Comparison of Chinese and Japanese A1 isolates of <Emphasis Type="Italic">Phytophthora infestans</Emphasis>

The mating type, glucose-6-phosphate isomerase (Gpi) and peptidase (Pep) genotypes, RG57 fingerprint, and mitochondrial DNA (mtDNA) haplotype of Chinese isolates of Phytophthora infestans collected in Hebei and Gansu in 1996 were compared with those of Japanese isolates collected during 1997–2000. The Chinese isolates were divided into four genotypes, one of which was identical to the dominant Japanese genotype, A1-A (mating type A1; Gpi 100/100; Pep 100/100; RG57 100010001100110100011001110: 1–25, 14a, and 24a; and mtDNA haplotype IIa). Comparison of the genotypes with reported data revealed that some completely and partially identical genotypes occur in Russia and parts of Europe. The other two A1 genotypes and one A2 genotype were also detected in Gansu (Gpi 100/100, Pep 100/100, and mtDNA haplotype Ia), which were regarded as unique to this region.     

Inheritance of resistance in <Emphasis Type="Italic">Solanum nigrum</Emphasis> to <Emphasis Type="Italic">Phytophthora infestans</Emphasis>

Solanum nigrum, black nightshade, is a wild non-tuber bearing hexaploid species with a high level of resistance to Phytophthora infestans (Colon et al. 1993), the causal agent of potato late blight, the most devastating disease in potato production. However, the genetic mode of resistance in S. nigrum is still poorly understood. In the present study, two S. nigrum accessions, 984750019 (N19) and #13, resistant (R) and susceptible (S), respectively, to three different isolates of P. infestans, were sexually crossed. The various kinds of progeny including F1, F2, F3, and backcross populations (BC₁; F1 × S), as well as two populations produced by self-pollinating the R parent and S parent, were each screened for susceptibility to P. infestans isolate MP 324 using detached leaf assays. Fifty seedling plant individuals of the F1 progeny were each resistant to this specific isolate, similarly to the seedling plants resulting from self-pollination of the resistant R parent. Thirty seedling plants obtained from self-pollination of the S parent were susceptible. Among a total of 180 F2 plants, the segregation ratio between resistant and susceptible plants was approximately 3: 1. Among the 66 seedling plants of the BC₁ progeny originating from crossing an F1 plant with the susceptible S parent, there were 26 susceptible and 40 resistant plants to P. infestans. The segregation patterns obtained indicated monogenic dominant inheritance of resistance to P. infestans isolate MP 324 in S. nigrum acc. 984750019. This gene, conferring resistance to P. infestans, may be useful for the transformation of potato cultivars susceptible to late blight.     

Post-infection activity of fungicides against <Emphasis Type="Italic">Phytophthora infestans</Emphasis> on tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.)

Late blight caused by Phytophthora infestans is the most devastating disease of tomato (Solanum lycopersicum L.) and causes important economically losses if not properly controlled. Control is achieved mainly by preventive fungicide applications. However, even if curative applications are discouraged because they increase the risk of resistance development in the target pathogens, in practice fungicides may be applied also when the disease is already present, a situation that commonly occurs in the field. The aim of this work was to study the curative activity of several fungicides toward P. infestans to determine their efficacy when applied after the infection process. Nine trials were performed in greenhouse using potted tomato plants that were treated 24 h after inoculation. Disease severity was assessed three times from the development of the symptoms on the untreated plants and data analysed using a linear mixed model. Differences in post-infection control between the different chemical classes were found. Metalaxyl-M and cymoxanil showed the best curative activity while among the CAA fungicides, a good efficacy was expressed by dimethomorph. Interestingly, evidence of synergy between active ingredients having different modes of action was observed such as in the mixtures containing dimethomorph?+?ametoctradin, dimethomorph?+?pyraclostrobin and fosetyl-Al?+?propamocarb. This study provided useful information on the post-infection activity of some fungicides used to control tomato late blight and should be taken into account to perform more in depth studies at the field level and to improve the management strategies of the disease.     

