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.     

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.     

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.     

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.     

<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.     

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.     

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.     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> against <Emphasis Type="Italic">Galleria mellonella</Emphasis>

The greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) is occasionally found in beehives and is a major pest of stored wax. Entomopathogenic fungi have recently received attention as possible biocontrol elements for certain insect pests. In this study, 90 isolates of Beauveria bassiana and 15 isolates of Metarhizium anisopliae were screened for proteases and lipases production. The results showed significant variations in the enzymatic action between the isolates. In the bioassay, the selected isolates evinced high virulence against the 4th instar of the G. mellonella larvae. The isolates BbaAUMC3076, BbaAUMC3263 and ManAUMC3085 realized 100% mortality at concentrations of 5.5 × 10⁶ conidia ml⁻¹, 5.86 × 10⁵ conidia ml⁻¹, and 4.8 × 10⁶ conidia ml⁻¹, respectively. Strong enzymatic activities in vitro did not necessarily indicate high virulence against the tested insect pest. The cuticle of the infected larvae became dark and black-spotted, indicating direct attack of fungus on the defense system of the insects. The LC₅₀ values were 1.43 × 10³, 1.04 × 10⁵ and 5.06 × 10⁴ for Bba3263AUMC, Bba3076AUMC and Man3085AUMC, respectively, and their slopes were determined by computerized probit analysis program as 0.738 ± 0.008, 0.635 ± 0.007 and 1.120 ± 0.024, respectively.     

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.     

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.     

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.     

Outcome of sexual reproduction in the <Emphasis Type="Italic">Phytophthora infestans</Emphasis> population in Estonian potato fields

In this study, the Estonian population of Phytophthora infestans was characterized with mating type, sensitivity to metalaxyl, virulence on 11 potato R-gene differentials and 12 SSR markers to show the outcome of potential sexual reproduction in the population. During the three years 2010–2012, 141 P. infestans isolates, collected from 23 potato fields, showed quite a high and stable frequency of the A2 mating type, 48% of the total population. In 87% of all sampled potato fields, both mating types were recorded, suggesting continuous sexual reproduction of P. infestans and possible oospore production. Metalaxyl-sensitive isolates prevailed in all three years (68 out of 99 isolates). Amongst the 95 isolates tested, 51 virulence races were found. The race structure was diverse, and most pathotypes were unique, appearing only once; the two most common pathotypes, 1.2.3.4.6.7.10.11 and 1.2.3.4.7.10.11, comprised 35% of the population. The P. infestans population was genetically highly diverse and most of the multilocus genotypes (MLGs) appeared only once. Furthermore, all of the MLGs appeared in only one of the three sampling years. Our results confirm that the high diversity in the Estonian P. infestans population is most likely the result of frequent sexual reproduction, which benefits the survival, adaptability and diversity of the pathogen in the climate of North-Eastern Europe.     

Population studies on <Emphasis Type="Italic">Phytophthora infestans</Emphasis> on potatoes and tomatoes in southern Germany

Fifty-seven isolates of Phytophthora infestans from blighted potato foliage were collected in 1995 in southern Germany and analysed for mating type and sensitivity to metalaxyl. Fifty-six of them were characterised as A1 and one as A2 mating types. Resistance to metalaxyl was observed frequently: 53 isolates were resistant, three were partially sensitive, and one was sensitive. In a subsequent field study in 1999, 84 isolates collected from blighted potato and tomato foliage were analysed for mating type. Seventy-two were characterised as A1 and twelve as A2 mating types. The response of 76 isolates to metalaxyl and to propamocarb was tested. The majority (42) of the 76 isolates was classified as resistant to metalaxyl; 31 were partially sensitive and only three isolates were sensitive. The results with propamocarb were less discrete; 10 isolates were classified as resistant and three were clearly sensitive. AFLP fingerprinting was used to examine the genetic structure of the southern German P. infestans population collected in 1999 and indicated that the tested population can be sub-divided into a tomato group, a potato group and a mixed group containing isolates collected from both crops. The presence of Ia and IIa mitochondrial DNA haplotypes indicates that the German P. infestans isolates belong to the new pathogen population that has also been reported in neighbouring regions of Europe. The present study indicates that at the beginning of the season only a few genotypes were present, and the population became genetically more variable at the end of the growing season.     

Nonhost resistance of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> against <Emphasis Type="Italic">Colletotrichum</Emphasis> species

Arabidopsis thaliana exhibits a durable resistance called nonhost resistance against nonadapted fungal pathogens. A. thaliana activates preinvasive resistance and terminates entry attempts by nonadapted fungi belonging to the genus Colletotrichum, which cause anthracnose disease in many plants. In the interaction between A. thaliana and nonadapted C. tropicale, the preinvasive resistance involves the PENETRATION 2-related antifungal secondary metabolite pathway and the ENHANCED DISEASE RESISTANCE 1-dependent antifungal peptide pathway. The development of invasive hyphae by C. tropicale owing to the reduction of preinvasive resistance then triggers the blockage of further hyphal expansion via the activation of the second layer of resistance, i.e., postinvasive resistance, which guarantees the robustness of the nonhost resistance of A. thaliana against Colletotrichum pathogens. Both the tryptophan-derived metabolic pathway and glutathione synthesis play critical roles in the postinvasive resistance against C. tropicale, although the molecular mechanism of postinvasive resistance remains to be elucidated. In this review, we describe the current understanding of the molecular background of the Arabidopsis nonhost resistance against Colletotrichum fungi and discuss perspectives for future research on this durable resistance.     

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.     

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.     

First report of blueberry red ringspot disease caused by <Emphasis Type="Italic">Blueberry</Emphasis><Emphasis Type="Italic">red</Emphasis><Emphasis Type="Italic">ringspot</Emphasis><Emphasis Type="Italic">virus</Emphasis> in Japan

Virus-like symptoms—red ringspots on stems and leaves, circular blotches or pale spots on fruit—were found on commercial highbush blueberry (Vaccinium corymbosum) cultivars Blueray, Weymouth, Duke and Sierra in Japan. In PCR testing, single DNA fragments were amplified from total nucleic acid samples of the diseased blueberry bushes using primers specific to Blueberry red ringspot virus (BRRV). Sequencing analysis of the amplified products revealed 95.7–97.7% nucleotide sequence identity with the BRRV genome. This paper is the first report of blueberry red ringspot disease caused by BRRV in Japan. The nucleotide sequence data reported in this paper are available in the GenBank/EMBL/DDBJ database as accessions AB469884 to AB469893 for BRRV isolates from Japan.     

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.