Gabriella Cirvilleri

Specific oligonucleotide primers for the rapid identification and detection of the agent of tomato pith necrosis, Pseudomonas corrugata, by PCR amplification : evidence for two distinct genomic groups

Unique DNA bands from strains representative of two groups of Pseudomonas corrugata, as shown by amplification of their genomic DNA by polymerase chain reaction using short random sequence oligonucleotide primers (RAPD-PCR), were isolated, cloned and sequenced. Two pairs of specific primer sequences, based on the ends of the cloned unique DNA bands from strains IPVCT10.3 and IPVCT8.1, were used in multiplex PCR with a range of P. corrugata strains. All strains produced one of the two specific bands, 1100 bp (from the IPVCT10.3-based primers) and 600 bp (from the IPVCT8.1-based primers), representing groups designated I and II, respectively. The primers were also tested on a wider range of Pseudomonas species, including the closely-related fluorescent Pseudomonas genomospecies FP1, FP2 and FP3: none of these bacteria produced any bands following amplification by PCR with these primers. The primer sets detected P. corrugata in tomato pith necrosis-infected plants providing a useful tool for rapid identification and epidemiological studies.

Evaluation of Trichoderma harzianum strain T22 as biological control agent of Calonectria pauciramosa

The objective of this research was to evaluate Trichoderma harzianum strain T22 as a biocontrol agent of collar and root rot caused by different Calonectria pauciramosa isolates. Thus, the microsclerotia-forming ability and virulence of twenty C. pauciramosa isolates were assessed. Microsclerotia production varied partially among the isolates and dual culture with T22 on carnation leaf agar revealed isolates with both high and low microsclerotia-forming ability. Inoculation tests on red clover (Triflolium pratense) demonstrated its susceptibility to the pathogen. On red clover, the degree of virulence and T22 effects in controlling infections were highly variable among the isolates tested. A nursery trial performed on Feijoa sellowiana seedlings confirmed previous results, clearly indicating virulence variability among C. pauciramosa isolates. For three isolates tested in nursery trial, T22 effectiveness in controlling infection was inversely related to their degree of virulence. Overall, T. harzianum strain T22 showed good antagonist activity in reducing microsclerotia production on carnation leaf and the incidence and severity of collar and root rot on both selected hosts. This data could be crucial in developing integrated pest management strategies in ornamental plant nurseries.

Postharvest efficacy of resistance inducers for the control of green mold on important Sicilian citrus varieties

The disease reducing activity of acibenzolar-S-methyl (ASM), chitosan and β-aminobutyric acid (BABA) against postharvest green mold (GM) of citrus fruit, Penicillium digitatum, was investigated by tests performed in in vitro and in vivo conditions. The inhibition of the mycelial growth of P. digitatum by ASM, chitosan and BABA at different concentrations was evaluated on potato dextrose agar and on orange peel extract agar. The pathogen was totally inhibited (100% growth inhibition) by chitosan at concentrations ≥ 0.1% and by BABA at 1000 mmol and only poorly inhibited by ASM at 0.5% (47% growth inhibition). Inhibitory effects of chitosan and BABA were confirmed by P. digitatum conidial germination tests. The performances of these chemical compounds were evaluated in vivo on artificially inoculated orange, lemon and grapefruit cultivars. On the whole, chitosan significantly reduced GM decay on ‘Valencia’ and ‘Washington Navel’ orange, ‘Femminello’ lemon and grapefruit cv. Marsh Seedless at the concentration of 0.5% (6–20% disease incidence), and BABA provided the best results on ‘Tarocco Scirè’and ‘Valencia’ orange and grapefruit cv. Marsh Seedless at the concentration of 1000 mmol (12–16% disease incidence). ASM did not show any efficacy. This study demonstrated that the effects of chitosan and BABA on GM in citrus fruits may be associated with their direct fungitoxic properties against the pathogen. Treatments with chitosan and BABA could be recommended for inclusion in postharvest decay management programs for citrus packinghouses and their use may be an effective method to improve the integrated pest management strategy.

Characterisation and pathogenicity of Pestalotiopsis uvicola and Pestalotiopsis clavispora causing grey leaf spot of mango (Mangifera indica L.) in Italy

During 2009 and 2010, twenty-one isolates of Pestalotiopsis spp. Associated with grey patches on the leaves, twigs, and panicles of mango were collected in six orchards located in Sicily (Italy). Morphological characteristics of colony (colour and mycelium appearance), and conidia (size, shape, septation, length and the number of apical and the basal appendages) as well as phylogenetic analysis of the Internal Transcribed Spacer (ITS) region (ITS1, 5.8S gene, and ITS2) of six representative isolates revealed the occurrence of P. uvicola and P. clavispora. The representative isolates of both species were pathogenic to the artificially inoculated detached mango leaves cv. Kensington Pride and showed significant variation in lesion size. This is the first report worldwide of P. uvicola and P. clavispora causing grey leaf spot of mango.

Occurrence and Pathogenicity of Pseudomonas Fluorescens and P. Putida on Tomato Plants in Italy

SUMMARY During 2006-2008, a survey conducted in soilless and soil-grown tomato crops in eastern and southeastern Sicily (Italy) disclosed the presence of widespread leaf chlorosis and necrosis associated with yellow, pink or brown discolouration of vascular and pith tissues. Bacteria were always isolated from symptomatic tissues on KB and NA media. A total of 158 isolates showed the LOPAT characters of group Va (-+-+-) and group Vb (++-+-) and 21 of these strains were identified as different biovars of Pseudomonas fluorescens and P. putida. Koch’s postulates showed that 42 fluorescent Pseudomonas isolates are able to induce vascular discolouration of tomato plants but not leaf chlorosis and necrosis. The interactions of vascular infections and abiotic stresses were studied in growth chamber experiments. Differences in virulence were found among P. fluorescens biovar I and P. putida bv. A strains on tomato plants grown under saline stress conditions. Both bacteria were able to induce leaf chlorosis, necrosis or death of tomato plants under saline stress conditions.

