Characterization and evaluation of <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> strain WF02 regarding its biocontrol activities and genetic responses against bacterial wilt in two different resistant tomato cultivars

Bacillus amyloliquefaciens strain WF02, isolated from soil collected at Wufeng Mountain, Taiwan, has siderophore-producing ability and in vitro antagonistic activity against bacterial wilt pathogen. To determine the impact of plant genotype on biocontrol effectiveness, we treated soil with this strain before infecting susceptible (L390) and moderately resistant (Micro-Tom) tomato cultivars with Ralstonia solanacearum strain Pss4. We also compared the efficacy of this strain with that of commercial Bacillus subtilis strain Y1336. Strain WF02 provided longer lasting protection against R. solanacearum than did strain Y1336 and controlled the development of wilt in both cultivars. To elucidate the genetic responses in these plants under WF02 treatment, we analyzed the temporal expression of defense-related genes in leaves. The salicylic acid pathway-related genes phenylalanine ammonia-lyase and pathogenesis-related protein 1a were up-regulated in both cultivars, whereas expression of the jasmonic acid pathway-related gene lipoxygenase was only elevated in the susceptible tomato cultivar (L390). These results suggest that WF02 can provide protection against bacterial wilt in tomato cultivars with different levels of disease resistance via direct and indirect modes of action.     

Central Role of Salicylic Acid in Resistance of Wheat Against <Emphasis Type="Italic">Fusarium graminearum</Emphasis>

Head blight caused by Fusarium graminearum (F. graminearum) is one of the major threats to wheat and barley around the world. The importance of this disease is due to a reduction in both grain yield and quality in infected plants. Currently, there is limited knowledge about the physiological mechanisms involved in plant resistance against this pathogen. To reveal the physiological mechanisms underlying the resistance to F. graminearum, spikes of resistant (Sumai3) and susceptible (Falat) wheat cultivars were analyzed 4 days after inoculation, as the first symptoms of pathogen infection appeared. F. graminearum inoculation resulted in a greater induction level and activity of salicylic acid (SA), callose, phenolic compounds, peroxidase, phenylalanine ammonia lyase (PAL), and polyphenol oxidase in resistant versus susceptible cultivars. Soil drench application to spikes of SA, 24 h before inoculation with F. graminearum alleviated Fusarium head blight symptoms in both resistant and susceptible cultivars. SA treated plants showed a significant increment in hydrogen peroxide (H₂O₂) production, lipid peroxidation, SA, and callose content. SA-induced H₂O₂ level seems to be related to increased superoxide dismutase and decreased catalase activities. In addition, real-time quantitative PCR analysis showed that SA pretreatment induced expression of PAL genes in both infected and non-infected head tissues of the susceptible and resistant cultivars. Our data showed that soil drench application of SA activates antioxidant defense responses and may subsequently induce systemic acquired resistance, which may contribute to the resistance against F. graminearum. These results provide novel insights about the physiological and molecular role of SA in plant resistance against hemi-biotrophic pathogen infection.     

Differential fungal colonization and physiological defense responses of new olive cultivars infected by the necrotrophic fungus <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

New olive cultivars adapted to Tunisia’s growing conditions were examined for their resistance to verticillium wilt (VWO) to determine whether differences in susceptibility among currently grown cultivars might contribute to the management of this disease. Based on the evaluation of 14 cultivars, 10 were classified as susceptible or extremely susceptible (Chetoui, Chemlali, Rkhami, Jarboui, Zalmati, Jarboui, Oueslati, Manzanille, Picholine and Frangivento), 2 as moderately susceptible (Koroneiki and Coratina), and 2 as resistant (Meski and Sayali) to VWO. Three cultivars with different susceptibility levels were selected to examine the levels of hydrogen peroxide (H₂O₂), soluble sugars (SS), soluble proteins (SP), total polyphenols (TP), lipid peroxidation, activities of antioxidant enzymes, and fungal biomass in planta. V. dahliae DNA occurred early in the roots at 15 dpi and reached a maximum of 3.507 and 2.52 ng/100 ng of plant DNA, respectively, in the extremely susceptible and resistant cultivars. Fungal DNA in the stems occurred at 30 dpi and increased slowly to reach a maximum of 0.23 ng/100 ng of total DNA in the extremely susceptible cultivars. We showed that the amount of fungal DNA in planta was roughly correlated with the susceptibility to VWO (P < 0.0001; r = 0.95). The comparison of cultivars at the physiological level indicated that olive resistance is roughly correlated with the antioxidant enzymes activity, H₂O₂ concentration, and TP and SP contents. The results of this study open new perspectives for olive genetic improvement programs aiming at developing new cultivars resistant to this wilt.     