Potato <Emphasis Type="Italic">R1</Emphasis> resistance gene confers resistance against <Emphasis Type="Italic">Phytophthora infestans</Emphasis> in transgenic tomato plants

Tomato is challenged by several pathogens which cause loss of production. One such pathogen is the oomycete Phytophthora infestans which is able to attack all the aerial parts of the plant. Although a wide range of resistance sources are available, genetic control of this disease is not yet successful. Pyramiding R-genes through genetic transformation could be a straightforward way to produce tomato and potato lines carrying durable resistance to P. infestans. In this work the R1 potato gene was transferred into tomato lines. The tomato transgenic lines were analyzed by using q-RT-PCR and progeny segregation to determine the gene copy number. To test the hypothesis that R1 represents a specifically regulated R-gene, transgenic tomato plants were inoculated with P. infestans isolate 88133 and IPO. All the plants containing the R1 gene were resistant to the late blight isolate IPO-0 and susceptible to isolate 88133. These results provide evidence for specific activation of the R1 gene during pathogen challenge. Furthermore, evidence for enhancement of PR-1 gene expression during P. infestans resistance response was obtained.     

BABA effects on the behaviour of potato cultivars infected by <Emphasis Type="Italic">Phytophthora infestans</Emphasis> and <Emphasis Type="Italic">Fusarium solani</Emphasis>

Since most plants possess resistance mechanisms which can be induced upon pre-treatment with a variety of chemical compounds, the use of β-aminobutyric acid (BABA) as a defence inducer without reported toxic effect on the environment was studied. The aim of this work was to analyse the effectiveness of BABA to induce resistance against Phytophthora infestans and Fusarium solani in potato cultivars differing in their level of resistance to late blight. The behaviour of some components of biochemical mechanisms by which BABA increases resistance against P. infestans, as well as the effect of BABA on the activity of a potential pathogenic factor of F. solani, were studied. Plants with four applications of BABA throughout the crop cycle produced tubers more resistant to P. infestans and F. solani than non-treated plants. In addition, tuber slices from treated plants, inoculated with P. infestans, showed an increase in phenol and phytoalexin content. The aspartyl protease StAP1 accumulation was also higher in tubers obtained from treated plants and inoculated with P. infestans. This result was observed only in the more resistant potato cv. Pampeana, early after infection. In the potato–F. solani interaction, infected tubers coming from BABA-treated plants showed minor fungal proteolytic activity than infected, non-treated ones. For potato cvs Pampeana and Bintje, the BABA treatment improved the yield of harvested tubers. The number of tubers per plant and total weight of harvested tubers was greater for those obtained from treated plants with two early or four applications of BABA. The results show that the BABA treatment increases the resistance of potatoes but the degree of increase depends on the original level of resistance present in each cultivar.     

GC-MS metabolite profiling of <Emphasis Type="Italic">Phytophthora infestans</Emphasis> resistant to metalaxyl

Phytophthora infestans is the most important potato pathogen worldwide. Various alternatives have been used to control the pathogen, including continuous applications of phenylamide fungicides which has caused a rapid development of resistance in populations of P. infestans. Despite the importance of the disease, metabolite profiling of fungicide-resistant P. infestans has not been reported. In vitro resistance of Phytophthora infestans isolates to metalaxyl was characterized and metabolic changes in resistant isolates were evaluated at low (0.5 mg/L) and high (100 mg/L) concentrations of the fungicide. About 70% of the isolates tested showed resistance to metalaxyl and a total of 49 metabolites were differently expressed in resistant isolates growing in the presence of the fungicide. Principal components analysis revealed a distinct metabolite profile of resistant isolates exposed to both low and high levels of metalaxyl. The main metabolites responsible for the clustering in both fungicide concentrations included fatty acids such as hexadecanoic and octadecanoic acids, sugars such as glucose and fructose, aminoacids such as proline and valine, and organic acids such as butanedioic and propanoic acids. Potential resistance-related metabolic pathways are mostly involved in the regulation of the pathogen’s membrane fluidity and included the fatty acid biosynthesis as well as the glycerophospholipid metabolism pathways. This is the first metabolomic-based characterization of fungicide resistance in plant pathogens.     

