Leonardo Schena

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.

The Top 10 oomycete pathogens in molecular plant pathology

Oomycetes form a deep lineage of eukaryotic organisms that includes a large number of plant pathogens which threaten natural and managed ecosystems. We undertook a survey to query the community for their ranking of plant-pathogenic oomycete species based on scientific and economic importance. In total, we received 263 votes from 62 scientists in 15 countries for a total of 33 species. The Top 10 species and their ranking are: (1) Phytophthora infestans; (2, tied) Hyaloperonospora arabidopsidis; (2, tied) Phytophthora ramorum; (4) Phytophthora sojae; (5) Phytophthora capsici; (6) Plasmopara viticola; (7) Phytophthora cinnamomi; (8, tied) Phytophthora parasitica; (8, tied) Pythium ultimum; and (10) Albugo candida. This article provides an introduction to these 10 taxa and a snapshot of current research. We hope that the list will serve as a benchmark for future trends in oomycete research.

Detection and quantification of Phytophthora ramorum, P. kernoviae, P. citricola and P. quercina in symptomatic leaves by multiplex real-time PCR

SUMMARY New species of Phytophthora such as Phytophthora ramorum, P. kernoviae and P. quercina together with P. citricola are plant pathogens which impact on forest health, natural ecosystem stability and international trade. A real-time multiplex PCR approach based on TaqMan PCR was developed to simultaneously identify and detect these four Phytophthora species. Specific primers and probes labelled with FAM (P. ramorum), Yakima Yellow (P. kernoviae), Rox (P. citricola) and Cy5 (P. quercina) were designed in different regions of the ras-related protein (Ypt1) gene. A new set of Black Hole Quenchers (BHQ), which dissipate energy as heat rather than fluorescence, were utilized. The method proved to be highly specific in tests with target DNA from 72 Phytophthora isolates (35 species). For all pathogens, the detection limit was 100 fg of target DNA and was not improved utilizing a nested approach to provide a first round of amplification with Phytophthora spp.-specific primers. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficients ranged from 0.947 to 0.996) and were not affected by the presence of plant extracts, indicating the appropriateness of the method for qualitative and quantitative analyses. Two universal primers and a TaqMan probe were also developed to evaluate the quality and quantity of extracted DNA and to avoid false negatives. The reliability of the entire procedure was assessed using both artificially and naturally infected leaves of a range of plant species. The method, combined with a rapid procedure for DNA extraction, proved to be rapid, reliable, sensitive and cost effective as multiple pathogens were detected within the same plant extract by using different primer/probe combinations.

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.

Biological control of Botrytis, Aspergillus and Rhizopus rots on table and wine grapes in Israel

Abstract One hundred and twenty-nine strains of epiphytic micro-organisms, isolated from table and wine grapes in Israel, were screened for antagonistic activity against Botrytis cinerea on table grapes. Two isolates (Candida guilliermondii, strain A42 and Acremonium cephalosporium, strain B11) were further evaluated for the control of decay in grapes caused by Aspergillus niger and Rhizopus stolonifer. Decay incidence caused by Botrytis cinerea, Aspergillus niger and Rhizopus stolonifer on wounded detached berries was reduced to 8, 14 and 22% respectively, by A42 and to 16, 82 and 60%, respectively, by B11. On small clusters with intact berries, decay was reduced to 30, 22 and 22%, respectively, by A42 and to 48, 39 and 30% respectively, by isolate B11. Both strains survived well under local vineyard conditions and during storage at 0°C and maintained relatively high cell counts on the berries. Field experiments were conducted in 1996, 1997 and 1998, with both table and wine grapes. Vines were sprayed with yeast suspension 2–5 times at 7–10 day intervals and decay was evaluated before harvest (wine grapes) or after storage (table grapes). A42 reduced decay caused by Botrytis cinerea in two of the three seasons in both table and wine grapes, and rots caused by Aspergillus niger in wine grapes were reduced significantly in 1997 and 1998. B11 reduced Botrytis cinerea development in the two years it was tested in wine grapes but in table grapes only in 1996. Morever, it did not control decay caused by Aspergillus niger.

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.

Molecular Detection of Strain L47 of Aureobasidium pullulans, a Biocontrol Agent of Postharvest Diseases

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.

Identification and Detection of Rosellinia Necatrix by Conventional and Real-time Scorpion-PCR

Several polymerase chain reaction (PCR) primers were designed from the internal transcribed spacer (ITS) regions of the rDNA genes of Rosellinia necatrix to develop a PCR-based identification method. Screening the primers against two isolates of R. necatrix and six other Rosellinia species resulted in the amplification of a single specific product from R. necatrix for most of the primer pairs. Two primer pairs (R2-R8 and R10-R7) confirmed their specificity when tested against 72 isolates of R. necatrix and 93 other fungi from different hosts and geographic areas. The R10 primer was modified to obtain a Scorpion primer for detecting a specific 112bp amplicon by fluorescence emitted from a fluorophore in a self-probing PCR assay. This assay specifically recognised the target sequence of R. necatrix over a large number of other fungal species. In conventional PCR, with primer pairs R2-R8 and R10-R7, 10-fold dilutions of R. necatrix DNA indicated a detection limit of 10pgμul-1 using a single set of primers and 10fgμl-1 in nested-PCR. For Scorpion-PCR, the detection limit was 1pgμl-1 and 1fgμl-1 in nested Scorpion-PCR, i.e. 10 times more sensitive than conventional PCR. A simple and rapid procedure for DNA extraction directly from soil was modified and developed to yield DNA of purity and quality suitable for PCR assays. Combining this protocol with the nested Scorpion-PCR procedure it has been possible to specifically detect R. necatrix from artificially inoculated soils in approximately 6h.

Real-time detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soil

Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg μl-1 for P. nicotianaeand 100 pg μl−1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg μl-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.