Roberto Faedda

DEVELOPMENT OF QUANTITATIVE PCR DETECTION METHODS FOR PHYTOPATHOGENIC FUNGI AND OOMYCETES

SUMMARY In recent years quantitative PCR (qPCR) detection methods have been widely utilised to detect phytopathogenic fungi and oomycetes and have greatly contributed to the advancement of knowledge in plant pathology. However, major drawbacks and common errors, most typical of earlier reports, still affect many methods currently available in the literature. Errors can be made throughout the entire process for the development of qPCR methods, at the level of selection of appropriate DNA extraction and purification protocols, identification of suitable target regions, choice of the chemistry, design and validation of specific primers and probes, analysis of sensitivity, choice of an absolute and/or relative quantification approach and analysis of the risk of detecting target DNA from dead sources. In the present review the above mentioned steps are analysed, highlighting their critical aspects and providing a practical guide for the users.

Insensitivity to Metalaxyl Among Isolates of Phytophthora capsici Causing Root and Crown Rot of Pepper in Southern Italy

Pepper (Capsicum annuum L.) has become an economically important crop in the coastal provinces of Catanzaro and Vibo Valentia, in Calabria (southern Italy). An old local selection Riggitano, very susceptible to root and crown rot caused by Phytophthora capsici Leonian, is the prevalent cultivar in this area. Although repeated applications of metalaxyl are used as a soil drench, severe outbreaks occur each year on greenhouse crops. To examine metalaxyl resistance in P. capsici, 60 single-hypha isolates of P. capsici were tested in vitro for their level of sensitivity to metalaxyl. The isolates were collected from 1992 to 1997, during epidemic outbreaks of root and crown rot, from two commercial greenhouse pepper crops, near Vibo Valentia and Lametia Terme (Catanzaro). Fungicide sensitivity was determined by plating mycelial plugs onto potato dextrose agar (PDA) amended with metalaxyl. The fungicide was added to PDA after autoclaving, at final concentrations of 0.1, 1, 5, 10, 50, 100, and 200 μg/ml a.i. The percentage of inhibition of radial growth on metalaxyl-amended medium compared with the growth on unamended medium was determined after 6 days of incubation in the dark at 25°C. Three replicate petri dishes were used per treatment and each test was performed twice. The isolates were paired in culture on V8 agar with isolates of P. capsici of known mating type and all proved to be A2 mating type. Significant variation was observed among the isolates tested in responce to metalaxyl. The ED50 values for in vitro inhibition of mycelial growth by metalaxyl ranged from 1 to 11 μg/ml, whereas an ED 50 value of 0.1 μg/ml had been reported for a wild-type isolate of P. capsici obtained from pepper in northern Italy (3). The variation observed among the isolates from Calabria appeared unrelated to both the geographical origin and the year of isolation. The isolates from Calabria were inhibited by between 1 and 12% at 0.1 μg/ml and by between 7 and 27% at 1 μg/ml, proving to be less sensitive to metalaxyl than isolates from Capsicum spp. originating from Central America, tested by other authors (1). According to the criterion used in a recent screening for sensitivity to metalaxyl (2), 19% of the isolates from Calabria should be considered sensitive, as they were inhibited by more than 60% at 5 μg/ml, while all the others were intermediate, as they were inhibited less than 60% at 5 μg/ml but more than 60% at 100 μg/ml. On the basis of this preliminary screening, we report the presence of insensitivity to metalaxyl in field isolates of P. capsici in southern Italy. Although no isolate tested appeared highly resistant to metalaxyl, the presence of a high proportion of isolates with an intermediate level of resistance should be a reason for the growers to use metalaxyl more cautiously to control root and collar rot. References: (1) M. D. Coffey and L. A. Bower. Phytopathology 74:502, 1984. (2) G. Parra and J. Ristaino. Plant Dis. 82:711, 1998. (3) M. L. Romano and A. Garibaldi. La difesa delle piante 3:153, 1984.