Phytophthora blight of southern star (<Emphasis Type="Italic">Oxypetalum caeruleum</Emphasis>) caused by <Emphasis Type="Italic">Phytophthora palmivora</Emphasis> in Japan

In 2004, a damping-off symptom was found on southern star, Oxypetalum caeruleum, in Kochi Prefecture, Japan. Two Phytophthora strains with different colony patterns on potato dextrose agar were isolated, and their pathogenicity was confirmed by inoculation of southern star plants and their reisolation from symptomatic plants. Both fungi were identified as Phytophthora palmivora based on morphology, physiology, and sequence analysis of the internal transcribed spacers of nuclear ribosomal DNA. This is the first report of Phytophthora blight of southern star in the world.     

<Emphasis Type="Italic">RB</Emphasis> and <Emphasis Type="Italic">Ph</Emphasis> resistance genes in potato and tomato minimize risk for oospore production in the presence of mating pairs of <Emphasis Type="Italic">Phytophthora infestans</Emphasis>

Composed mostly of fungivorous species, the genus Aphelenchoides also comprises 14 plant-parasitic species. The most common and devastating, A. besseyi, A. fragariae, A. ritzemabosi and A. subtenuis have been reported on more than 900 plant species. The combination of low inter-specific and high intra-specific morphological variability makes morphology-based identification extremely difficult within this genus, and has led to molecular tools being employed to ensure accurate diagnoses. rDNA markers are widely used for the identification of nematodes while the Cytochrome Oxidase I gene (COI) remains relatively unexplored despite its role as the standard barcode for almost all animal groups. To explore its suitability as a diagnostic tool, we studied a fragment of the mtCOI region of the four main plant-parasitic Aphelenchoides within a phylogenetic framework. We generated 69 mtCOI and 123 rDNA sequences of diverse Aphelenchoides taxa; 67 belong to the main plant-parasitic species including the first mtCOI sequence of A. fragariae and the first mtCOI and 28S sequences of A. subtenuis. mtCOI had a similar success rate for PCR amplification. Phylogenetic trees based on the three studied markers are largely in agreement with one another, validating their use for Aphelenchoides diagnosis; additionally, we were able to locate several misidentified sequences of plant-parasitic Aphelenchoides in existing databases. The concatenated analysis from the three markers resulted in a more robust insight into the phylogeny and evolution of Aphelenchoides, revealing that plant-parasitism has evolved independently at least three times within this genus, presumably from fungal-feeding ancestors.     

Effects of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis>) juice containing allicin on <Emphasis Type="Italic">Phytophthora infestans</Emphasis> and downy mildew of cucumber caused by <Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis>

The volatile antimicrobial substance allicin (diallylthiosulphinate) is produced in garlic when the tissues are damaged and the substrate allicin (S-allyl-l-cysteine sulphoxide) mixes with the enzyme alliin-lyase (E.C.4.4.1.4). Allicin undergoes thiol-disulphide exchange reactions with free thiol groups in proteins and it is thought that this is the basis of its antimicrobial action. At 50 μg ml^-1, allicin in garlic juice inhibited the germination of sporangia and cysts and subsequent germ tube growth by Phytophthora infestans both in vitro and in vivo on the leaf surface. Disease severity in P. infestans-infected tomato seedlings was also reduced by spraying leaves with garlic juice containing allicin over the range tested (55–110 μg ml⁻¹) with an effectiveness ranging from approximately 45–100%. Similarly, in growth room experiments at concentrations from 50–1,000 μg ml⁻¹, allicin in garlic juice reduced the severity of cucumber downy mildew caused by Pseudoperonospora cubensis by approximately 50–100%. These results suggest a potential for developing preparations from garlic for use in specialised aspects of organic farming, e.g. for reducing pathogen inoculum potential and perhaps for use under glass in horticulture.     

