Genome-Wide Association Analysis of Resistance to Pseudoperonospora cubensis in Citron Watermelon.

Cultivated sweet watermelon (Citrullus lanatus) is an important vegetable crop for millions of people around the world. There are limited sources of resistance to economically important diseases within C. lanatus, whereas C. amarus has a reservoir of traits that can be exploited to improve C. lanatus for resistance to biotic and abiotic stresses. Cucurbit downy mildew (CDM), caused by Pseudoperonospora cubensis, is an emerging threat to watermelon production. We screened 122 C. amarus accessions for resistance to CDM over two tests (environments). The accessions were genotyped by whole-genome resequencing to generate 2,126,759 single nucleotide polymorphic (SNP) markers. A genome-wide association study was deployed to uncover marker-trait associations and identify candidate genes underlying resistance to CDM. Our results indicate the presence of wide phenotypic variability (1.1 to 57.8%) for leaf area infection, representing a 50.7-fold variation for CDM resistance across the C. amarus germplasm collection. Broad-sense heritability estimate was 0.55, implying the presence of moderate genetic effects for resistance to CDM. The peak SNP markers associated with resistance to P. cubensis were located on chromosomes Ca03, Ca05, Ca07, and Ca11. The significant SNP markers accounted for up to 30% of the phenotypic variation and were associated with promising candidate genes encoding leucine-rich repeat receptor-like protein kinase and the WRKY transcription factor. This information will be useful in understanding the genetic architecture of the P. cubensis-Citrullus spp. patho-system as well as development of resources for genomics-assisted breeding for resistance to CDM in watermelon.

Garlic Volatile Diallyl Disulfide Induced Cucumber Resistance to Downy Mildew.

Allicin compositions in garlic are used widely as fungicides in modern agriculture, in which diallyl disulfide (DADS) is a major compound. Downy mildew, caused by Pseudoperonospora cubensis (P. cubensis), is one of the most destructive diseases and causes severe yield losses in cucumbers. To explore the potential mechanism of DADS-induced cucumber resistance to downy mildew, cucumber seedlings were treated with DADS and then inoculated with P. cubensis at a 10-day interval. Symptom observation showed that DADS significantly induced cucumber resistance to downy mildew. Furthermore, both lignin and H2O2 were significantly increased by DADS treatment to responding P. cubensis infection. Simultaneously, the enzyme activities of peroxidase (POD) in DADS-treated seedlings were significantly promoted. Meanwhile, both the auxin (IAA) and salicylic acid (SA) contents were increased, and their related differentially expressed genes (DEGs) were up-regulated when treated with DADS. Transcriptome profiling showed that many DEGs were involved in the biological processes of defense responses, in which DEGs on the pathways of 'phenylpropanoid biosynthesis', 'phenylalanine metabolism', 'MAPK signaling', and 'plant hormone signal transduction' were significantly up-regulated in DADS-treated cucumbers uninoculated with the pathogen. Based on the results of several physiological indices and transcriptomes, a potential molecular mechanism of DADS-induced cucumber resistance to downy mildew was proposed and discussed. The results of this study might give new insight into the exploration of the induced resistance mechanism of cucumber to downy mildew and provide useful information for the subsequent mining of resistance genes in cucumber.

Fatty Acid Desaturases: Uncovering Their Involvement in Grapevine Defence against Downy Mildew.

