Phytophthora alticola and P. boodjera associated with decline of young Eucalyptus smithii trees in Uruguay

Eucalyptus production mainly destined to cellulose pulp production has expanded strongly in the last 30 years in Uruguay. Eucalyptus smithii has recently emerged as a promising species for cellulose pulp production. However, an average of 40% of young trees die during the first and second summer of post-planting. In this study, 32 Phytophthora isolates were obtained from 132 E. smithii young trees with root and collar rot symptoms, confirming the association of Phytophthora to E. smithii decline. Based on phylogenetic analysis of ITS, TUB2, cox1 and HSP90 gene regions and phenotypical characteristics, two species belonging to the genera Phytophthora clade 4 were identified, P. alticola (96%) and P. boodjera (4%). Tested isolates of both species significantly reduced both shoot and root dry weights of inoculated E. smithii seedlings compared to control plants. To our best knowledge, this is the first time that P. alticola and P. boodjera are recovered from young symptomatic E. smithii trees in commercial plantations as well as the first time these species are found in the Americas.     

Effect of light and dark on the growth and development of downy mildew pathogen Hyaloperonospora arabidopsidis

Disease development in plants requires a susceptible host, a virulent pathogen, and a favourable environment. Oomycete pathogens cause many important diseases and have evolved sophisticated molecular mechanisms to manipulate their hosts. Day length has been shown to impact plant–oomycete interactions but a need exists for a tractable reference system to understand the mechanistic interplay between light regulation, oomycete pathogen virulence, and plant host immunity. Here we present data demonstrating that light is a critical factor in the interaction between Arabidopsis thaliana and its naturally occurring downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). We investigated the role of light on spore germination, mycelium development, sporulation, and oospore formation of Hpa, along with defence responses in the host. We observed abundant Hpa sporulation on compatible Arabidopsis under day lengths ranging from 10 to 14 hr. In contrast, exposure to constant light or constant dark suppressed sporulation. Exposure to constant dark suppressed spore germination, mycelial development, and oospore formation, whereas exposure to constant light stimulated these three stages of development. A biomarker of plant immune system activation was induced under both constant light and constant dark. Altogether, these findings demonstrate that Hpa has the molecular mechanisms to perceive and respond to light and that both the host and pathogen responses are influenced by the light regime. Therefore, this pathosystem can be used for investigations to understand the molecular mechanisms through which oomycete pathogens like Hpa perceive and integrate light signals, and how light influences pathogen virulence and host immunity during their interactions.     

Response of accessions within tomato wild species,Solanum pimpinellifolium to late blight

Late blight (LB), caused by the oomycete Phytophthora infestans, is one of the most devastating diseases of the cultivated tomato (Solanum lycopersicum) worldwide. Most commercial cultivars of tomato are susceptible to LB. Previously, three major LB resistance genes (Ph‐1, Ph‐2, Ph‐3) were identified and incorporated into a few commercial cultivars of tomato. Reduced effectiveness and potential breakdown of the resistance genes has necessitated identification, characterization and utilization of new sources of resistance. We evaluated the response of 67 accessions of the wild tomato species, S. pimpinellifolium to LB, under multiple field and greenhouse (GH) conditions and compared them with six control genotypes. Sixteen accessions were identified with strong LB resistance in both field and GH experiments. However, 12 accessions exhibited resistance similar to a control line which was homozygous for Ph‐2 + Ph‐3. Genotyping accessions with molecular markers for Ph‐2 and Ph‐3 were not conclusive, indicating that resistance in these accessions could be due to these or other resistance genes. Strong correlations were observed between field and GH disease response and between foliar and stem infection.     

Characterization of Phytophthora cinnamomi from common walnut in Southern Europe environment

Forty‐eight isolates of Phytophthora cinnamomi obtained from common walnut were analysed according to their variability in growth at different temperatures, virulence, sensitivity to metalaxyl and in genomic DNA. Isolates were obtained from commercial common walnut orchards located in northern Italy and in southern France. Inter‐simple sequence repeat (ISSR) were analysed for the 49 isolates, 43 of which were Italian, 6 French; an isolate of the same species obtained from Viburnum spp. was used as an outgroup. ANOVA on phenotypic characters showed a significant impact of the geographic location of the orchard on isolate variability in terms of reaction to temperatures and aggressiveness. In turn, clustering obtained with UPGMA analysis on genetic data was almost exclusively dependant on isolate variability, nevertheless the 48 isolates seem to share a common variability that differentiates the group from the isolate from Viburnum spp. Correlation between phenotypic and genetic traits was not statistically significant. In conclusion, phenotypic variability like virulence seemed to be conditioned from geographic origin while the genetic variability of P. cinnamomi isolates from walnut was associated to the single genotype.     

Specific and Sensitive Detection of Phytophthora nicotianae By Simple and Nested-PCR

Phytophthora nicotianae Breda de Haan is one of the most important soil-borne plant pathogens. The identification of this pathogen based on morphological or physiological characters is time-consuming and labour-intensive and requires comprehensive knowledge of fungi. Molecular analysis of the internal transcribed spacer (ITS) regions of rDNA is a novel and very effective method of species determination. Based on this concept, conventional and single closed tube nested-PCRs were developed for the specific and sensitive detection of P. nicotianae. Two new specific primers, designed from the spacer regions ITS1 and ITS2, internal to the nucleotide sequence flanked by universal primers ITS4 and ITS6, were used. To evaluate the specificity of the method, 36 morphologically characterized isolates were tested. A positive reaction, characterized by an amplification product of 737 bp, was shown by all P. nicotianae isolates and two P. nicotianae/cactorum hybrids. No amplification product was observed when other Phytophthora species and genera were assayed. The sensitivity of this method was analysed by serial dilutions of a defined amount of fungal DNA in a healthy root extract. Nested-PCR was at least 1000 times more sensitive than conventional PCR. In addition, samples from different infection sites, origins and crops, samples from nutrient solution, water and the rockwool used in hydroponic cultures, were analysed to validate this method.     

