Liberomycespistaciae sp. nov., the causal agent of pistachio cankers and decline in Italy.

A new canker and decline disease of pistachio (Pistaciavera) is described from Sicily (Italy). Observations of the disease and sampling of the causal agent started in spring 2010, in the area where this crop is typically cultivated, Bronte and Adrano (Catania province) and later extended to the Agrigento and Caltanissetta provinces. Isolations from the margins of twig, branch and stem cankers of declining plants resulted in fungal colonies with the same morphology. Pathogenicity tests on 5-year-old potted plants of Pistaciavera grafted on P.terebinthus reproduced similar symptoms to those observed in nature and the pathogen was confirmed to be a coloniser of woody plant tissue. Comparison of our isolates with the type of the apparently similar Asteromellapistaciarum showed that our isolates are morphologically and ecologically different from A.pistaciarum, the latter being a typical member of Mycosphaerellaceae. Asteromellapistaciarum is lectotypified, described and illustrated and it is considered to represent a spermatial morph of Septoriapistaciarum. Multi-locus phylogenies based on two (ITS and LSU rDNA) and three (ITS, rpb2 and tub2) genomic loci revealed isolates of the canker pathogen to represent a new species of Liberomyces within the Delonicicolaceae (Xylariales), which is here described as Liberomycespistaciae sp. nov. (Delonicicolaceae, Xylariales). The presence of this fungus in asymptomatic plants with apparently healthy woody tissues indicates that it also has a latent growth phase. This study improves the understanding of pistachio decline, but further studies are needed for planning effective disease management strategies and ensuring that the pathogen is not introduced into new areas with apparently healthy, but infected plants.

Environmental and Pathogenic Factors Inducing Brown Apical Necrosis on Fruit of English (Persian) Walnut.

Brown apical necrosis (BAN) is a most recently described disease affecting English (Persian) walnut fruit. BAN was only recorded in intensively managed walnut orchards and was found to be a disease complex mainly caused by Fusarium species. All fungi associated with this disease are polyphagous and ubiquitous, not specific to walnut. Consequently, BAN occurrence is more strictly dependent, than generally, on the interaction between pathological features and environmental conditions. Environmental variables identified with regression analysis showed that maximum temperature, angle of main wind direction versus tree row orientation, and orchard distance to the closest river/canal, all representative of climatic conditions occurring in the orchard, were related to fruit drop. The factor displaying the highest influence on severity of BAN fruit drop was maximum temperature and only subordinately factors are associated with relative humidity. BAN symptoms were reproduced with in planta artificial inoculation, and fruit drop of symptomatic fruit was significantly higher than that of the noninoculated trees for each type of inoculum (Fusarium semitectum, F. graminearum, and Alternaria spp.). F. semitectum and F. graminearum were more aggressive than Alternaria species, and the earliest artificial inoculations in mid-May resulted in the highest fruit drop. The extension of walnut fruit susceptibility and the conducive environmental factors to BAN are discussed.

A newly developed real-time PCR assay for detection and quantification of Fusarium oxysporum and its use in compatible and incompatible interactions with grafted melon genotypes.

A reliable and species-specific real-time quantitative polymerase chain reaction (qPCR) assay was developed for detection of the complex soilborne anamorphic fungus Fusarium oxysporum. The new primer pair, designed on the translation elongation factor 1-α gene with an amplicon of 142 bp, was highly specific to F. oxysporum without cross reactions with other Fusarium spp. The protocol was applied to grafted melon plants for the detection and quantification of F. oxysporum f. sp. melonis, a devastating pathogen of this cucurbit. Grafting technologies are widely used in melon to confer resistance against new virulent races of F. oxysporum f. sp. melonis, while maintaining the properties of valuable commercial varieties. However, the effects on the vascular pathogen colonization have not been fully investigated. Analyses were performed on 'Charentais-T' (susceptible) and 'Nad-1' (resistant) melon cultivars, both used either as rootstock and scion, and inoculated with F. oxysporum f. sp. melonis race 1 and race 1,2. Pathogen development was compared using qPCR and isolations from stem tissues. Early asymptomatic melon infections were detected with a quantification limit of 1 pg of fungal DNA. The qPCR protocol clearly showed that fungal development was highly affected by host-pathogen interaction (compatible or incompatible) and time (days postinoculation). The principal significant effect (P ≤ 0.01) on fungal development was due to the melon genotype used as rootstock, and this effect had a significant interaction with time and F. oxysporum f. sp. melonis race. In particular, the amount of race 1,2 DNA was significantly higher compared with that estimated for race 1 in the incompatible interaction at 18 days postinoculation. The two fungal races were always present in both the rootstock and scion of grafted plants in either the compatible or incompatible interaction.

Distinct colonization patterns and cDNA-AFLP transcriptome profiles in compatible and incompatible interactions between melon and different races of Fusarium oxysporum f. sp. melonis.

