<Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> upregulates marker genes of the salicylic acid and ethylene signaling pathways but not those of the jasmonic acid pathway in leaflets of <Emphasis Type="Italic">Solanum</Emphasis> lines during early stage of infection

Real-Time PCR assay was used to quantify the expression of marker genes of the salicylic acid, jasmonic acid and ethylene signaling pathways in seven Solanum lines after inoculation with a Ralstonia solanacearum phylotype I strain, R008. Four Solanum lycopersicum lines (CRA 66, Hawaii 7996, MST 32/1, Quatre carrées), one S. tuberosum line (Spunta), the wild Lycopersicon cerasiforme and Solanum commersonii were used for this investigation. Results revealed very little activation of the jasmonic acid pathway marker genes, lipoxygenase A (LoxA) and protease inhibitor II (Pin2), with no significant difference (p > 0.05) in fold change expression among the Solanum lines. In contrast the salicylic acid pathway marker genes, glucanase A (GluA) and PR-1a, and the ethylene pathway marker genes, osmotin-like (Osm) and PR-1b, were expressed at higher levels with a statistically significant difference (p < 0.05) in fold change expression among the Solanum lines. The resistant lines L. cerasiforme, CRA 66, Hawaii 7996 and S. commersonii showed stronger activation of the salicylic acid and ethylene marker genes than the moderately resistant cultivar (MST 32/1) and the susceptible lines (Quatre carrées and Spunta). The marker genes reached their highest expression levels earlier (4 h.p.i) in the resistant and moderately resistant lines than in the susceptible lines (48 h.p.i.). These results indicate that salicylic acid and ethylene signaling pathways have a significant role in defense against R. solanacearum. The timing and magnitude of the upregulation of gene expression may determine the plant ability to put up a defense response against the pathogen.     

Alteration of gene expression profile in the roots of wild diploid Arachis duranensis inoculated with Ralstonia solanacearum

Bacterial wilt caused by Ralstonia solanacearum is a serious disease of peanut (Arachis hypogaea) in China. However, the molecular basis of peanut resistance to R. solanacearum is poorly understood. Arachis duranensis, a wild diploid species of the genus Arachis, has been proven to be resistant to bacterial wilt, and thus holds valuable potential for understanding the mechanism of resistance to bacterial wilt and genetic improvement of peanut disease resistance. Here, suppression subtractive hybridization (SSH) and macroarray hybridization were employed to detect differentially expressed genes (DEGs) in the roots of A. duranensis after R. solanacearum inoculation. A total of 317 unique genes were obtained, 265 of which had homologues and functional annotations. KEGG analysis revealed that a large proportion of these unigenes are mainly involved in the biosynthesis of phytoalexins, particularly in the biosynthetic pathways of terpenoids and flavonoids. Subsequent real‐time polymerase chain reaction (PCR) analysis showed that the terpenoid and flavonoid synthesis‐related genes showed higher expression levels in a resistant genotype of A. duranensis than in a susceptible genotype, indicating that the terpenoids and flavonoids probably played a fundamental role in the resistance of A. duranensis to R. solanacearum. This study provides an overview of the gene expression profile in the roots of wild Arachis species in response to R. solanacearum infection. Moreover, the related candidate genes are also valuable for the further study of the molecular mechanisms of resistance to R. solanacearum.     

Expression of biotic stress response genes to Phytophthora infestans inoculation in White Lady,a potato cultivar with race-specific resistance to late blight

Expression changes of biotic stress response genes were analyzed during a 65 h period post inoculation with Phytophthora infestans in potato cultivar White Lady that possesses race-specific resistance to this pathogen. All analyzed respiratory burst oxidase homologs, the PR proteins, the serine-, cysteine- and aspartic protease inhibitors, as well as the Rpi-bt1 gene homolog were up-regulated in the biotrophic phase. The R1 and R2 gene homologs showed up-regulation only at 65 hpi, and interestingly, the R3a gene showed only a very slight expressional increase. It is concluded, that beside the constitutively expressed R genes a number of non-specific stress response genes contribute to the successful resistance response in race-specific defense.     

