photoperiod conditions, showed a high degree of flowering, but did not produce pollen. In addition, somatic hybrids were generally more resistant to frost and Verticillium wilt than the cultivated parent, indicating the introgression of relevant resistance genes from the wild species into the genetic background of S. tuberosum.

Simultaneous detection of cucumber mosaic virus, tomato mosaic virus and potato virus Y by flow cytometry.

The simultaneous detection is described of cucumber mosaic virus (CMV), potato virus Y (PVY) and tomato mosaic virus (ToMV) by flow cytometry. Extracts from leaves of healthy and CMV or PVY infected plants were incubated with latex particles, each with a diameter of 3 microm. Extracts from ToMV infected or uninfected plants, however, were incubated with particles, each with a diameter of 6 microm. Beads were washed and incubated in succession with primary and secondary antibodies, the latter labeled with phycoerythrin (PE) or fluorescein (FITC). CMV and PVY were distinguished on the basis of the fluorescence emitted by FITC and PE; ToMV was distinguished from CMV and PVY on the basis of the different diameter (6 microm) of the particles on which it was adsorbed. The three viruses were detected also by another approach. Latex particles with a diameter of 3, 6 and 10 microm were separately sensitized with antibodies specific for CMV, PVY and ToMV. An equal number of sensitized particles was mixed and incubated with the plant extracts containing the three viruses and then with anti-CMV, anti-PVY and anti-ToMV antibodies labeled with FITC. The study describes also a virus purification method based on the use of antibody coated latex particles. The method is simple technically and applicable to the purification of large as well as minute amounts of different viruses (CMV, PVY and ToMV).

Potential of genes and gene products fromTrichoderma sp. andGliocladium sp. for the development of biological pesticides

Fungal cell wall degrading enzymes produced by the biocontrol fungiTrichoderma harzianum andGliocladium virens are strong inhibitors of spore germination and hyphal elongation of a number of phytopathogenic fungi. The purified enzymes include chitinolytic enzymes with different modes of action or different substrate specificity and glucanolytic enzymes with exo-activity. A variety of synergistic interactions were found when different enzymes were combined or associated with biotic or abiotic antifungal agents. The levels of inhibition obtained by using enzyme combinations were, in some cases, comparable with commercial fungicides. Moreover, the antifungal interaction between enzymes and common fungicides allowed the reduction of the chemical doses up to 200-fold. Chitinolytic and glucanolytic enzymes fromT. harzianum were able to improve substantially the antifungal ability of a biocontrol strain ofEnterobacter cloacae. DNA fragments containing genes encoding for different chitinolytic enzymes were isolated from a cDNA library ofT. harzianum and cloned for mechanistic studies and biocontrol purposes. Our results provide additional information on the role of lytic enzymes in processes of biocontrol and strongly suggest the use of lytic enzymes and their genes for biological control of plant diseases.

Resistance to Ralstonia solanacearum of Sexual Hybrids Between Solanum commersonii and S. tuberosum

This research was carried out to study the levels of bacterial wilt resistance and genetic diversity of (near)pentaploid sexual hybrids between S. commersonii (2n = 2x = 24, 1EBN) and cultivated S. tuberosum. Following artificial inoculations with Ralstonia solanacearum, wilting degree was estimated on a scale from 0 to 4, and seven genotypes of 26 (27%) displaying a S. commersonii like behavior were identified. Latent bacterial colonizations were detected in roots of symptomless S. commersonii and hybrids, whereas no bacterial populations were detected within stems. This suggests that the movement and/or growth of the bacterium in the aerial part were strongly inhibited. A molecular study with AFLP markers clustered hybrids into nine groups and provided evidence that resistant hybrids were slightly more similar to cultivated S. tuberosum than to the wild parent. This is important in view of the re-establishment of the cultivated genetic background through backcrosses. Hybrids displayed good fertility and are being used for further breeding efforts.ResumenEsta investigación fue realizada para estudiar los niveles de resistencia a la marchitez bacteriana y la diversidad genética de híbridos sexuales (casi) pentaploides entre S. commersoni y S. tuberosum cultivado. Después de las inoculaciones artificiales con Ralstonia solanacearum, la marchitez fue estimada en una escala de 0 a 4 y se identificaron siete genotipos de 26 (27%) que se comportaron como S. commersoni. Se detectaron colonizaciones bacterianas latentes en raíces que no presentaban síntomas de S. commersoni e híbridos mientras que no se detectó población bacteriana dentro de los tallos. Esto sugiere que el movimiento y/o crecimiento de la bacteria en la parte aérea fue fuertemente inhibido. Un estudio molecular con marcadores AFLP agrupó los híbridos en nueve grupos y dio la evidencia de que los híbridos fueron ligeramente más similares al S. tuberosum cultivado que sus progenitores silvestres. Esto es importante en vista del restablecimiento de los antecedentes genéticos de la especie cultivada por medio de retro cruzamientos. Los híbridos mostraron buena fertilidad y están siendo usados para mejoramiento.

Insights on the susceptibility of plant pathogenic fungi to phenazine-1-carboxylic acid and its chemical derivatives

Pseudomonas chlororaphis subsp. aureofaciens strain M71 produced two phenazine compounds as main secondary metabolites. These metabolites were identified as phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH P). In this study, the spectrum of the activity of PCA and 2-OH P was evaluated against a group of crop and forestal plant pathogenic fungi by an agar plate bioassay. PCA was active against most of the tested plant pathogens, while 2-OH P slightly inhibited a few fungal species. Furthermore, four semisynthesised derivatives of PCA (phenazine-1-carboxymethyl, phenazine-1-carboxamide, phenazine-1-hydroxymethyl and phenazine-1-acetoxymethyl) were assayed for their antifungal activity against 11 phytopathogenic species. Results showed that the carboxyl group is a structural feature important for the antifungal activity of PCA. Since the activity of phenazine-1-carboxymethyl and phenazine-1-carboxamide, the two more lipophilic and reversible PCA derivatives remained substantially unaltered compared with PCA.

