Phytophthora pseudocryptogea, P. nicotianae and P. multivora Associated to Cycas revoluta: First Report Worldwide.

A dieback was observed on three-year-old pot-grown plants of Cycas revoluta in Sicily (Italy). Symptoms, including stunting, yellowing and blight of the leaf crown, root rot and internal browning and decay of the basal stem, closely resembled the Phytophthora root and crown rot syndrome, common in other ornamentals. Isolations from rotten stem and roots, using a selective medium, and from rhizosphere soil of symptomatic plants, using leaf baiting, yielded three Phytophthora species, P. multivora, P. nicotianae and P. pseudocryptogea, were obtained. Isolates were identified by both morphological characters and DNA barcoding analysis, using three gene regions: ITS, ß-tub and COI. Phytophthora pseudocryptogea was the sole species isolated directly from the stem and roots. The pathogenicity of the isolates of the three Phytophthora species was tested on one-year-old potted plants of C. revoluta, using both stem inoculation by wounding, and root inoculation through infested soil. Phytophthora pseudocryptogea was the most virulent and, like P. nicotianae, reproduced all the symptoms of natural infections, while P. multivora was the least virulent and induced solely very mild symptoms. Phytophthora pseudocryptogea was identified as the causal agent of the decline of C. revoluta, as it was re-isolated from both the roots and stems of artificially infected symptomatic plants, thus fulfilling Koch's postulates.

Characterization of Colletotrichum ocimi Population Associated with Black Spot of Sweet Basil (Ocimum basilicum) in Northern Italy.

Black spot is a major foliar disease of sweet basil (Ocimum basilicum) present in a typical cultivation area of northern Italy, including the Liguria and southern Piedmont regions, where this aromatic herb is an economically important crop. In this study, 15 Colletotrichum isolates obtained from sweet basil plants with symptoms of black spot sampled in this area were characterized morphologically and by nuclear DNA analysis using internal transcribed spacers (ITS) and intervening 5.8S nrDNA as well as part of the ß-tubulin gene (TUB2) regions as barcode markers. Analysis revealed all but one isolate belonged to the recently described species C. ocimi of the C. destructivum species complex. Only one isolate was identified as C. destructivum sensu stricto (s.s.). In pathogenicity tests on sweet basil, both C. ocimi and C. destructivum s.s. isolates incited typical symptoms of black spot, showing that although C. ocimi prevails in this basil production area, it is not the sole causal agent of black spot in northern Italy. While no other hosts of C. ocimi are known worldwide, the close related species C. destructivum has a broad host range, suggesting a speciation process of C. ocimi within this species complex driven by adaptation to the host.

Evaluation of High-Resolution Melting for Rapid Differentiation of Phytophthora Hybrids and Their Parental Species.

Phytophthora species hybrids have been repeatedly reported as causing damaging diseases to cultivated and wild plants. Two known hybrids, P. andina and P. × pelgrandis, are pathogens of Solanaceae and ornamentals, respectively, although the extent of their host ranges are unknown. P. andina emerged from hybridization of P. infestans and an unidentified related species, whereas P. × pelgrandis emerged from P. nicotianae and P. cactorum. Considering that hybrids and parental species can coexist in the same regions and to distinguish them usually requires cloning or whole genome sequencing, we aimed to develop a rapid tool to distinguish them. Specifically, we used high-resolution melting (HRM) assays to differentiate genotypes based on their amplicon melting profiles. We designed primers for P. × pelgrandis and parental species based on available sequences of P. nicotianae and P. cactorum nuclear genes containing polymorphisms between species. For P. andina, heterozygous sites from Illumina short reads were used for the same purpose. We identified multiple amplicons exhibiting differences in melting curves between parental species and hybrids. We propose HRM as a rapid method for differentiation of P. andina and P. × pelgrandis hybrids from parental species that could be employed to advance research on these pathogens.

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.

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.

Cadmium induces cadmium-tolerant gene expression in the filamentous fungus Trichoderma harzianum.

The filamentous fungus Trichoderma harzianum, strain IMI 393899, was able to grow in the presence of the heavy metals cadmium and mercury. The main objective of this research was to study the molecular mechanisms underlying the tolerance of the fungus T. harzianum to cadmium. The suppression subtractive hybridization (SSH) method was used for the characterization of the genes of T. harzianum implicated in cadmium tolerance compared with those expressed in the response to the stress induced by mercury. Finally, the effects of cadmium exposure were also validated by measuring the expression levels of the putative genes coding for a glucose transporter, a plasma membrane ATPase, a Cd(2+)/Zn(2+) transporter protein and a two-component system sensor histidine kinase YcbA, by real-time-PCR. By using the aforementioned SSH strategy, it was possible to identify 108 differentially expressed genes of the strain IMI 393899 of T. harzianum grown in a mineral substrate with the addition of cadmium. The expressed sequence tags identified by SSH technique were encoding different genes that may be involved in different biological processes, including those associated to primary and secondary metabolism, intracellular transport, transcription factors, cell defence, signal transduction, DNA metabolism, cell growth and protein synthesis. Finally, the results show that in the mechanism of tolerance to cadmium a possible signal transduction pathway could activate a Cd(2+)/Zn(2+) transporter protein and/or a plasma membrane ATPase that could be involved in the compartmentalization of cadmium inside the cell.

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 ß-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.

Gene silencing and gene expression in phytopathogenic fungi using a plant virus vector.

RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.

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.

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 ß-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.

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.

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.

Expression of genes of Trichoderma harzianum in response to the presence of cadmium in the substrate.

Trichoderma harzianum is a very important fungus for the reduction of the amount of heavy metals resulting from agricultural and industrial activities. This filamentous fungus has been shown to be tolerant towards several heavy metals (e.g. Cd, Pb, Zn, Ni and Mn), but the mechanism underlying this tolerance is not entirely understood. In this study, we confirmed the ability of T. harzianum to grow in the presence of cadmium and observed a significant stimulation of its growth in the presence of mercury. A molecular approach to investigate the cadmium tolerance mechanisms was carried out by the application of the suppression subtractive hybridisation (SSH) technique. To this end, we have used a particular strategy to discriminate cadmium-induced differentially expressed genes from those generally expressed upon heavy metal treatment. Several genes (109) were found to be overexpressed in the presence of cadmium, confirming the dramatic metabolic modification underlying the metal stress response of the fungus.

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.