The nucleotide sequence of the putative coat protein open reading frame of seven previously uncharacterized AMV strains from Italy and France was determined and aligned with comparable sequences of other AMV strains (425 L, 425 M, YSMV, S, VRU, 15/64 and Da). The data set of AMV sequences was used to determine phylogenetic relationships by both a stochastic (stationary Markov model) and a deterministic method (maximum-parsimony) of analysis. The topology of the trees obtained with the two methods was essentially the same showing that all AMV strains clustered in two monophyletic groups. Close clustering of Italian strains in subgroup I and of French strains in subgroup II seems to suggests the effect of geographic distinctiveness of evolutionary dynamics of these AMV strains. This separation did not correlate with differences in host range or symptoms (necrotic or non necrotic) induced in tomato but rather it reflected variations in the amino acid sequence of their CP, which might be related to structural properties of virus particles. A simple and rapid procedure based on the reverse transcriptase-polymerase chain reaction (RT-PCR) followed by ezymatic digestion (RFLP) was developed to identify and classify AMV isolates into the two subgroups. The method applied to a number of other AMV isolates from Italy and France supported their division in two distinct subgroups. This RT-PCR RFLP method may be useful way to investigate the dynamics of AMV populations in nature.

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The strain L47 of Aureobasidium pullulans is an effective biocontrol agent of postharvest diseases. When applied in the field before harvesting it requires a specific monitoring method to evaluate colonization and dispersal in the environment. The randomly amplified polymorphic DNA technique (RAPD) was used for a preliminary screening of A. pullulans genetic variability among 205 isolates. This approach allowed the selection of a 1.3-kb fragment (L4) present solely in isolates L47 and 633. In Southern blots, a digoxigenin (DIG)-labeled L4 amplicon specifically recognized the corresponding fragment present in the polymorphic pattern of L47 and 633. The L4 fragment was cloned, sequenced, and used to design two sequence-characterized amplification region (SCAR) primers and a 242-bp riboprobe. Both the SCAR primers and the 242-bp DIG-labeled riboprobe were highly specific for L47. In classical polymerase chain reaction (PCR), with a series of 10-fold dilutions of L47 DNA, the limit of detection was 20 pg/μl. The Ap13 primer was also modified to obtain a Scorpion primer for detecting a 150-bp amplicon by fluorescence emitted from a fluorophore through a self-probing PCR assay. This assay specifically recognized the target sequence of L47 strain over a number of other A. pullulans isolates in field-treated grape berry washings. The limit of detection was 105 cells per ml, i.e. 10 times higher than the limit of the CFU method. The method is also proposed as a way to demonstrate the ability of L47 strain to penetrate the epidermis of sweet cherry fruits and to track it in the mesocarp.

Mixed infection with the SON41 strain of Potato virus Y (PVY-SON41) in tomato increased accumulation of RNAs of strains Fny and LS of Cucumber mosaic virus (CMV-Fny and CMV-LS, respectively) and enhanced disease symptoms. By contrast, replication of PVY-SON41 was downregulated by CMV-Fny and this was due to the CMV-Fny 2b protein. The CMV-FnyΔ2b mutant was unable to systemically invade the tomato plant because its movement was blocked at the bundle sheath of the phloem. The function needed for invading the phloem was complemented by PVY-SON41 in plants grown at 22°C whereas this complementation was not necessary in plants grown at 15°C. Mutations in the 2b protein coding sequence of CMV-Fny as well as inhibition of translation of the 2a/2b overlapping region of the 2a protein lessened both the accumulation of viral RNAs and the severity of symptoms. Both of these functions were complemented by PVY-SON41. Infection of CMV-Fny supporting replication of the Tfn-satellite RNA reduced the accumulation of CMV RNA and suppressed symptom expression also in plants mixed-infected with PVY-SON41. The interaction between CMV and PVY-SON41 in tomato exhibited different features from that documented in other hosts. The results of this work are relevant from an ecological and epidemiological perspective due to the frequency of natural mixed infection of CMV and PVY in tomato.

Expression profiles were identified in the fungus Pochonia chlamydosporia, a biological control agent of plant parasitic nematodes, through a cDNA-amplified fragment length polymorphism approach. Two isolates with different host ranges, IMI 380407 and IMI 331547, were assayed in conditions of saprotrophic-to-parasitic transition, through in vitro assays. Gene expression profiles from three different nutritional conditions and four sampling times were generated, with eggs of host nematodes Globodera pallida and Meloidogyne incognita. Expression of transcripts changed in RNA fingerprints obtained under different nutritional stresses (starvation in presence/absence of eggs, or rich growth media). Transcript derived fragments (TDFs) obtained from the expression profiles corresponded to 6,800 products. A subset was sequenced and their expression profile confirmed through RT PCR. A total of 57 TDFs were selected for further analysis, based on similarities to translated or annotated sequences. Genes expressed during egg parasitism for both IMI 380407 and IMI 331547 were involved in metabolic functions, cellular signal regulation, cellular transport, regulation of gene expression, DNA repair, and other unknown functions. Multivariate analysis of TDF expression showed three groups for IMI 380407 and one for IMI 331547, each characterized by expression of genes related to eggs parasitism. Common amplification profiles among TDF clusters from both isolates also reflected a pool of constitutive genes, not affected by the nutritional conditions and nematode associations, related to general metabolic functions. The differential expression of parasitism related genes suggest a network of induced/repressed products, playing a role in fungal signaling and infection, with partial overlaps in host infection and parasitism traits.