Biochemical and molecular studies on the resistance mechanisms in tea [<Emphasis Type="Italic">Camellia sinensis</Emphasis> (L.) O. Kuntze] against blister blight disease

Tea (Camellia sinensis) plantations are exposed to biotic and abiotic stresses. Among the biotic factors, blister blight (BB), caused by Exobasidium vexans, affects the quality and quantity of the product and demands high fungicide application. A long term solution for disease resistance would require the knowledge of the basic molecular and biochemical changes occurring in plant as an attempt to resist the pathogen and limit the spread of the disease which can further help in developing resistant cultivars using biotechnological tools. Thus, gene expression studies using the cDNA based suppressive subtractive hybridization library, characterization of genes for pathogenesis related (PR) proteins [chitinase (CsCHIT), glucanase (CsGLUC), phenylalanine ammonia lyase (CsPAL)] and genes in flavonoid pathway were accessed in the BB resistant and susceptible cultivars, SA6 and TES34, respectively. Further, biochemical analysis of PR and antioxidant enzymes (POX, APX, SOD) involved in BB resistance have been carried out to investigate the potential molecular and biochemical changes. Various stages of pathogen development had varied impact on PR protein, flavonoid pathway and anti-oxidative enzymes and indicates the possible role of reactive oxygen species, lignins, flavonoids, anthocyanins and other synthesized compounds in acting as antimicrobial/antifungal agents in tea cultivars.     

Assay development and marker validation for marker assisted selection of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">niveum</Emphasis> race 1 in watermelon

Fusarium wilt, Fusarium oxysporum f. sp. niveum (FON), of watermelon (Citrullus lanatus) is a fungal pathogen that causes significant yield losses in the US watermelon industry. FON damages watermelon through invasion of the root system and remains a difficult pathogen to manage due to its long-lasting survival spores which persist in the soil. Chemical control options for this pathogen are lacking, making development of genetic resistance the best option. There are four known races of FON (0, 1, 2, and 3) which are distinguished based on their pathogenicity of differential cultivars. Most modern cultivar releases have FON race 1 (FON-1) resistance, which has been mapped on the end of chromosome 1. Application of marker assisted selection (MAS) would improve the efficiency of FON-1 resistance breeding. In order to identify markers for selection in the FON-1 region, the QTL-seq method was utilized on an F₂ population segregating for FON-1 resistance. Single nucleotide polymorphism (SNP) markers in the region were developed into Kompetitive allele-specific PCR (KASP?) assays and tested for trait association on the segregating F_2:3 population. Marker validation was done using an F₂ population from a cross between FON-1 susceptible “New Hampshire Midget” and FON-1-resistant “Calhoun Gray.” Further validation on a panel of susceptible and resistant cultivars and Plant Introductions identified SNP marker UGA1_502161 as a useful marker for selection of FON-1 resistance from Calhoun Gray.     

Identification of a molecular marker tightly linked to bacterial wilt resistance in tomato by genome-wide SNP analysis

Key message

Genotyping of disease resistance to bacterial wilt in tomato by a genome-wide SNP analysis

Abstract

Bacterial wilt caused by Ralstonia pseudosolanacearum is one of the destructive diseases in tomato. The previous studies have identified Bwr-6 (chromosome 6) and Bwr-12 (chromosome 12) loci as the major quantitative trait loci (QTLs) contributing to resistance against bacterial wilt in tomato cultivar ‘Hawaii7996’. However, the genetic identities of two QTLs have not been uncovered yet. In this study, using whole-genome resequencing, we analyzed genome-wide single-nucleotide polymorphisms (SNPs) that can distinguish a resistant group, including seven tomato varieties resistant to bacterial wilt, from a susceptible group, including two susceptible to the same disease. In total, 5259 non-synonymous SNPs were found between the two groups. Among them, only 265 SNPs were located in the coding DNA sequences, and the majority of these SNPs were located on chromosomes 6 and 12. The genes that both carry SNP(s) and are near Bwr-6 and Bwr-12 were selected. In particular, four genes in chromosome 12 encode putative leucine-rich repeat (LRR) receptor-like proteins. SNPs within these four genes were used to develop SNP markers, and each SNP marker was validated by a high-resolution melting method. Consequently, one SNP marker, including a functional SNP in a gene, Solyc12g009690.1, could efficiently distinguish tomato varieties resistant to bacterial wilt from susceptible varieties. These results indicate that Solyc12g009690.1, the gene encoding a putative LRR receptor-like protein, might be tightly linked to Bwr-12, and the SNP marker developed in this study will be useful for selection of tomato cultivars resistant to bacterial wilt.

