Franco Nigro

Real-time quantitative PCR: a new technology to detect and study phytopathogenic and antagonistic fungi

Real-time PCR technologies open increasing opportunities to detect and study phytopathogenic and antagonistic fungi. They combine the sensitivity of conventional PCR with the generation of a specific fluorescent signal providing real-time analysis of the reaction kinetics and allowing quantification of specific DNA targets. Four main chemistries are currently used for the application of this technique in plant pathology. These chemistries can be grouped into amplicon sequence non-specific (SYBR Green I) and sequence specific (TaqMan, Molecular beacons, and Scorpion-PCR) methods. Amplicon sequence non-specific methods are based on the use of a dye that emits fluorescent light when intercalated into double-stranded DNA. Amplicon sequence specific methods are based on the use of oligonucleotide probes labelled with a donor fluorophore and an acceptor dye (quencher). The fluorescent signal eliminates the requirement for post-amplification processing steps, such as gel electrophoresis and ethidium bromide staining. This significantly reduces time and labour required for the analysis and greatly increases the throughput of PCR testing as an automated diagnostic system, making it suitable for large-scale analyses. Furthermore, the use of different fluorescent dyes facilitates the detection of several target microrganisms in a single reaction (multiplex-PCR). Real-time PCR makes possible an accurate, reliable and high throughput quantification of target fungal DNA in various environmental samples, including hosts tissues, soil, water and air, thus opening new research opportunities for the study of diagnosis, inoculum threshold levels, epidemiology and host–pathogen interactions. Moreover, the quantification of specific mRNA transcription by real-time PCR is being increasingly applied to the study of changes in gene expression in response to phytopathogenic and antagonistic fungi.

Impact of preharvest application of biological control agents on postharvest diseases of fresh fruits and vegetables

Abstract During the storage of harvested commodities, environmental parameters are quite stable. For this and other reasons, it is generally believed that biological control by means of microbial antagonists may have a greater potential for success when applied postharvest. However, one of the major obstacles to the development of postharvest biocontrol agents is that they are unable to control previously established infections, such as latent and quiescent infections and incipient infections occurring through wounds resulting from harvesting operations. Field application of biocontrol agents may enable early colonisation of fruit surfaces, thus protecting from these infections. Moreover, preharvest applications can be an appropriate strategy for fruits and vegetables subject to damage in postharvest handling. To be successful in preharvest applications, putative biocontrol agents must be able to tolerate low-nutrient availability, UV radiation, high temperature and dry conditions. Some reports of postharvest biological control accomplished by preharvest applications, include apples, avocados, sweet cherries, grapes, and strawberries. This paper provides a brief overview on particular aspects of preharvest application of biocontrol agents to reduce postharvest decay. Research areas relevant for the development of this strategy are also indicated.

Effectiveness of Aureobasidium pullulans and Candida oleophila against postharvest strawberry rots

Abstract Many yeasts, including yeast-like fungi, were selectively isolated from fruits and vegetables. In several assays performed on strawberries, table grape berries and kiwifruit, the yeast-like fungus Aureobasidium pullulans L47 and the yeasts Candida vanderwaltii L60 and C. oleophila L66 were the most effective antagonists of Botrytis cinerea and Rhizopus stolonifer . Isolates L47 and L66 were utilized in trials on strawberries grown under plastic tunnels. They were applied at flowering (full bloom and late petal fall) and at fruit maturity (just before or after harvest). Isolate L47 was the most effective against both B. cinerea and R. stolonifer . Both antagonists were more active when applied at the flowering stage, with isolate L47 more effective than vinclozolin. The antagonist population was consistently high on flowers and developing fruits, and on cold-stored strawberries. Isolates L47 and L66 showed a low sensitivity towards some fungicides in culture and were able to grow at temperatures between those of cold storage and 33 °C. Competition for nutrients seems to be the main mode of action.

Use of UV-C light to reduce Botrytis storage rot of table grapes

Abstract Single table grape berries ( Vitis vinifera L. cv. Italia) were irradiated with ultraviolet-C (UV-C) doses ranging from 0.125 to 4 kJ m −2 and inoculated with Botrytis cinerea . The pathogen was inoculated on artificial wounds at different times (0, 24, 48, 72, 96 and 144 h) after irradiation and the berries were stored either at 21 or 3°C. To check the influence of UV-C irradiation on the wound-healing processes, trials using berries wounded just before the UV-C irradiation and inoculated at different times were also performed. Significantly lower numbers of infected berries and lesion diameter were found in berries treated with UV-C doses ranging from 0.125 to 0.5 kJ m −2 . There was also a significantly lower level of disease in berries inoculated after 24–48 h than in those inoculated just after (10–15 min) the UV-C treatment. Thus, pretreatment with low UV-C doses followed by artificial inoculation with B. cinerea reduces postharvest grey mould of table grapes, suggesting induced resistance to the disease, both in berries wounded before and after irradiation. The microbial epiphytic population on UV-C-treated berries was also monitored. Results showed a significantly higher increase in the population of yeasts (including yeast-like fungi) and bacteria on berries irradiated with 0.25 and 0.5 kJ m −2 than on unirradiated control berries.