Phytophthora diversity and the population structure of Phytophthora ramorum in Swiss ornamental nurseries

Invasive oomycete pathogens have been causing significant damage to native ecosystems worldwide for over a century. A recent well‐known example is Phytophthora ramorum, the causal agent of sudden oak death, which emerged in the 1990s in Europe and North America. In Europe, this pathogen is mainly restricted to woody ornamentals in nurseries and public greens, while severe outbreaks in the wild have only been reported in the UK. This study presents the results of the P. ramorum survey conducted in Swiss nurseries between 2003 and 2011. In all 120 nurseries subjected to the plant passport system, the main P. ramorum hosts were visually checked for above ground infections. Phytophthora species were isolated from tissue showing symptoms and identified on the basis of the morphological features of the cultures and sequencing of the ribosomal ITS region. Phytophthora was detected on 125 plants (66 Viburnum, 58 Rhododendron and one Pieris). Phytophthora ramorum was the most frequent species (59·2% of the plants), followed by P. plurivora, P. cactorum, P. citrophthora, P. cinnamomi, P. cactorum/P. hedraiandra, P. multivora and P. taxon PgChlamydo. The highest incidence of P. ramorum was observed on Viburnum × bodnantense. Microsatellite genotyping showed that the Swiss P. ramorum population is highly clonal and consists of seven genotypes (five previously reported in Europe, two new), all belonging to the European EU1 clonal lineage. It can therefore be assumed that P. ramorum entered Switzerland through nursery trade. Despite sanitation measures, repeated P. ramorum infections have been recorded in seven nurseries, suggesting either reintroduction or unsuccessful eradication efforts.     

Characterization of accessions and species of Macadamia to stem infection by Phytophthora cinnamomi

Phytophthora cinnamomi is a major pathogen in most macadamia plantations worldwide. Due to stem lesions, stem cankers and leaf defoliation, it results in loss of productivity and tree death. This study examined accessions of the four Macadamia species and their hybrids, produced via rooted stem cuttings or germinated seeds, for susceptibility to stem canker and necrotic lesions caused by P. cinnamomi. Plants were wound‐inoculated with agar containing P. cinnamomi. The symptoms produced in inoculated plants were used to characterize host susceptibility variation within and among the population. Lesion length and severity of stem canker were recorded. The four species and hybrids differed significantly in stem canker severity (P < 0.001) and lesion length (P = 0.04). Macadamia integrifolia and M. tetraphylla hybrids were the most susceptible. Macadamia integrifolia had the greatest stem canker severity and the most extensive lesions above and below the site of inoculation. Restricted lesion sizes were observed in M. ternifolia and M.  jansenii. The effects of basal stem diameter and the method of propagation either from cuttings or from seed were not significant. The genetic variation in the reaction of macadamia accessions to stem infection by P. cinnamomi is discussed.     

Molecular detection of Phytophthora capsici in infected plant tissues, soil and water

A species-specific PCR assay was developed for rapid and accurate detection of the pathogenic oomycete Phytophthora capsici in diseased plant tissues, soil and artificially infested irrigation water. Based on differences in internal transcribed spacer (ITS) sequences of Phytophthora spp. and other oomycetes, one pair of species-specific primers, PC-1/PC-2, was synthesized. After screening 15 isolates of P. capsici and 77 isolates from the Ascomycota, Basidiomycota, Deuteromycota and Oomycota, the PC-1/PC-2 primers amplified only a single PCR band of c . 560 bp from P. capsici . The detection sensitivity with primers PC-1/PC-2 was 1 pg genomic DNA (equivalent to half the genomic DNA of a single zoospore) per 25- µ L PCR reaction volume; traditional PCR could detect P. capsici in naturally infected plant tissues, diseased field soil and artificially inoculated irrigation water. Using ITS1/ITS4 as the first-round primers and PC-1/PC-2 in the second round, nested PCR procedures were developed, increasing detection sensitivity to 1 fg per 25- µ L reaction volume. The results suggested that the assay detected the pathogen more rapidly and accurately than standard isolation methods. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring, as well as guiding plant disease management.     

Disease complexes involving plant parasitic nematodes and soilborne pathogens

This review discusses the mechanisms underlying synergistic interactions between phytophagous nematodes and soilborne pathogens, and identifies biotic and abiotic factors affecting these interactions. Approaches towards the resolution and management of nematode–pathogen complexes are considered and discussed.     

Specific detection of Phytophthora nicotianae using the polymerase chain reaction and primers based on the DNA sequence of its elictin gene ParA1

Six primers based on the sequence of the flanking and coding regions of the elicitin gene ParA1 of Phytophthora nicotianae were tested for specific detection of the fungus by the polymerase chain reaction (PCR). One combination, IL7/IL8, with IL7 in a flanking region and IL8 in a coding region of the gene, gave an intense 378 bp signal with a diverse collection of isolates of P. nicotianae, that included some from black shank disease of tobacco and others from a variety of hosts. The sequence of the amplification product obtained with an isolate that produces elicitin and one that does not, was homologous with the known sequence of the ParA1 gene. The same primer combination gave no signal with sixteen other Phytophthora species tested except for two isolates P. palmivora with which it gave a weak 800 bp signal. It gave no signal with DNA from healthy tobacco and tomato plants but P. nicotianae was detected in inoculated tobacco and tomato plants. Small numbers of zoospores (>100) trapped onto a nitrocellulose membrane after filtration from suspension were also detected after two successive rounds of PCR.