Phytophthora niederhauserii sp. nov., a polyphagous species associated with ornamentals, fruit trees and native plants in 13 countries

A non-papillate, heterothallic Phytophthora species first isolated in 2001 and subsequently from symptomatic roots, crowns and stems of 33 plant species in 25 unrelated botanical families from 13 countries is formally described here as a new species. Symptoms on various hosts included crown and stem rot, chlorosis, wilting, leaf blight, cankers and gumming. This species was isolated from Australia, Hungary, Israel, Italy, Japan, the Netherlands, Norway, South Africa, Spain, Taiwan, Turkey, the United Kingdom and United States in association with shrubs and herbaceous ornamentals grown mainly in greenhouses. The most prevalent hosts are English ivy (Hedera helix) and Cistus (Cistus salvifolius). The association of the species with acorn banksia (Banksia prionotes) plants in natural ecosystems in Australia, in affected vineyards (Vitis vinifera) in South Africa and almond (Prunus dulcis) trees in Spain and Turkey in addition to infection of shrubs and herbaceous ornamentals in a broad range of unrelated families are a sign of a wide ecological adaptation of the species and its potential threat to agricultural and natural ecosystems. The morphology of the persistent non-papillate ellipsoid sporangia, unique toruloid lobate hyphal swellings and amphigynous antheridia does not match any of the described species. Phylogenetic analysis based on sequences of the ITS rDNA, EF-1α, and β-tub supported that this organism is a hitherto unknown species. It is closely related to species in ITS clade 7b with the most closely related species being P. sojae. The name Phytophthora niederhauserii has been used in previous studies without the formal description of the holotype. This name is validated in this manuscript with the formal description of Phytophthora niederhauserii Z.G. Abad et J.A. Abad, sp. nov. The name is coined to honor Dr John S. Niederhauser, a notable plant pathologist and the 1990 World Food Prize laureate.

Analyses of the Population Structure in a Global Collection of Phytophthora nicotianae Isolates Inferred from Mitochondrial and Nuclear DNA Sequences

Genetic variation within the heterothallic cosmopolitan plant pathogen Phytophthora nicotianae was determined in 96 isolates from a wide range of hosts and geographic locations by characterizing four mitochondrial (10% of the genome) and three nuclear loci. In all, 52 single-nucleotide polymorphisms (SNPs) (an average of 1 every 58 bp) and 313 sites with gaps representing 5,450 bases enabled the identification of 50 different multilocus mitochondrial haplotypes. Similarly, 24 SNPs (an average of 1 every 69 bp), with heterozygosity observed at each locus, were observed in three nuclear regions (hyp, scp, and β-tub) differentiating 40 multilocus nuclear genotypes. Both mitochondrial and nuclear markers revealed a high level of dispersal of isolates and an inconsistent geographic structuring of populations. However, a specific association was observed for host of origin and genetic grouping with both nuclear and mitochondrial sequences. In particular, the majority of citrus isolates from Italy, California, Florida, Syria, Albania, and the Philippines clustered in the same mitochondrial group and shared at least one nuclear allele. A similar association was also observed for isolates recovered from Nicotiana and Solanum spp. The present study suggests an important role of nursery populations in increasing genetic recombination within the species and the existence of extensive phenomena of migration of isolates that have been likely spread worldwide with infected plant material.