     

Genome-Wide Analysis of Genes Induced by <Emphasis Type="Italic">Fusarium graminearum</Emphasis> Infection in Resistant and Susceptible Wheat Cultivars

Fusarium head blight (FHB) caused by Fusarium graminearum is one of the most serious diseases in wheat (Triticum aestivum) and barley (Hordeum vulgare). Dahongmil is an elite Korean wheat cultivar with relatively high resistance to FHB. To identify differentially expressed genes in the resistant cultivar Dahongmil and the susceptible cultivar Urimil after inoculation of F. graminearum, we used the Affymetrix GeneChip® Wheat Genome Array to identify 328 ESTs that were differentially expressed in inoculated seedling tissues of the two cultivars. From these, we selected 16 induced genes and found that they have defense functions, such as genes encoding pathogen resistance proteins, oxidative stress-related proteins, metabolism, and proteins involved in defense mechanisms. To verify the DNA microarray results, we tested seven of these genes by semiquantitative RT-PCR and confirmed that these defense- and stress-related genes were expressed at much higher levels in the resistant Dahongmil cultivar. We next developed a hypothetical functional gene network and identified 89 interaction pairs mediated by four of the differentially expressed genes in the hypothetical network. We further refined the network by identifying nine genes showing significant up- or down-regulation after FHB challenge in the resistant cultivar and two genes having multiple interactions with queried proteins. We hope that the set of induced genes identified in this study can be used for development of new wheat and barley cultivars with improved resistance to FHB.     

Allele-specific CAPS marker in a <Emphasis Type="Italic">Ve1</Emphasis> homolog of <Emphasis Type="Italic">Capsicum annuum</Emphasis> for improved selection of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> resistance

Verticillium wilt (Verticillium dahliae) is an economically important disease for many high-value crops. The pathogen is difficult to manage due to the long viability of its resting structures, wide host range, and the inability of fungicides to affect the pathogen once in the plant vascular system. In chile pepper (Capsicum annuum), breeding for resistance to Verticillium wilt is especially challenging due to the limited resistance sources. The dominant Ve locus in tomato (Solanum lycopersicum) contains two closely linked and inversely oriented genes, Ve1 and Ve2. Homologs of Ve1 have been characterized in diverse plant species, and interfamily transfer of Ve1 confers race-specific resistance. Queries in the chile pepper WGS database in NCBI with Ve1 and Ve2 sequences identified one open reading frame (ORF) with homology to the tomato Ve genes. Comparison of the candidate CaVe (Capsicum annuum Ve) gene sequences from susceptible and resistant accessions revealed 16 single nucleotide polymorphisms (SNPs) and several haplotypes. A homozygous haplotype was identified for the susceptible accessions and for resistant accessions. We developed a cleaved amplified polymorphic sequence (CAPS) molecular marker within the coding region of CaVe and screened diverse germplasm that has been previously reported as being resistant to Verticillium wilt in other regions. Based on our phenotyping using the New Mexico V. dahliae isolate, the marker could select resistance accessions with 48% accuracy. This molecular marker is a promising tool towards marker-assisted selection for Verticillium wilt resistance and has the potential to improve the efficacy of chile pepper breeding programs, but does not eliminate the need for a bioassay. Furthermore, this work provides a basis for future research in this important pathosystem.     

Development of a CAPS marker for the verticillium wilt resistance in tomatoes

The nucleotide sequences of the Verticillium wilt resistance locus of resistant and susceptible tomato genotypes were cloned and analyzed. The nucleotide sequences displaying high degree of homology to the earlier cloned Ve1 and Ve2 genes were detected in the tomato forms susceptible to Verticillium wilt. The polymorphism at this locus between the resistant and susceptible plants was detected and used to elaborate a highly efficient CAPS marker.     