Identification of differentially expressed genes in tolerant and susceptible potato cultivars in response to Spongospora subterranea f. sp. subterranea tuber infection

Powdery scab caused by Spongospora subterranea f. sp. subterranea (Sss) has recently become one of the most devastating potato diseases of economic importance in South Africa. The use of resistant cultivars has long been considered the most effective and sustainable strategy to manage the pathogen. However, little is known about the molecular mechanisms underlying resistance of potato tubers to Sss. Using RNA-sequencing (RNA-seq), 2058 differentially expressed genes (DEGs) were identified from two potato cultivars (tolerant and susceptible) in response to Sss infection. Analysis of the expression patterns of 10 selected defence-response genes was carried out at two different stages of tuber growth using RT-qPCR to validate the RNA-seq data. Several defence-related genes showed contrasting expression patterns between the tolerant and susceptible cultivars, including marker genes involved in the salicylic acid hormonal response pathway (StMRNA, StUDP and StWRKY6). Induction of six defence-related genes (StWRKY6, StTOSB, StSN2, StLOX, StUDP and StSN1) persisted until harvest of the tubers, while three other genes (StNBS, StMRNA and StPRF) were highly up-regulated during the initial stages of disease development. The results of this preliminary study suggest that the tolerant potato cultivar employs quantitative resistance and salicylic acid pathway hormonal responses against tuber infection by Sss. The identified genes have the potential to be used in the development of molecular markers for selection of powdery scab resistant potato lines in marker-assisted breeding programmes.     

Analysis of proteins differentially accumulated during potato late blight resistance mediated by the RB resistance gene

The RB gene, cloned from the wild diploid potato species Solanum bulbocastanum, confers resistance against the late blight pathogen, Phytophthora infestans. We examined changes in the proteome of potato leaves in response to inoculation with P. infestans. A nearly isogenic system comprised of susceptible Solanum tuberosum cultivar ‘Katahdin’ and resistant transgenic potato (cv. ‘Katahdin’) carrying a single copy of RB was utilized for this study. Comparative proteomic analysis revealed the presence of only 12 protein spots with a significant difference (≥ twofold) in relative abundance in resistant and susceptible potato plants after inoculation with P. infestans. Five out of the 12 identified proteins have putative roles in photosynthesis and stress responses. Silencing of these genes in Nicotiana benthamiana had no impact on RB-mediated induction of the hypersensitive response (HR). However, we found that silencing of molecular chaperone HSP90 led to the inability of RB to induce the HR after recognition of the P. infestans effector IpiO in planta.     

Multiple Selection of Potato Cyst Nematode Globodera pallida Virulence on a Range of Potato Species. I. Serial Selection on Solanum-hybrids

A series of selection experiments on potato cyst nematode Globodera pallida, pathotype Pa1, tested the virulence response of the nematode to a range of resistant potato Solanum genotypes. Alleles conferring virulence against all four Solanum sources used in the study (i.e. Solanum vernei, S. multidissectum, S. sanctae-rosae and S. tuberosum ssp. andigena) were detected. Selection for multiple virulence against a combination of resistant sources resulted in the originally-selected virulence genes being retained or lost. The mechanism, or basis, of potato cyst nematode resistance differs between the Solanum species. The appropriate use of resistance cultivars produced from a range of Solanum-resistant species offers a management tool for controlling potato cyst nematode levels in infested land.     

<Emphasis Type="Italic">Trichoderma harzianum</Emphasis> elicits defence response genes in roots of potato plantlets challenged by <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Biological control of Rhizoctonia solani with Trichoderma harzianum has been demonstrated in several studies. However, none have reported the dynamics of expression of defence response genes. Here we investigated the expression of these genes in potato roots challenged by R. solani in the presence/absence of T. harzianum Rifai MUCL 29707. Analysis of gene expression revealed an induction of PR1 at 168 h post-inoculation (hpi) and PAL at 96 hpi in the plants inoculated with T. harzianum Rifai MUCL 29707, an induction of PR1, PR2 and PAL at 48 hpi in the plants inoculated with R. solani and an induction of Lox at 24 hpi and PR1, PR2, PAL and GST1 at 72 hpi in the plants inoculated with both organisms. These results suggest that in the presence of T. harzianum Rifai MUCL 29707, the expression of Lox and GST1 genes are primed in potato plantlets infected with R. solani at an early stage of infection. Mycothèque de l’Université catholique de Louvain of S. Cranenbrouck's affiliation is part of the Belgian Coordinated Collections of Micro-organisms (BCCM).     