Metabarcoding Analysis of Phytophthora Diversity Using Genus-Specific Primers and 454 Pyrosequencing

A metabarcoding method based on genus-specific primers and 454 pyrosequencing was utilized to investigate the genetic diversity of Phytophthora spp. in soil and root samples of potted plants, from eight nurseries. Pyrosequencing enabled the detection of 25 Phytophthora phylotypes distributed in seven different clades and provided a much higher resolution than a corresponding cloning/Sanger sequencing approach. Eleven of these phylotypes, including P. cactorum, P. citricola s.str., P. palmivora, P. palmivora-like, P. megasperma or P. gonapodyides, P. ramorum, and five putative new Phytophthora species phylogenetically related to clades 1, 2, 4, 6, and 7 were detected only with the 454 pyrosequencing approach. We also found an additional 18 novel records of a phylotype in a particular nursery that were not detected with cloning/Sanger sequencing. Several aspects confirmed the reliability of the method: (i) many identical sequence types were identified independently in different nurseries, (ii) most sequence types identified with 454 pyrosequencing were identical to those from the cloning/Sanger sequencing approach and/or perfectly matched GenBank deposited sequences, and (iii) the divergence noted between sequence types of putative new Phytophthora species and all other detected sequences was sufficient to rule out sequencing errors. The proposed method represents a powerful tool to study Phytophthora diversity providing that particular attention is paid to the analysis of 454 pyrosequencing raw read sequences and to the identification of sequence types.

Covalent selection of the thiol proteome on activated thiol sepharose: A robust tool for redox proteomics

Protein thiols contribute significantly to antioxidant defence and selective oxidation of cysteines is important in signal transduction even in sub-stress scenarios. However, cysteine is the second rarest residue in proteins and it can be difficult to target low-abundance thiol (-SH)-containing proteins in proteomic separations. Activated thiol sepharose (ATS) allows covalent selection of -SH-containing proteins which can then be recovered by reduction with mercaptoethanol or dithiothreitol. This is a robust method for enriching -SH-containing proteins. We have used ATS to estimate the percentage (by weight) of thiol-containing proteins in cell extracts from a range of biological sources: a bacterium, Escherichia coli; a fungus, Trichoderma harzianum; and a bivalve mollusc Mytilus edulis. -SH-containing proteins account for 2.52% (E. coli), 1.4% (T. harzianum) and 1.4% (M. edulis) of total protein. Exposure to pro-oxidants did not materially alter these values. On removal of low M(r) thiols such as glutathione, the values for M. edulis did not significantly change but those for T. harzianum increased threefold. The two-dimensional electrophoresis profiles of ATS-selected proteins for each organism were compared in control and pro-oxidant-exposed preparations. This revealed that some proteins present in controls were absent in pro-oxidant-treated extracts which we attribute to thiol oxidation. ATS has significant potential in enrichment for -SH-containing proteins in redox proteomics.

Identification of differentially expressed genes in response to mercury I and II stress in Trichoderma harzianum.

Filamentous fungi are very promising organisms in both the control and the reduction of the amount of heavy metal released by human and industrial activities. In particular, Trichoderma harzianum demonstrated to be tolerant towards different heavy metals, such as mercury and cadmium, even though the mechanism underlying this tolerance is not fully understood. By using a particular strategy of the suppression subtractive hybridization technique, we were able to identify in the strain IMI 393899 of T. harzianum eight different genes up-regulated in the presence of mercury II with respect to cadmium. Among the genes identified, a possible role in the tolerance mechanism could be envisaged for hydrophobin, due to its ability to dissolve hydrophobic molecules into aqueous media. We also show that IMI 393899 grows at the same rate of control culture in the presence of mercury I and that all eight genes isolated were also up-regulated in this condition.

Phytophthora × pelgrandis Causes Root and Collar Rot of Lavandula stoechas in Italy

