Romano Locci

Actinomycetes as plant pathogens

Biology, taxonomy, pathogenicity and control of plant disease inducing actinomycetes are reviewed. Recent progress in the study of potato, sweet potato, blueberry and fruit and forest tree diseases is illustrated. The role in potato scab pathogenesis of the newly discovered phytotoxins, thaxtomins, is discussed.

Molecular characterization of a phytoplasma causing Phyllody in Clover and other herbaceous hosts in Northern Italy

Red clover (Trifolium pratense) and Ladino clover (Trifolium repens) plants showing phytoplasma-associated symptoms (yellowing/reddening, virescence and phyllody) have been recovered in Friuli-Venezia Giulia, Italy. Using AluI RFLP analysis of PCR amplified 16S rDNA we showed that the disease can be caused independently by two phylogenetically distinct phytoplasmas. One of them showed the very typical 16S rDNA RFLP pattern of the agent of Clover Phyllody in Canada (CCPh). The 16S rDNA of the other phytoplasma (Italian Clover Phyllody phytoplasma, ICPhp) has been PCR amplified, cloned and sequenced. The sequence revealed high similarity (>98%) with phytoplasmas belonging to the X disease cluster, which includes organisms not reported to cause phyllody on their hosts. The analysis by AluI RFLP of the PCR amplified pathogen 16S rDNA from other herbaceous plants (Crepis biennis, Taraxacum officinale, Leucanthemum vulgare) collected nearby with phytoplasma-associated symptoms showed similar patterns. Southern blot hybridization of their EcoRI digested total DNA revealed identical RFLP patterns, suggesting that the causative agent may be the same organism.

Growth kinetics of Botrytis cinerea on organic acids and sugars in relation to colonization of grape berries

The relationship between hyphal growth and branching of the grape pathogen Botrytis cinerea was determined on solid media containing either glucose, fructose, sucrose, tartaric acid or malic acid. The concentration of the carbon source had little effect on specific growth rate or the specific rate of tip formation, but growth was inhibited at high concentrations of tartaric and malic acids. Hyphal growth unit length and hyphal extension rate increased with increasing sugar concentration and were always significantly greater than values on tartaric or malic acids. The data provide an explanation for colonization patterns of grape berries. Growth will be poor during the period from setting to the onset of ripening, when organic acids are the main carbon source produced by the berry. Following the onset of ripening, the production of sugars provides more favourable carbon sources for the fungus, enabling achievement of higher specific growth rates, greater hyphal extension rates and, hence, greater colonizing potential.

Evolutionary analysis of endopolygalacturonase-encoding genes of Botrytis cinerea

Sequence analysis of five of the six endopolygalacturonase-encoding genes (Bcpg1, Bcpg2, Bcpg3, Bcpg4, Bcpg5) from 32 strains of Botrytis cinerea showed marked gene to gene differences in the amount of among-strains diversity. Bcpg4 was almost invariable in all strains; Bcpg3 and Bcpg5 showed a moderate variability, similar to that of non-pathogenicity-associated genes examined in other studies. Conversely, Bcpg1 and Bcpg2 were highly variable and were shown to be under positive selection based on the McDonald-Kreitman test and likelihood ratio test. The evolution of the five endopolygalacturonase genes is explained by their different ecophysiological role. Diversification and balancing selection, as detected in Bcpg1 and Bcpg2, can be used by the pathogen to escape recognition by the host and delay plant reaction in the early phases of infection. The analysis of the polymorphisms and the location of the sites with high probability of being positively selected highlighted the relevance of variability of the BcPG1 and BcPG2 proteins at their C-terminal end. By contrast, the absence of variability in Bcpg4 suggests that the efficiency of the product of this gene is critical for B. cinerea growth in late phases of infection or during intraspecific competition, thus markedly affecting strain fitness.