Short hypobaric treatments potentiate the effect of chitosan in reducing storage decay of sweet cherries

The effectiveness of chitosan and short hypobaric treatments, alone or in combination, to control storage decay of sweet cherries, was investigated over 2 years. In single treatments, chitosan was applied by postharvest dipping or preharvest spraying at 0.1, 0.5, and 1.0% concentrations; hypobaric treatments at 0.50 and 0.25 atm were applied for 4 h. In combined treatments, sweet cherries were dipped in 1.0% chitosan and then exposed to 0.50 and 0.25 atm, or sprayed with chitosan (0.1, 0.5, and 1.0%) 7 days before harvest and exposed to 0.50 atm soon after harvest. Untreated sweet cherries kept at normal pressure (near 1.00 atm) were used as controls. Rot incidence was evaluated after 14 days storage at 0±1 °C, followed by a 7 day shelf life. In both years, chitosan and hypobaric treatments applied alone significantly reduced brown rot, grey mould, and total rots, the latter also including blue mould, Alternaria, Rhizopus and green rots. A combined treatment with 1.0% chitosan and 0.50 atm was the best in controlling decay, showing in the first year, a synergistic effect in the reduction of brown rot and total rots. The results indicate that the combination of hypobaric and chitosan treatments is a valid strategy for increasing the effectiveness of the treatments in controlling postharvest decay of sweet cherries.

Control of postharvest rots of sweet cherries and table grapes with endophytic isolates of Aureobasidium pullulans

Abstract Fifty-one endophytic isolates of Aureobasidium pullulans were obtained from the flesh of sweet cherries and extensively screened to evaluate their biocontrol activity against postharvest rots of sweet cherries and table grapes. Preliminary analysis of all isolates by randomly amplified polymorphic DNA (RAPD) with three different primers showed the presence of a high genetic variability and enabled isolates not showing any genetic difference to be discarded. Thirty-five isolates with different RAPD electrophoresis patterns had a wide range of biocontrol activity against Botrytis cinerea and Monilinia laxa on single-wounded berries of sweet cherries and table grapes with a reduction of decay from 10 to 100%. Two isolates (533 and 547) significantly reduced B. cinerea on table grape berries also when applied 6, 12, and 24 h after the pathogen inoculation. In a 2-year period of investigation (1998–1999), a reduction of total rots ranging from 32 to 80% (sweet cherries) and from 59 to 64% (table grape) was achieved with isolates 533 and 547 applied after harvest. Preharvest applications of isolate 547 significantly reduced postharvest rots of sweet cherries and table grapes by 47 and 38%, respectively. On the whole, isolates 533 and 547 were more effective than A. pullulans L47, a biocontrol agent of postharvest diseases with a known activity. Population studies demonstrated that isolate 547 was able to survive under field conditions, to increase its population during cold storage, and to penetrate the flesh of sweet cherries when applied during flowering.

Genetic diversity and biocontrol activity of Aureobasidium pullulans isolates against postharvest rots

Aureobasidium pullulans, a cosmopolitan yeast-like fungus, colonises leaf surfaces and is a potential biocontrol agent for plant pathogens. Forty-one isolates of the fungus from the surface of several fruits and vegetables cultivated in Southern Italy were compared by molecular analysis and biocontrol activity. Characterisation of the isolates by using arbitrarily primed PCR (ap-PCR) confirmed the presence of a high genetic variability within the species. All the isolates were evaluated for their ability to control postharvest grey mold of apples, and two of them (SL250 and SL236), plus a proven antagonist (isolate L47), were able to control Penicillium digitatum on grapefruit, Botrytis cinerea, Rhizopus stolonifer and Aspergillus niger on table grape and B. cinerea and R. stolonifer on cherry tomato. Furthermore, preharvest application of isolate L47 on table grape resulted in a significant reduction of grey mold ranging from 27.1 to 49.5% compared to the untreated control. Random amplified polymorphic DNA technique (RAPD) was a useful method for the identification and evaluation of the survival rate of the applied antagonist.