Grapevine downy mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most severe and devastating diseases in viticulture. Unravelling the grapevine defence mechanisms is crucial to develop sustainable disease control measures. Here we provide new insights concerning fatty acid's (FA) desaturation, a fundamental process in lipid remodelling and signalling. Previously, we have provided evidence that lipid signalling is essential in the establishment of the incompatible interaction between grapevine and Plasmopara viticola. In the first hours after pathogen challenge, jasmonic acid (JA) accumulation, activation of its biosynthetic pathway and an accumulation of its precursor, the polyunsaturated α-linolenic acid (C18:3), were observed in the leaves of the tolerant genotype, Regent. This work was aimed at a better comprehension of the desaturation processes occurring after inoculation. We characterised, for the first time in Vitis vinifera, the gene family of the FA desaturases and evaluated their involvement in Regent response to Plasmopara viticola. Upon pathogen challenge, an up-regulation of the expression of plastidial FA desaturases genes was observed, resulting in a higher content of polyunsaturated fatty acids (PUFAs) of chloroplast lipids. This study highlights FA desaturases as key players in membrane remodelling and signalling in grapevine defence towards biotrophic pathogens.

Investigation of long non-coding RNAs as regulatory players of grapevine response to powdery and downy mildew infection.

BACKGROUND: Long non-coding RNAs (lncRNAs) are regulatory transcripts of length > 200 nt. Owing to the rapidly progressing RNA-sequencing technologies, lncRNAs are emerging as considerable nodes in the plant antifungal defense networks. Therefore, we investigated their role in Vitis vinifera (grapevine) in response to obligate biotrophic fungal phytopathogens, Erysiphe necator (powdery mildew, PM) and Plasmopara viticola (downy mildew, DM), which impose huge agro-economic burden on grape-growers worldwide. RESULTS: Using computational approach based on RNA-seq data, 71 PM- and 83 DM-responsive V. vinifera lncRNAs were identified and comprehensively examined for their putative functional roles in plant defense response. V. vinifera protein coding sequences (CDS) were also profiled based on expression levels, and 1037 PM-responsive and 670 DM-responsive CDS were identified. Next, co-expression analysis-based functional annotation revealed their association with gene ontology (GO) terms for 'response to stress', 'response to biotic stimulus', 'immune system process', etc. Further investigation based on analysis of domains, enzyme classification, pathways enrichment, transcription factors (TFs), interactions with microRNAs (miRNAs), and real-time quantitative PCR of lncRNAs and co-expressing CDS pairs suggested their involvement in modulation of basal and specific defense responses such as: Ca2+-dependent signaling, cell wall reinforcement, reactive oxygen species metabolism, pathogenesis related proteins accumulation, phytohormonal signal transduction, and secondary metabolism. CONCLUSIONS: Overall, the identified lncRNAs provide insights into the underlying intricacy of grapevine transcriptional reprogramming/post-transcriptional regulation to delay or seize the living cell-dependent pathogen growth. Therefore, in addition to defense-responsive genes such as TFs, the identified lncRNAs can be further examined and leveraged to candidates for biotechnological improvement/breeding to enhance fungal stress resistance in this susceptible fruit crop of economic and nutritional importance.

Genetic variation for tolerance to the downy mildew pathogen Peronospora variabilis in genetic resources of quinoa (Chenopodium quinoa).

BACKGROUND: Quinoa (Chenopodium quinoa Willd.) is an ancient grain crop that is tolerant to abiotic stress and has favorable nutritional properties. Downy mildew is the main disease of quinoa and is caused by infections of the biotrophic oomycete Peronospora variabilis Gaüm. Since the disease causes major yield losses, identifying sources of downy mildew tolerance in genetic resources and understanding its genetic basis are important goals in quinoa breeding. RESULTS: We infected 132 South American genotypes, three Danish cultivars and the weedy relative C. album with a single isolate of P. variabilis under greenhouse conditions and observed a large variation in disease traits like severity of infection, which ranged from 5 to 83%. Linear mixed models revealed a significant effect of genotypes on disease traits with high heritabilities (0.72 to 0.81). Factors like altitude at site of origin or seed saponin content did not correlate with mildew tolerance, but stomatal width was weakly correlated with severity of infection. Despite the strong genotypic effects on mildew tolerance, genome-wide association mapping with 88 genotypes failed to identify significant marker-trait associations indicating a polygenic architecture of mildew tolerance. CONCLUSIONS: The strong genetic effects on mildew tolerance allow to identify genetic resources, which are valuable sources of resistance in future quinoa breeding.