Races of <Emphasis Type="Italic">Phytophthora infestans</Emphasis> isolated from potato in Hokkaido,Japan

The virulence of 29 isolates of Phytophthora infestans collected in potato fields in Hokkaido, Japan, in 2013 and 2014, was tested for race identification. Thirteen different races were identified, each of which had five to eight virulence factors. All of the isolates caused a virulent reaction against plants with R1 and R7, and most of the isolates caused a virulent reaction against plants with R3, R4, R10, and R11. On the other hand, no isolate was virulent against plants with R9. These results demonstrate that the current Japanese P. infestans population is more complex than the population in the 1990s from the viewpoint of race.     

Reduced efficacy of fluazinam against <Emphasis FontCategory="SansSerif" Type="Italic">Phytophthora infestans</Emphasis> in the Netherlands

Phytophthora infestans is the causal organism of potato late blight, the most important disease in potato, the second most important arable crop in Europe. The P. infestans population in Europe is well known for its sudden changes in composition. Currently it is composed of a wide variety of genotypes, some of which are dominant clonal lines while others are rare or even unique to a year or location. Fungicides play a crucial role in the integrated control of late blight. Since its introduction in the Netherlands in 1992, fluazinam has been used in late blight control strategies in ware and starch potatoes. It has a broad spectrum of activity and is effective against a range of diseases including potato late blight. Fluazinam interrupts the pathogen cell’s energy production process by an uncoupling effect on oxidative phosphorylation. It is considered to have a low resistance risk. Until recently, reduced efficacy against fluazinam was not detected in P. infestans surveys in Europe. In this paper we present the finding of a new clonal lineage (EU_33_A2) of P. infestans in the Netherlands and the reduced efficacy of fluazinam to control one of the EU_33_A2 isolates in field experiments carried out in 2011 and 2015 under high disease pressure. The potential effects of this finding on practical late blight control strategies are discussed.     

The structure of <Emphasis Type="Italic">Phytophthora infestans</Emphasis> populations from organic and conventional crops

The characteristics of populations of Phytophthora infestans from organic farms, small conventional farms and large conventional farms were determined from isolates collected in northern Estonia in 2004 and 2005. For the population as a whole 41% were A2; all virulence factors to the 11 R genes from Solanum demissum were found; and more than 70% had high or intermediate resistance to metalaxyl. Isolates from organic farms tended to have more complex pathotypes than isolates from either large or small conventional farms, but there was a higher proportion of metalaxyl resistant isolates from large conventional farms than from small conventional farms or from organic farms.     

Host Specialization not Detected Among Isolates of the EC-1 Lineage of <Emphasis Type="Italic">Phytophthora infestans</Emphasis> Attacking Wild and Cultivated Potatoes in Peru

To determine whether populations of Phytophthora infestans attacking wild and cultivated potatoes in the highlands of Peru are specialized on their hosts of origin, we characterized isolates using several neutral markers, metalaxyl resistance and for aggressiveness in a detached leaf assay. One hundred and fifty-three isolates were collected from the northern and central highlands of Peru from different potato cultivars (both modern and native cultivars) and from different species of wild, tuber-bearing potatoes. All the isolates analyzed belonged to one of four clonal lineages that had been described previously in Peru: EC-1, US-1, PE-3 and PE-7. The EC-1 lineage (n = 133) was dominant and present in similar frequencies on wild and cultivated potatoes. PE-3 (n = 14) was found primarily on cultivated potatoes, with only one isolate coming from a wild host. US-1 (n = 2) and PE-7 (n = 4) were rare; all but one (PE-7) occurred on wild potatoes. Isolates from the EC-1 lineage from modern cultivars were compared in three separate detached leaf inoculation assays with EC-1 isolates from the wild potato species S. sogarandinum, S. bill-hookerii or S. huancabambense, respectively. No significant interactions between isolate type (from wild or cultivated potato) and host type (wild or cultivated) were measured for any assay. It appears that the pathogen genotypes in the EC-1 lineage indiscriminately attack both wild and cultivated tuber-bearing solanaceous hosts in Peru, and breeders should be able to select for resistance using the common EC-1 lineage.     