Detection of Phytophthora nicotianae and P. citrophthora in Citrus Roots and Soils by Nested PCR

The polymerase chain reaction (PCR) was used for the specific detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils. Primers were based on the nucleotide sequences of the internal transcribed space regions (ITS1 and ITS2) of 16 different species of Phytophthora. Two primer pairs, Pn5B–Pn6 and Pc2B–Pc7, were designed specifically to amplify DNA from P. nicotianae and P. citrophthora, respectively. Another primer pair (Ph2–ITS4) was designed to amplify DNA from many Phytophthora species. All primer pairs were assessed for specificity and absence of cross-reactivity, using DNA from 118 isolates of Phytophthora and 82 of other common soil fungi. In conventional PCR, with a 10-fold dilution series of template DNA, the limit of detection was of 1pgμl−1 DNA for all the primer pairs (Ph2–ITS4, Pn5B–Pn6, and Pc2B–Pc7). In nested PCR, with primers Ph2–ITS4 in the first round, the detection limit was of 1fgμl−1 for both the primer sets (Pn5B–Pn6 and Pc2B–Pc7). Simple, inexpensive and rapid procedures for direct extraction of DNA from soil and roots were developed. The method yielded DNA of a purity and quality suitable for PCR within 2–3h. DNA extracted from soil and roots was amplified by nested PCR utilizing primers Ph2–ITS4 in the first round. In the second round the primer pairs Pn5B–Pn6 and Pc2B–Pc7 were utilized to detect P. nicotianae and P. citrophthora, respectively. Comparison between the molecular method and pathogen isolation by means of a selective medium did not show any significant differences in sensitivity.

IDENTIFICATION OF DNA SEQUENCES RELATED TO XYLELLA FASTIDIOSA IN OLEANDER, ALMOND AND OLIVE TREES EXHIBITING LEAF SCORCH SYMPTOMS IN APULIA (SOUTHERN ITALY)

Xylella fastidiosa is an important pathogen of commercial crops, landscape trees and ornamentals in North and South America. In Europe, symptoms resembling those caused by X. fastidiosa have occasionally been observed, but the presence of this EPPO quarantinable pathogen has never been confirmed. Recently, a rapidly spreading decline of aged olive trees has taken place in a large area of the Salento peninsula (Apulia, southern Italy). PCR assays on extracts from leaf veins and petioles of diseased trees gave positive reactions using X. fastidiosa gene-specific primers. In particular, PCR amplicons were generated by primers targeting the conserved hypothetical HL protein (Francis et al., 2006), the RNA polymerase sigma-70 factor, and the 16S rDNA genes (Rodrigues et al., 2003). Furthermore, molecular tests extended to almond and oleander trees with leaf scorching symptoms, growing next to diseased olive orchards, were also positive for X. fastidiosa. PCR products amplified from diseased olive trees were sequenced in duplicate and the sequences (EMBL-EBI provisional accession Nos HX2000034932- HX2000035003) showed 95 to 99% identity with the homologous genomic regions of X. fastidiosa. Tests for ascertaining the presence of X. fastidiosa by DAS-ELISA using two commercial kits (Agadia, USA and Bio-Rad, USA) were also positive, thus confirming molecular tests. Studies aimed at isolating the bacterium, determining the strain, evaluating its pathogenicity, and identifying the putative local vector(s) are currently in progress. X. fastidiosa has an extensive natural host range, including olive, from which the bacterial genotype A, pathogenic to oleander and almond, but not to grapevine, has been isolated in California (Krugner et al., 2010).

Effect of short hypobaric treatments on postharvest rots of sweet cherries, strawberries and table grapes

The effectiveness of short hypobaric treatments against postharvest rots was investigated by exposing sweet cherries, strawberries and table grapes to sub-atmospheric pressures (0.25, 0.50, and 0.75 atm) for different times (from 1 to 24 h). Postharvest rots of sweet cherries and strawberries arose from natural infections, whereas small table grape bunches and artificially wounded single berries were inoculated with Botrytis cinerea after hypobaric treatment. Sweet cherries exposed to 0.50 atm for 4 h had the lowest incidence of gray mould, brown rot and total rots, while a 1 h treatment was not effective. On strawberries, the greatest reductions of gray mould and Rhizopus rot were observed on fruits treated for 4 h at 0.25 and 0.50 atm, respectively. On table grape bunches treatment with 0.25 atm applied for 24 h significantly reduced the incidence of gray mould. In experiments performed with artificially wounded single table grape berries exposed to 0.50 atm for 24 h and then inoculated, the percentage of infected fruits and the diameter of the lesions were significantly reduced, in comparison with the controls. As a sub-atmospheric pressure of 0.25 atm did not affect radial growth of B. cinerea and Monilinia laxa, induced resistance was likely to be responsible for the observed reduction in decay.