In 2007, Phytophthora isolates with atypical morphological and biological characteristics were found associated with root and collar rot of potted plants of Stoechas lavender (Lavandula stoechas) in an ornamental nursery in Italy. A polyphasic approach, including morphological and cultural observations, sequencing the ITS-rDNA region, the Pheca and the mitochondrial coxI genes, multiplex PCRs with primers specific for P. nicotianae or P. cactorum, as well as random amplified polymorphic DNA-polymerase chain reaction, was used to characterize these isolates. On the basis of morpho-cultural and molecular analyses, the isolates from Stoechas lavender were identified as Phytophthora × pelgrandis, a natural hybrid of P. nicotianae × P. cactorum previously reported in other European countries, the Americas, and Taiwan, as a pathogen of ornamentals and loquat plants. In pathogenicity tests using potted plants of Stoechas lavender, the P. × pelgrandis isolates, similarly to the parental species P. nicotianae, induced the symptoms observed on plants with natural infections and were reisolated only from artificially inoculated plants. Dispersal of P. × pelgrandis on this host could exacerbate the damage caused by Phytophthora root and collar rot, of which the main causal agent presently is P. nicotianae on lavender in Europe. Application of hygienic measures is important to reduce the proliferation and spread of the Phytophthora hybrids.

Two previously unknown Phytophthora species associated with brown rot of Pomelo (Citrus grandis) fruits in Vietnam

Two distinct Phytophthora taxa were found to be associated with brown rot of pomelo (Citrus grandis), a new disease of this ancestral Citrus species, in the Vinh Long province, Mekong River Delta area, southern Vietnam. On the basis of morphological characters and using the ITS1-5.8S-ITS2 region of the rDNA and the cytochrome oxidase subunit 1 (COI) as barcode genes, one of the two taxa was provisionally named as Phytophthora sp. prodigiosa, being closely related to but distinct from P. insolita, a species in Phytophthora Clade 9, while the other one, was closely related to but distinct from the Clade 2 species P. meadii and was informally designated as Phytophthora sp. mekongensis. Isolates of P. sp. prodigiosa and P. sp. mekongensis were also obtained from necrotic fibrous roots of Volkamer lemon (C. volkameriana) rootstocks grafted with ‘King’ mandarin (Citrus nobilis) and from trees of pomelo, respectively, in other provinces of the Mekong River Delta, indicating a widespread occurrence of both Phytophthora species in this citrus-growing area. Koch’s postulates were fulfilled via pathogenicity tests on fruits of various Citrus species, including pomelo, grapefruit (Citrus x paradisi), sweet orange (Citrus x sinensis) and bergamot (Citrus x bergamia) as well as on the rootstock of 2-year-old trees of pomelo and sweet orange on ‘Carrizo’ citrange (C. sinensis ‘Washington Navel’ x Poncirus trifoliata). This is the first report of a Phytophthora species from Clade 2 other than P. citricola and P. citrophthora as causal agent of fruit brown rot of Citrus worldwide and the first report of P. insolita complex in Vietnam. Results indicate that likely Vietnam is still an unexplored reservoir of Phytophthora diversity.

Characterization of Basidiomycetes associated with wood rot of citrus in southern Italy.

The characterization of Basidiomycetes associated with wood rots in commercial citrus orchards in southern Italy revealed that both white and brown rot fungi are implicated in this disease. Fomitiporia mediterranea was the most prevalent species causing a white rot, followed by Fomitopsis sp. which, by contrast, was associated with brown rot wood decay. Furthermore, Phellinus spp. and other nonidentified basidiomycetous fungi showing genetic affinity with the genera Phellinus and Coniophora were occasionally isolated. Artificial inoculations on lemon (Citrus limon) branches showed a faster wood colonization by Fomitopsis sp. compared with F. mediterranea, indicating that the former species as a potentially serious pathogen of citrus trees. The analysis of F. mediterranea internal transcribed spacer (ITS) sequences revealed a high level of genetic variability, with 13 genotypes which were both homozygous (6 genotypes) and heterozygous (7 genotypes). The presence of heterozygous genomes based on ITS sequences has never been reported before for F. mediterranea. This, together with the high frequency of basidiomata on infected wood, unambiguously confirms the outcrossing nature of reproduction in F. mediterranea and the primary role of basidiospores in the dissemination of inoculum. Similarly, high genetic variability was observed analyzing Fomitopsis sp. Because basidiomata of this fungus have not been observed on citrus trees, it can be hypothesized that basidiospores are produced on alternative host plants.