Characterisation of Neofusicoccum species causing mango dieback in Italy

Species of Botryosphaeriaceae are important fungal pathogens of mango worldwide. A survey of 11 mango orchards located in the provinces of Catania, Messina, Palermo and Ragusa (Sicily, southern Italy), resulted in the isolation of a large number (76) of Neofusicoccum isolates associated with decline and dieback symptoms. Isolates were identified based on morphology and DNA sequence data analyses of the internal transcribed spacer region of the nrDNA and partial translation of the elongation factor 1-alpha gene regions. Two species of Neofusicoccum were identified, which included N. parvum and N. australe, the former of which was the dominant species. The high incidence in local orchards and the pathogenicity results indicate that N. parvum and N. australe are important pathogens of mango in Sicily where they may significantly limit mango production.

Molecular characterisation and pathogenicity of Aspergillus Sect. Nigri causing Aspergillus vine canker of table grapes in Italy

Thirty-two isolates belonging to black aspergilli (Aspergillus section Nigri) associated to vine canker disease of grapevine were collected in seven vineyards located in southeastern Sicily (Italy). Molecular analysis was performed to identify the isolates by multilocus sequence analysis. Amplification of part of the β-tubulin gene (benA) and partial calmodulin (CaM) gene were performed using the Bt2a, Bt2b and CL1, CL2A primers, respectively. Molecular characterisation showed a high distribution of Aspergillus niger “aggregate” species on grapes in Sicily and in particular of A. niger (21 isolates), A. tubingensis (9 isolates), and A. carbonarius (2 isolates). The 21 isolates of A. niger found to belong within the newly described cryptic species A. awamori. Six isolates (3 of A. tubingensis, 2 of A. carbonarius, and 1 of A. niger) were used in pathogenicity studies on mature canes of cv. Italia grape. All species caused Aspergillus vine canker equally well, with no differences in virulence.

CHARACTERISATION OF COLLETOTRICHUM SPECIES CAUSING ANTHRACNOSE DISEASE OF MANGO IN ITALY

Anthracnose symptoms consisting of necrotic spots on the leaves, twigs and branches were observed on mango trees of cv. Kensington Pride in orchards located in the countryside of Palermo and Milazzo (southern Italy). Based on morphological observations and phylogenetic analysis of the β-tubulin (benA) and histone H3 (HIS3) genes, three Colletotrichum species were identified and recovered from diseased plants, i.e. C. karstii (nine isolates), C. kahawae subsp. ciggaro (six isolates) and C. gloeosporioides (six isolates). Following artificial inoculation, all species induced symptoms on the leaves and fruits of cv. Kensington Pride. To our knowledge, this is the first report of mango anthracnose caused by C. karstii, C. kahawae subsp. ciggaro and C. gloeosporioides in Italy.

Potential Role of Exoglucanase Genes (WaEXG1 and WaEXG2) in the Biocontrol Activity of Wickerhamomyces anomalus

The use of yeasts, including Wickerhamomyces anomalus, as biocontrol agents of fungi responsible for postharvest diseases of fruits and vegetables has been investigated for the past two decades. Among a variety of mechanisms, the production of glucanases coded by the “killer genes” WaEXG1 and WaEXG2 have been reported to play a role in the ability of yeast to inhibit other fungi. The objective of the present study was to determine the expression of these genes by RT-qPCR, utilizing gene-specific primers, when W. anomalus was grown on grape berries and oranges that were either non-inoculated or inoculated with Botrytis cinerea or Penicillium digitatum, or in minimal media supplemented with cell walls of various plant pathogens and different amounts of glucose. Results indicated that WaEXG2 was more responsive than WaEXG1 to the nutritional environment (including the addition of glucose to cell wall-amended media) in vitro and appeared to play a greater role in the cellular metabolism of W. anomalus. WaEXG2 expression also appeared to be more responsive to the presence of cell walls of P. digitatum and B. cinerea than other fungal species, whereas the same level of induction was not seen in vivo when the yeast was grown in wounded/pathogen-inoculated fruits.

Characterization of Pseudomonas syringae pv. syringae isolated from mango in Sicily and occurrence of copper-resistant strains.

Mango (Mangifera indica) is grown throughout a wide range of frost-free climates and is one of the world’s most important fruit crops. During 2010-2014, severe symptoms of bacterial apical necrosis (BAN) caused by Pseudomonas syringae pv. syringae (Pss) were observed on mango plants throughout all mango-growing areas in Sicily (southern Italy). The causal agent was identified based on phenotypic, genotypic and pathogenicity tests. The genetic variability of strains obtained from different areas and cultivars were assessed by rep- PCR and allowed clear differentiation of the Pss strains isolated from mango from other representative strains of the pathovar. In addition, the respective production of syringomycin, syringopeptin and mangotoxin was checked by in vitro tests and PCR detection. All tested strains showed the presence of mgoA and mgoB of the mangotoxin operon. Some copper- resistant Pss strains showing cusCBA genes were also found in some orchards. This feature could explain the failure of the disease control using copper compounds. Among 71 strains tested using in vitro assay, no copper-sensitive strains were detected, whereas 44 strains (62%) had high resistance, 16 strains (22.5%) were resistant and 11 strains (15.5%) had low resistance to copper sulfate. To our knowledge, this is the first report of copper resistance among Pss strains causing BAN on mango in Sicily.