Nitric Oxide Treatment and Induced Genes Role Against <Emphasis Type="Italic">Phytophthora infestans</Emphasis> in Potato

Effects of sodium nitroprusside (SNP; nitric oxide donor) treatments on enhancement of secondary metabolites production, oxidative stress mediators (\(\mathrm{O}_{2}^{-}\)) accumulation and antioxidant defense enzymes of Potato Spunta Sp. suspension culture cells elicited by a fungal extract from phytophthora infestans mycelium. The obtained data confirmed the significant increase in various oxidative burst (super oxide anion, hydrogen peroxide and total glutathione) contents. The administration of various NO concentrations strongly decreased hydrogen peroxide concentration and superoxide anion levels. Moreover, the SNP treatments regulate elicitor-induced activation of phenylalanine ammonium-lyase and total soluble phenols accumulation. The highest concentrations of NO donor sodium nitroprusside potentiated elicitor-induced H₂O₂ production. On the other hand, the lowest H₂O₂ contents coincided with elicitation regulated various activities of enzymes superoxide-dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and Phenyl alanine ammonia lyase (PAL) activity, the decrease in H₂O₂ concentration was probably due to a direct reduction interaction of NO-H₂O₂. On the other hand, the addition of these previous NO treatments affects mRNA peroxidase gene expression using RT-PCR techniques. In general, the addition of lower concentrations of nitric oxide reduce the mRNA peroxidase activity on contrary, the higher concentrations induced the mRNA peroxidase activity, which induce the hypersensitive reactions against fungus infection.     

Characterization of <Emphasis Type="Italic">Phytophthora clandestina</Emphasis> races on <Emphasis Type="Italic">Trifolium subterraneum</Emphasis> in Western Australia

Phytophthora clandestina is a causal agent of root rot disease of subterranean clover in Western Australia (W.A). As a significant number of isolates of P. clandestina from W.A. could not previously be designated using existing differentials, a comprehensive set of subterranean clover (Trifolium subterraneum) cultivars was used as differentials to delineate a broader range of races of the pathogen. One hundred and one isolates of the pathogen collected from W.A. were screened on nine subterranean clover cultivars, of which seven were found to be useful as host differentials. A total of 10 races (in contrast to the five recognized previously) were defined and differentiated using octal nomenclature, presenting a clearer picture of the racial distribution of P. clandestina among W.A. isolates. Differences were found in the race populations between Australian states and are therefore important to the selection/breeding of cultivars for specific regions of Australia to counter the predominant race populations and for enforcing quarantine measures in relation to seed movements within and outside Australia. The octal nomenclature used provides a sound basis for follow-up studies and future race designations. Races 173 and 177 in this study were widely distributed and were the most common races in W.A., and together constitute 80% of the isolates characterized. While six of the seven host differentials were resistant to isolates belonging to race 001 and all were resistant to 000, it is of concern that only one differential was resistant to 157 and 173 and that none of the host differentials were resistant to 177. Our approach to P. clandestina race delineation is clearly conservative and is different from previous studies. The octal nomenclature we applied in this study is not only scientifically sound but also will facilitate rapid recognition and characterization of the races.     