Differentially expressed genes in dicamba‐resistant and dicamba‐susceptible biotypes

In an ongoing effort to investigate the mechanism of auxinic herbicide resistance in Kochia scoparia (kochia), polymerase chain reaction‐based cDNA suppression subtractive hybridization was used to identify genes that are differentially expressed between dicamba‐resistant (HRd) and dicamba‐susceptible (S1) kochia biotypes in response to herbicide treatment. Both the HRd and S1 adaptor‐ligated cDNAs were used in separate hybridizations in order to generate biotype‐specific clones. A total of 710 cDNAs, representing putative differentially expressed mRNAs, were isolated and subjected to further screening. The false‐positive cDNAs were removed by conducting two colony hybridizations and at least one Northern hybridization. Differential or biotype‐specific expression was confirmed for six clones each from the HRd and S1 plants. The S1‐related genes were constitutively expressed at higher levels than in the HRd plants, but none had significant sequence similarity to known genes. Among the HRd‐related genes, HRd‐88 had 42% amino acid sequence identity to a conserved domain within thiol peptidases, which might be involved in auxin‐regulated gene expression. The constitutively expressed and inducible (by the dicamba treatment) HRd‐39 had 40% identity and 60% similarity to a domain from the Fe(II)/α‐ketoglutarate‐dependent hydroxylase superfamily. The HRd‐39 gene product had the characteristics of an enzyme that is able to detoxify dicamba via oxidative hydroxylation and thus its overexpression might confer the dicamba resistance phenotype.     

Compost enhances plant resistance against the bacterial wilt pathogen Ralstonia solanacearum via up-regulation of ascorbate-glutathione redox cycle

The interactions between the pathogen Ralstonia solanacearum and potato Solanum tuberosum plants were studied to investigate the reactive oxygen species metabolic system and ascorbate (ASC)-glutathione (GSH) redox cycle in response to compost application. Single potato eyepieces were germinated and grown in pots containing sandy soil with or without compost at a rate of 7.5 g kg^?1 soil. Non-compost- and compost-treated plants (CTP) were inoculated with R. solanacearum 25 days after planting and then analyzed after 10 days, unless otherwise stated. The present results revealed that pathogen infection caused a remarkable decrease in plant growth related parameters and productivity and an increase in disease incidence. However, under these conditions compost had substantially improved plant growth and decreased disease incidence and bacterial population. R. solanacearum resulted in significant enhancement in the activities of NADPH oxidase, lipoxygenase, the production rate of superoxide and hydroxyl radicals, levels of hydrogen peroxide, membrane lipid peroxidation, and protein oxidation indicating the induction of oxidative stress in potato roots. However, the pathogen-mediated enhancement in indices of oxidative stress was considerably decreased by compost application, which enhanced the activities of ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) in infected potato plants, implying a better ROS-scavenging activity. Data also indicated that there were general increases in ASC and GSH content in infected compost treated plants, but non-compost treated ones significantly had lower levels of such redox metabolites. In addition, significantly higher ratios of ASC/DHA (dehydroascorbate) and GSH/GSSG (glutathione disulphide) were generally found in CTP than in non-compost treated-ones. The obtained results suggest that compost provides effective protection against the Ralstonia bacterial pathogen via up-regulation of the capacity of the ASC-GSH cycle and modulation of the cellular redox status, thereby eliminating ROS damage and sustaining membrane stability.     