The influence of local factors on the prediction of fumonisin contamination in maize

BACKGROUND Contamination by mycotoxins is a major concern to the maize industry in north-east Italy where maize grain is often spoiled by Fusarium spp. In this work, fumonisins, deoxynivalenol and zearalenone were determined and an artificial neural network (ANN) model suitable for predicting mycotoxin contamination of maize at harvest time was developed. RESULTS The occurrence of deoxynivalenol and zearalenone was very limited, while fumonisins concentration ranged from 163 and to 3663 µg kg(-1) in 2007, and from 333 to 11473 µg kg(-1) in 2008. Statistical data analysis of factors affecting fumonisins concentration revealed that irrigation, chemical treatment against the European corn borer and harvest date significantly affected the level of contamination (P < 0.05), although the relevance of the factors was different in 2007 and 2008. The neural network approach showed a significant correlation between ascertained values and predictions based on agronomic data. CONCLUSION This is the first time that an artificial neural network has been used to predict fumonisin accumulation in maize: the prediction has been shown to have the potential for the development of a new approach for the rapid cataloging of grain lots.

Air analysis in the assessment of fumonisin contamination risk in maize

BACKGROUND In maize-growing areas where fumonisin contamination is endemic, there is an urgent need for novel methods to assess the quality of grain lots before their delivery to common drying and storage collection centres. Aerobiological samples of fungal spores released during harvest were analysed to establish a relationship between fumonisin contamination and the abundance of pathogen propagules collected in the combine harvester using a cyclone and membrane filters. Filter-captured propagules were analysed by direct plating, immunoenzymatic assay of specific Fusarium extracellular polysaccharides and real time polymerase chain reaction of the extracted DNA using fum1, a gene involved in the biosynthesis of fumonisin, as a target. RESULTS The results showed that time of harvest and environmental conditions strongly influenced the efficiency and performance of the collection system. The data obtained were informative in comparing individual samples collected under similar conditions. The immunoenzymatic assay provided the most reliable data, which improved the ability of a neural network to predict the fumonisin content of lots, when added to agronomic, environmental and phytosanitary data. CONCLUSION This is the first attempt to evaluate the Fusarium propagules dispersed during harvesting as a predictive means to assess maize quality. A method based on cyclone/filter capture and immunological detection has been shown to be feasible and to have the potential for the development of a continuous monitoring system, but the prediction capabilities in the present implementation were limited.

Mitochondrial plasmids of the pCp family are spread worldwide in Cryphonectria parasitica populations

A worldwide collection of strains of Cryphonectria parasitica was examined to draw a precise picture of the incidence and diversity of mitochondrial plasmids related to the plasmid pUG1. Amplification by specific PCR of 199 strains showed the presence of pUG1-like plasmids in 22% of the populations examined. The entire plasmid molecules were amplified by multiplex PCR and the products showed different RFLP patterns. The variability was mostly in a non-coding region of the molecule that has been sequenced in some representative strains, enabling the molecular evolution of the molecule to be elucidated. The data show that mitochondrial plasmids of C. parasitica comprise an almost homogeneous family (designated pCp) that can be divided into two clusters based on the presence/absence respectively of a 60 nucleotide region in North American and European plasmids.

Sequence analysis of domains III and IV of the 23S rRNA gene of verticillate streptomycetes

Domains III and IV of the 23S rRNA gene of 25 strains of verticillate streptomycetes were sequenced. None of the sequences was identical to any other, with regions of variability being restricted to parts of helices 54 and 64. No relationships were detected between the similarities in the sequence and the assignment to phenetic clusters as defined by the numerical taxonomy studies. Limited agreement was also found between similarity of the sequences and DNA-DNA homology values. However, species (> 70% DNA-DNA homology values)-specific diagnostic oligonucleotides generally could be defined, except for Streptomyces baldaccii. Therefore the determination of the 23S rRNA sequence may be of greater value for fingerprinting individual strains than for taxonomic or identification purposes.