Proteomic investigation of response to forl infection in tomato roots

Fusarium oxysporum f. sp. radicis-lycopersici (FORL) leading to fusarium crown and root rot is considered one of the most destructive tomato soilborne diseases occurring in greenhouse and field crops. In this study, response to FORL infection in tomato roots was investigated by differential proteomics in susceptible (Monalbo) and resistant (Momor) isogenic tomato lines, thus leading to identify 33 proteins whose amount changed depending on the pathogen infection, and/or on the two genotypes. FORL infection induced accumulation of pathogen-related proteins (PR proteins) displaying glucanase and endochitinases activity or involved in redox processes in the Monalbo genotype. Interestingly, the level of the above mentioned PR proteins was not influenced by FORL infection in the resistant tomato line, while other proteins involved in general response mechanisms to biotic and/or abiotic stresses showed significant quantitative differences. In particular, the increased level of proteins participating to arginine metabolism and glutathione S-transferase (GST; EC 2.5.1.18) as well as that of protein LOC544002 and phosphoprotein ECPP44-like, suggested their key role in pathogen defence.

Evaluation for chipping and tuber soft rot (Erwinia carotovora) resistance in potato clones from unilateral sexual polyploidization (2x × 4x)

The aim of this work is the evaluation of tetraploid potato clones with germplasm fromS. chacoense andS. phureja for chipping and resistance to tuber soft rot (Erwinia carotovora subsp.carotovora). Tetraploid potato clones were obtained via 2n eggs through 2x–4x crosses. Cytological mechanisms were investigated in the diploid hybrid 2n-egg producer used as parent in the unilateral sexual polyploidization scheme; the omission of the second meiotic division and cytokinesis failure at the end of meiosis were identified with a genetic consequence equivalent to second division restitution (SDR). Tuber marketable yield, specific gravity and chip-processing ability evaluated at harvest and after cold storage with and without reconditioning were higher in some clones as compared to theS. tuberosum tetraploid parent. Tuber soft rot resistance, contributed byS. chacoense, was successfully transferred to the4x level. After inoculation withErwinia carotovora subsp.carotovora and estimation of the rotted area, one tetraploid clone proved to be resistant and three partially resistant. The genetic value of exotic germplasm and sexual polyploidization strategy are discussed in relation to potato improvement.ResumenEl objetivo de este trabajo es la evaluatión de clones tetraploides de papa con germoplasma deS. chacoense yS. phureja para hojuelas y para resistencia a la pudrición blanda del tubérculo (Erwinia carotovora subsp.carotovora). Los clones tetraploides fueron obtenidos mediante cruces 2x–4x, utilizando gametos 2n. Los mecanismos citológicos fueron investigados en el productor diploide del híbrido del gameto2n usado como padre en el esquema sexual unilateral de poliploidización; la omisión de la segunda división meiótica y la falla de citocinesis al final de la meiosis fueron identificadas con una consecuencia genética equivalente a la segunda division de restitución (SDR).Los tubérculos con rendimiento comercial, la gravedad específica y la habilidad para el procesamiento de hojuelas evaluados al momento de la cosecha y después del almacenaje en frío con y sin reacondicionamiento, fueron altos en algunos clones comparados con el padre tetraploide deS. tuberosum. La resistencia a la pudrición blanda del tubérculo, aportada porS. chacoense, fue transferida exitosamente al nivel4x. Después de la inoculación conErwinia carotovora subsp.carotovora y la estimación del área podrida, un clon tetraploide probó ser resistente y otros tres, parcialmente resistentes. El valor genético del germoplasma exótico y la estrategia de poliploidización sexual son discutidas en relación al mejoramiento de papa.

Host and non-host plant response to bacterial wilt in potato: role of the lipopolysaccharide isolated from Ralstonia solanacearum and molecular analysis of plant-pathogen interaction.

Ralstonia solanacearum is one of the most devastating phytopathogenic bacteria, in particular its race 3. This microorganism is the causal agent of destructive diseases of different crops including tomato and potato. An important aspect of the interaction between this pathogen, and the host and non‐host plants was its biochemical and molecular basis. Thus, the lipopolysaccharides (LPS) were extracted from the R. solanacearum cell wall, purified, and the O‐specific polysaccharide (OPS) was isolated and chemically characterized by compositional analyses and NMR spectroscopy. The OPS was constituted of two linear polymers of an approximate ratio of 3 : 1, both of which were built up from three rhamnose and one N‐acetylglucosamine residues and differed only in the substitution of one rhamnose residue. The LPS inhibited the hypersensitivity reaction (HR) in non‐host tobacco plants and induced localized resistance in host potato plants, both of which were pre‐treated with the LPS before being inoculated with the pathogen. A cDNA‐AFLP approach was used to study transcriptome variation during the resistant and susceptible interactions. This revealed the presence of metabolites specifically expressed in the S. commersonii‐resistant genotypes, which could be involved in the plant–pathogen incompatible reaction. Furthermore, a specific EST collection of the Ralstonia–potato interaction has been built up.