Potato bacterial wilt in India caused by strains of phylotype I, II and IV of Ralstonia solanacearum

Bacterial wilt or brown rot is one of the most devastating diseases of potato caused by a bacterium Ralstonia solanacearum (Smith 1986) Yabuuchi et al. (Microbiol Immunol 39:897–904 1995). Traditionally, R. solanacearum is classified into five races (r) on the basis of differences in host range and six biovars (bvs) on the basis of biochemical properties. Recently using molecular methods, R.?solanacearum has been classified into phylotypes based on the intergenic transcribed sequence of the ribosomal RNA genes 16S and 23S and into sequevars based on the endoglucanase gene (egl) sequence. In the present study, 75 bacterial strains, isolated from wilt infected potatoes from various potato growing regions of India, were classified by traditional and molecular methods. The identity of all the strains was confirmed as R. solanacearum as expected single 280-bp fragment resulted in all the strains following PCR amplification using R. solanacearum specific universal primer pair 759/760. Biovar (bv) analysis, based on utilization of disaccharide sugars and hexose alcohols, categorised the 75 strains into bv2 (78.7 %), 2 T (5.3 %), 3 (5.3 %) and 4 (10.7 %). The phylotype specific multiplex PCR assigned 78.7 % strains to phylotype II, 16.0 % to phylotype I and 5.3 % to phylotype IV. Phylogenetic analysis of egl gene sequences clustered all fifty nine phylotype II (bv2) strains with reference strain IPO1609 (IIB-1), all four phylotype IV (bv2T) strains with reference strain MAFF301558 (IV-8), three phylotype I (bv3) strains with reference strain MAFF211479 (I-30) and all eight phylotype I (bv4) and one phylotype I (bv3) strain with reference strain CIP365 (I-45). The study concluded that the Indian potato strains of R. solanacearum belong to three out of four phylotypes namely: the Asian phylotype I, the American phylotype II, and the Indonesian phylotype IV. This is the first study to address the diversity of R. solanacearum from potato in India using phylotype and sequevar scheme. We also report here for the first time the occurrence of phylotype IV sequevar 8 (bv2T) strain of R. solanacearum causing potato bacterial wilt in mid hills of Meghalaya in India.     

Expression of defence‐related genes in stems and leaves of resistant and susceptible field pea (Pisum sativum) during infection by Phoma koolunga

The differential expression of 13 defence‐related genes during Phoma koolunga infection of stems and leaves of susceptible versus resistant field pea (Pisum sativum) was determined using qRT‐PCR. Expression, in terms of relative mRNA level ratios, of genes encoding ferredoxin NADP oxidoreductase, 6a‐hydroxymaackiain methyltransferase (hmm6), chalcone synthase (PSCHS3) and ascorbate peroxidase in leaves and stems differed during 6–72 hours post‐inoculation (hpi) and reflected known host resistance levels in leaves versus stems. In comparison to the susceptible genotype, at 24, 48 and 72 hpi, two genes, hmm6 (122.43‐, 206.99‐ and 32.25‐fold, respectively) and PSCHS3 (175.00‐, 250.13‐ and 216.24‐fold, respectively), were strongly up‐regulated in leaves of the resistant genotype, highlighting that resistance against P. koolunga in field pea is governed by the early synthesis of pisatin. At 24 hpi, leaves infected by P. koolunga showed clear differences in expression of target genes. For example, the gene encoding a precursor of the defensin ‘disease resistance response protein 39’ was substantially down‐regulated in leaves of both the susceptible and the resistant genotypes inoculated with P. koolunga. This contrasts with other studies on another pea black spot pathogen, Didymella pinodes, where this same gene is strongly up‐regulated in leaves of resistant and susceptible genotypes. The current study provides the first understanding of defence‐related genes involved in the resistance against P. koolunga, opening novel avenues to engineer new field pea cultivars with improved leaf and stem black spot disease resistance as the basis for developing more effective and sustainable management strategies.     

Identification of genes associated with phytoplasma resistance through suppressive subtraction hybridization in Chinese jujube

Jujube witches' broom (JWB) is a destructive disease for Chinese jujube caused by phytoplasma. A suppression subtractive hybridization library of resistant cultivar ‘Xingguang’ was constructed under phytoplasma stress to identify genes related to JWB resistance. 77 of 200 unique expressed sequence tags had significant sequence homologies and were classified into 10 functional groups. The most abundant group was disease/defense (20.8%), which was consistent with the phytoplasma stress. These differentially expressed genes provide the groundwork for addressing the plant–phytoplasma interaction. Meanwhile, the expression of five selected genes (TLP, PR10, HSP70, ERF, kinase-related protein) was confirmed to upregulate at different infection periods.     