BACKGROUND: Fusarium oxysporum f. sp. melonis Snyd. & Hans. (FOM) causes Fusarium wilt, the most important infectious disease of melon (Cucumis melo L.). The four known races of this pathogen can be distinguished only by infection on appropriate cultivars. No molecular tools are available that can discriminate among the races, and the molecular basis of compatibility and disease progression are poorly understood. Resistance to races 1 and 2 is controlled by a single dominant gene, whereas only partial polygenic resistance to race 1,2 has been described. We carried out a large-scale cDNA-AFLP analysis to identify host genes potentially related to resistance and susceptibility as well as fungal genes associated with the infection process. At the same time, a systematic reisolation procedure on infected stems allowed us to monitor fungal colonization in compatible and incompatible host-pathogen combinations. RESULTS: Melon plants (cv. Charentais Fom-2), which are susceptible to race 1,2 and resistant to race 1, were artificially infected with a race 1 strain of FOM or one of two race 1,2 w strains. Host colonization of stems was assessed at 1, 2, 4, 8, 14, 16, 18 and 21 days post inoculation (dpi), and the fungus was reisolated from infected plants. Markedly different colonization patterns were observed in compatible and incompatible host-pathogen combinations. Five time points from the symptomless early stage (2 dpi) to obvious wilting symptoms (21 dpi) were considered for cDNA-AFLP analysis. After successful sequencing of 627 transcript-derived fragments (TDFs) differentially expressed in infected plants, homology searching retrieved 305 melon transcripts, 195 FOM transcripts expressed in planta and 127 orphan TDFs. RNA samples from FOM colonies of the three strains grown in vitro were also included in the analysis to facilitate the detection of in planta-specific transcripts and to identify TDFs differentially expressed among races/strains. CONCLUSION: Our data suggest that resistance against FOM in melon involves only limited transcriptional changes, and that wilting symptoms could derive, at least partially, from an active plant response.We discuss the pathogen-derived transcripts expressed in planta during the infection process and potentially related to virulence functions, as well as transcripts that are differentially expressed between the two FOM races grown in vitro. These transcripts provide candidate sequences that can be further tested for their ability to distinguish between races.Sequence data from this article have been deposited in GenBank, Accession Numbers: HO867279-HO867981.

The need for culture collections to support plant pathogen diagnostic networks.

Plant-pathogenic microorganisms, by virtue of their size, similarity in disease symptoms and closely related morphologies, are notoriously difficult to diagnose and detect. Diagnosis gives proof as to the causal agent of disease and is important for developing appropriate control measures. Detection shows the presence of a microorganism and is of importance for safeguarding national and international trade. Live reference collections are required to characterize the taxonomy and function of microorganisms as a prerequisite to development of tools for diagnosis and detection. Two case studies will be presented in this paper to demonstrate the importance of microorganism collections for facilitating knowledge sharing and the development of identification methods. Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense and sharka disease of stone fruits caused by plum pox virus (PPV) are considered. Both diseases consist of different races/strains with different host specificities, but Fusarium wilt poses a threat to food security, while PPV poses a threat to trade due to its classification as a quarantine pest, since there is no anti-virus treatment available to control sharka disease in orchards. It is only through comprehensive collections of correctly identified and well-maintained strains representing the genetic diversity of a target organism that robust, specific, reliable and efficient diagnostic and detection tools can be developed.

Polyphasic classification of Alternaria isolated from hazelnut and walnut fruit in Europe.

Brown apical necrosis of English walnut and grey necrosis of hazelnut are destructive fruit diseases caused by a complex of opportunistic fungi including several small-spored catenulate Alternaria taxa. Thirty Alternaria isolates recovered from walnut and hazelnut fruit that were pathogenic on their respective host were compared along with type or representative isolates of A. alternata, A. tenuissima, A. arborescens, and A. infectoria using morphological and molecular criteria. Morphological examination using standardized procedures separated the walnut and hazelnut isolates into three morphological groups: the A. alternata group, the A. tenuissima group, and the A. arborescens group based upon common characteristics of the conidium and the sporulation apparatus. To evaluate genetic relationships among these groups, AFLP markers, inter simple sequence repeat (ISSR) markers, and histone gene sequence data were compared. Based upon AFLP data, the A. alternata and A. tenuissima groups comprised a single lineage, and the A. arborescens group comprised a separate lineage. ISSR data supported the grouping by AFLP data except for three isolates of the A. alternata group that clustered with the A. arborescens group. Base substitution of the H4 gene supported the discrimination of the A. arborescens group from the A. alternata and A. tenuissima groups. Tests of hypotheses based upon groupings derived from the various data sets supported the discrimination of the A. arborescens group but did not support the discrimination of the A. alternata group from the A. tenuissima group.