QTL mapping for bacterial wilt resistance in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Bacterial wilt (BW) caused by Ralstonia solanacearum is a serious, global, disease of peanut (Arachis hypogaea L.), but it is especially destructive in China. Identification of DNA markers linked to the resistance to this disease will help peanut breeders efficiently develop resistant cultivars through molecular breeding. A F₂ population, from a cross between disease-resistant and disease-susceptible cultivars, was used to detect quantitative trait loci (QTL) associated with the resistance to this disease in the cultivated peanut. Genome-wide SNPs were identified from restriction-site-associated DNA sequencing tags using next-generation DNA sequencing technology. SNPs linked to disease resistance were determined in two bulks of 30 resistant and 30 susceptible plants along with two parental plants using bulk segregant analysis. Polymorphic SSR and SNP markers were utilized for construction of a linkage map and for performing the QTL analysis, and a moderately dense linkage map was constructed in the F₂ population. Two QTL (qBW-1 and qBW-2) detected for resistance to BW disease were located in the linkage groups LG1 and LG10 and account for 21 and 12 % of the bacterial wilt phenotypic variance. To confirm these QTL, the F₈ RIL population with 223 plants was utilized for genotyping and phenotyping plants by year and location as compared to the F₂ population. The QTL qBW-1 was consistent in the location of LG1 in the F₈ population though the QTL qBW-2 could not be clarified due to fewer markers used and mapped in LG10. The QTL qBW-1, including four linked SNP markers and one SSR marker within 14.4-cM interval in the F₈, was closely related to a disease resistance gene homolog and was considered as a candidate gene for resistance to BW. QTL identified in this study would be useful to conduct marker-assisted selection and may permit cloning of resistance genes. Our study shows that bulk segregant analysis of genome-wide SNPs is a useful approach to expedite the identification of genetic markers linked to disease resistance traits in the allotetraploidy species peanut.     

Over-expression of the <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> harpin-encoding gene <Emphasis Type="Italic">hrpZm</Emphasis> confers enhanced tolerance to Phytophthora root and stem rot in transgenic soybean

Phytophthora root and stem rot (PRR) caused by Phytophthora sojae is one of the most devastating diseases reducing soybean (Glycine max) production all over the world. Harpin proteins in many plant pathogenic bacteria were confirmed to enhance disease and insect resistance in crop plants. Here, a harpin protein-encoding gene hrpZpsta from the P. syringae pv. tabaci strain Psta218 was codon-optimized (renamed hrpZm) and introduced into soybean cultivars Williams 82 and Shennong 9 by Agrobacterium-mediated transformation. Three independent transgenic lines over-expressing hrpZm were obtained and exhibited stable and enhanced tolerance to P. sojae infection in T₂–T₄ generations compared to the non-transformed (NT) and empty vector (EV)-transformed plants. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of salicylic acid-dependent genes PR1, PR12, and PAL, jasmonic acid-dependent gene PPO, and hypersensitive response (HR)-related genes GmNPR1 and RAR was significantly up-regulated after P. sojae inoculation. Moreover, the activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO), peroxidase, and superoxide dismutase also increased significantly in the transgenic lines compared to the NT and EV-transformed plants after inoculation. Our results suggest that over-expression of the hrpZm gene significantly enhances PRR tolerance in soybean by eliciting resistance responses mediated by multiple defense signaling pathways, thus providing an alternative approach for development of soybean varieties with improved tolerance against the soil-borne pathogen PRR.     

MiR395 Overexpression Increases Eggplant Sensibility to <Emphasis Type="Italic">Verticillium dahliae</Emphasis> Infection

Verticillium wilt (V. wilt), a notorious wilt disease caused by Verticillium dahliae, often leads to the reduction of eggplant (Solanum melongena L.) production. MiRNAs, as a class of small RNAs, can regulate gene expression and then affect growth and development in plants. MiR395 has been proven to respond to sulfate-deficient stress in Arabidopsis thaliana and sulfate is well known to have a close relationship with plant disease resistance. To explore the function of eggplant miR395, we examined its expression in V. dahliae-infected eggplant by qRT-PCR and found miR395 exhibited a gradual reduction trend with time after infection. We then expressed pre-miR395 from Arabidopsis thaliana in Suqi eggplant and resistance analysis showed that miR395 overexpressed plants were hypersensitive to V. dahliae infection. We further measured the content of GSH and activities of POD and SOD and the results indicated that the index of GSH/POD/SOD in the overexpressed plants was lower than that of the wild-type control under V. dahliae infection. These results suggest that miR395 plays a negative role in eggplant response to V. dahliae infection.     