Ralstonia solanacearum Biovar 2/Rasse 3 (Erreger der Schleimkrankheit der Kartoffel) in Oberflächengewässern und Solanum dulcamara – Ergebnisse eines mehrjährigen Monitorings in Bayern

The potato brown rot, caused by the bacterium Ralstonia solanacearum biovar 2/race 3 (potato race) was classified as quarantine pathogen by the European Union (EU) due to the risks it would pose for the cultivation of potatoes and tomatoes. Quarantine regulations stipulate control surveys and tests on potatoes used as seed, for food as well as industrial purposes and in surface water to check for contamination with R. solanacearum. Toward this end, the Institute for Plant Protection of the Bavarian State Research Center for Agriculture has been conducting an intensive survey for the presence of the potato brown rot pathogen in Bavarian rivers since 1997. An important component of this monitoring is the testing of potential weed hosts growing near river banks. Every year, from June to September, water- and plant samples have been collected from rivers in potato cultivation areas and near potato processing plants. Since the start of the survey, a total of 70 rivers has been tested. The presence of the pathogen was checked by immunofluorescence antibody staining (IFAS) and polymerase chain reaction (PCR). Positive IFAS or PCR tests were checked again by biotests and pathogenicity tests on eggplant (Solanum melongena). So far R. solanacearum could be found in nine Bavarian rivers. Concerning wild plants growing near the river banks the bacterium could be isolated only from roots of the bittersweet nightshade (Solanum dulcamara). However, from stinging nettle (Urtica dioica), nodding beggartick (Bidens cernua), black nightshade (Solanum nigrum), great yellowcress (Rorippa amphibia) and gipsywort (Lycopus europaeus) R. solanacearum could not be isolated. In contaminated rivers, with S. dulcamara growing on the river banks, R. solanacearum could be detected repetitively for several years. In 1997 it was found that rivers can be contaminated by the pathogen, if insufficiently treated sewage from potato processing plants is released into waterways. Potato cultivation is threatened by surface water only, if contaminated water is used to irrigate potato fields. Consequently, surface water should never be used for irrigation of potato fields or application of plant protection products.     

The role of jasmonic acid signalling in wheat (Triticum aestivum L.) powdery mildew resistance reaction

Jasmonic acid (JA) signalling plays an important role in plant resistance to pathogens. Previously, JA has been found to play a role in induced disease resistance to necrotrophic pathogens in various plant species, but current researches showed that JA also enhanced resistance to biotrophic pathogens. However, its role in wheat (Triticum aestivum L.) powdery mildew (Blumeria graminis f. sp. tritici, Bgt) resistance reaction is largely unknown. To settle this issue, several typical powdery mildew resistant and susceptible wheat varieties were employed. The sensitivity to exogenous methyl jasmonate (MeJA) to wheat powdery mildew resistance, the concentration fluctuation of endogenous JAs after Bgt inoculation, and the expression profiles of nine pathogenesis-related protein genes (PR genes) after MeJA and Bgt treatments were studied systematically. Exogenous MeJA significantly enhanced the powdery mildew resistance of the susceptible varieties. After inoculation with Bgt, endogenous JAs accumulated rapidly, reached the maxima at 2 to 5 h post-inoculation (hpi), then decreased rapidly, and the concentration was almost the same as that of un-inoculated control at 96 hpi. The expression levels of the nine PRs were measured by real time quantitative RT-PCR (qRT-PCR) at different time points after MeJA application or Bgt inoculation respectively. The MeJA and Bgt strongly activated PR1, PR2, PR3, PR4, PR5, PR9, PR10 and Ta-JA2, but almost didn’t affect Ta-GLP2a. The induced powdery mildew resistance was positively correlated with the activated PR genes. JA plays a positive role in defence against Bgt. JA is a signalling molecule in wheat powdery mildew resistance and future manipulation of this pathway may improve powdery mildew resistance in wheat breeding.