Characterization of Egyptian <Emphasis Type="Italic">Phytophthora infestans</Emphasis> population using simple sequence repeat markers

The plant pathogenic oomycete, Phytophthora infestans, is the causal agent of late blight disease in tomato and potato. For characterizing Egyptian P. infestans isolates by DNA marker analysis, 40 isolates of P. infestans were collected from different locations in Egypt during two growing seasons (2012/2013 and 2013/2014). The 40 isolates were grouped into seven genotypes, in which 24 alleles were detected. The identified genotypes were not completely associated with geographic location and sample collection years. These results provide genetic and geographical information for developing a program to manage late blight disease.     

Distribution of <Emphasis Type="Italic">Dickeya</Emphasis> spp. and <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> in naturally infected seed potatoes

Detailed studies were conducted on the distribution of Pectobacterium carotovorum subsp. carotovorum and Dickeya spp. in two potato seed lots of different cultivars harvested from blackleg-diseased crops. Composite samples of six different tuber sections (peel, stolon end, and peeled potato tissue 0.5, 1.0, 2.0 and 4.0 cm from the stolon end) were analysed by enrichment PCR, and CVP plating followed by colony PCR on the resulting cavity-forming bacteria. Seed lots were contaminated with Dickeya spp. and P. carotovorum subsp. carotovorum (Pcc), but not with P. atrosepticum. Dickeya spp. and Pcc were found at high concentrations in the stolon ends, whereas relatively low densities were found in the peel and in deeper located potato tissue. Rep-PCR, 16S rDNA sequence analysis and biochemical assays, grouped all the Dickeya spp. isolates from the two potato seed lots as biovar 3. The implications of the results for the control of Pectobacterium and Dickeya spp., and sampling strategies in relation to seed testing, are discussed.     

Mechanical transmission of <Emphasis Type="Italic">Potato spindle tuber viroid</Emphasis> between plants of <Emphasis Type="Italic">Brugmansia suaveoles,Solanum jasminoides</Emphasis> and potatoes and tomatoes

Potato spindle tuber viroid (PSTVd) has been recently found in many solanaceous ornamental plant species. This study reports on the effectiveness of mechanical transmission between Brugmansia suaveolens, Solanum jasminoides, potato and tomato. Inoculation with ‘infected’ plant sap diluted in water, rubbing with contaminated finger tips and cutting with contaminated razor blades all resulted in transmission of PSTVd. Temperature, plant species and source of inoculum were found to be critical factors. An average temperature of 15°C only resulted in a few infections, whereas transmission at 20 and 25°C was more successful. Tomatoes were more susceptible to PSTVd than B. suaveolens, S. jasminoides and potatoes. Furthermore, S. jasminoides was a better source of inoculum than B. suaveolens. No transmission was obtained after repeated addition of inocula to tomato roots. These results indicate that PSTVd can be transmitted between plant species in practice by crop handling.     

<Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">brasiliensis</Emphasis> causing blackleg on potatoes in South Africa

In South Africa during the 2006/2007 potato growing season, outbreaks of blackleg occurred, causing severe economic losses in commercial potato production fields. Symptoms were initially observed on only one stem per plant, on which the top leaves rolled upwards, wilted and became necrotic. As symptoms progressed to the lower leaves with subsequent leaf desiccation, a light to dark brown discolouration of the vascular system at the stem base developed, followed by external darkening. Under prevailing wet and humid conditions stems became slimy and pale. In the stems, the pith became necrotic and hollow. These symptoms were similar to those described in Brazil, where the causal agent was identified as a new subspecies, Pectobacterium carotovorum subsp. brasiliensis (Pbcb). Isolations from plants showing typical blackleg symptoms were made on CVP medium. Sequences and phylogenetic analysis of the partial 16S–23S rDNA intergenic spacer region indicated that the isolates were Pbcb. Comparison of PCR-RFLP patterns of the 16S–23S rDNA of isolates to reference cultures confirmed the identity of the South African blackleg strains as Pbcb, identical to strain 8 isolated in Brazil. This is the first report of Pbcb in South Africa and it appears to be the most important causal agent of blackleg in South Africa. The disease poses a major potential threat to the South African potato industry especially in terms of seed exports, tuber quality and yield.