A Graft Mimic Strategy for Verticillium Resistance in Tomato

Grafting vegetables for disease resistance has increased greatly in popularity over the past 10 years. Verticillium wilt of tomato is commonly controlled through grafting of commercial varieties on resistant rootstocks expressing the Ve1 R-gene. To mimic the grafted plant, proteomic analyses in tomato were used to identify a suitable root-specific promoter (TMVi), which was used to express the Ve1-allele in susceptible Craigella (Cs) tomato plants. The results indicate that when infected with Verticillim dahliae, race 1, the transformed plants are comparable to resistant cultivars (Cr) or grafted plants.     

Selected reactive oxygen species and antioxidant enzymes in common bean after <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">phaseolicola</Emphasis> and <Emphasis Type="Italic">Botrytis cinerea</Emphasis> infection

Phaseolus vulgaris cv. Korona plants were inoculated with the bacteria Pseudomonas syringae pv. phaseolicola (Psp), necrotrophic fungus Botrytis cinerea (Bc) or with both pathogens sequentially. The aim of the experiment was to determine how plants cope with multiple infection with pathogens having different attack strategy. Possible suppression of the non-specific infection with the necrotrophic fungus Bc by earlier Psp inoculation was examined. Concentration of reactive oxygen species (ROS), such as superoxide anion (O₂ ^?) and H₂O₂ and activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were determined 6, 12, 24 and 48 h after inoculation. The measurements were done for ROS cytosolic fraction and enzymatic cytosolic or apoplastic fraction. Infection with Psp caused significant increase in ROS levels since the beginning of experiment. Activity of the apoplastic enzymes also increased remarkably at the beginning of experiment in contrast to the cytosolic ones. Cytosolic SOD and guaiacol peroxidase (GPOD) activities achieved the maximum values 48 h after treatment. Additional forms of the examined enzymes after specific Psp infection were identified; however, they were not present after single Bc inoculation. Subsequent Bc infection resulted only in changes of H₂O₂ and SOD that occurred to be especially important during plant–pathogen interaction. Cultivar Korona of common bean is considered to be resistant to Psp and mobilises its system upon infection with these bacteria. We put forward a hypothesis that the extent of defence reaction was so great that subsequent infection did not trigger significant additional response.     

<Emphasis Type="Italic">STAYGREEN</Emphasis> (<Emphasis Type="Italic">CsSGR</Emphasis>) is a candidate for the anthracnose (<Emphasis Type="Italic">Colletotrichum orbiculare</Emphasis>) resistance locus <Emphasis Type="Italic">cla</Emphasis> in Gy14 cucumber

Key message

Map-based cloning identified a candidate gene for resistance to the anthracnose fungal pathogen Colletotrichum orbiculare in cucumber, which reveals a novel function for the highly conserved STAYGREEN family genes for host disease resistance in plants.

Abstract

Colletotrichum orbiculare is a hemibiotrophic fungal pathogen that causes anthracnose disease in cucumber and other cucurbit crops. No host resistance genes against the anthracnose pathogens have been cloned in crop plants. Here, we reported fine mapping and cloning of a resistance gene to the race 1 anthracnose pathogen in cucumber inbred lines Gy14 and WI 2757. Phenotypic and QTL analysis in multiple populations revealed that a single recessive gene, cla, was underlying anthracnose resistance in both lines, but WI2757 carried an additional minor-effect QTL. Fine mapping using 150 Gy14?×?9930 recombinant inbred lines and 1043 F₂ individuals delimited the cla locus into a 32 kb region in cucumber Chromosome 5 with three predicted genes. Multiple lines of evidence suggested that the cucumber STAYGREEN (CsSGR) gene is a candidate for the anthracnose resistance locus. A single nucleotide mutation in the third exon of CsSGR resulted in the substitution of Glutamine in 9930 to Arginine in Gy14 in CsSGR protein which seems responsible for the differential anthracnose inoculation responses between Gy14 and 9930. Quantitative real-time PCR analysis indicated that CsSGR was significantly upregulated upon anthracnose pathogen inoculation in the susceptible 9930, while its expression was much lower in the resistant Gy14. Investigation of allelic diversities in natural cucumber populations revealed that the resistance allele in almost all improved cultivars or breeding lines of the U.S. origin was derived from PI 197087. This work reveals an unknown function for the highly conserved STAYGREEN (SGR) family genes for host disease resistance in plants.