Genome reconstruction of the non-culturable spinach downy mildew Peronospora effusa by metagenome filtering.

Peronospora effusa (previously known as P. farinosa f. sp. spinaciae, and here referred to as Pfs) is an obligate biotrophic oomycete that causes downy mildew on spinach (Spinacia oleracea). To combat this destructive many disease resistant cultivars have been bred and used. However, new Pfs races rapidly break the employed resistance genes. To get insight into the gene repertoire of Pfs and identify infection-related genes, the genome of the first reference race, Pfs1, was sequenced, assembled, and annotated. Due to the obligate biotrophic nature of this pathogen, material for DNA isolation can only be collected from infected spinach leaves that, however, also contain many other microorganisms. The obtained sequences can, therefore, be considered a metagenome. To filter and obtain Pfs sequences we utilized the CAT tool to taxonomically annotate ORFs residing on long sequences of a genome pre-assembly. This study is the first to show that CAT filtering performs well on eukaryotic contigs. Based on the taxonomy, determined on multiple ORFs, contaminating long sequences and corresponding reads were removed from the metagenome. Filtered reads were re-assembled to provide a clean and improved Pfs genome sequence of 32.4 Mbp consisting of 8,635 scaffolds. Transcript sequencing of a range of infection time points aided the prediction of a total of 13,277 gene models, including 99 RxLR(-like) effector, and 14 putative Crinkler genes. Comparative analysis identified common features in the predicted secretomes of different obligate biotrophic oomycetes, regardless of their phylogenetic distance. Their secretomes are generally smaller, compared to hemi-biotrophic and necrotrophic oomycete species. We observe a reduction in proteins involved in cell wall degradation, in Nep1-like proteins (NLPs), proteins with PAN/apple domains, and host translocated effectors. The genome of Pfs1 will be instrumental in studying downy mildew virulence and for understanding the molecular adaptations by which new isolates break spinach resistance.

Effect of physio-chemical seed treatments on opium poppy downy mildews caused by Peronospora meconopsidis and P. somniferi.

Downy mildew of opium poppy is the single biggest disease constraint afflicting the Australian poppy industry. Within the pathosystem, the transmission of infections via infested seed is of major concern. Both downy mildew pathogens of poppy; Peronospora meconopsidis and P. somniferi, are known contaminants of commercial seed stocks. Using seed naturally infested with these pathogens, the effect of physio-chemical seed treatments on seedling health and disease transmission were evaluated. Individual seed treatments were tested to determine optimal treatment parameters for each; including incubation time, temperature and treatment concentration. Optimised physiochemical treatments were then compared. The most effective treatment methods were seed washes in acidified electrolytic water (400 ppm hypochlorous acid for 5 min) and hypochlorite solution (2% NaOCI for 5 min). In seed to seedling transmission assays, these two treatments reduced transmission of P. somniferi by 88.8% and 74.61%, and P. meconopsidis by 93.3% and 100%, respectively. These methods are recommended for seed treatment of commercial opium poppy seed to assist in the control of the downy mildew diseases.

Identification and Validation of Candidate Genes Conferring Resistance to Downy Mildew in Maize (Zea mays L.).

Downy mildew (DM) is a major disease of maize that causes significant yield loss in subtropical and tropical regions around the world. A variety of DM strains have been reported, and the resistance to them is polygenically controlled. In this study, we analyzed the quantitative trait loci (QTLs) involved in resistance to Peronosclerospora sorghi (sorghum DM), P. maydis (Java DM), and Sclerophthora macrospora (crazy top DM) using a recombinant inbred line (RIL) from a cross between B73 (susceptible) and Ki11 (resistant), and the candidate genes for P. sorghi, P. maydis, and S. macrospora resistance were discovered. The linkage map was constructed with 234 simple sequence repeat (SSR) and restriction fragment length polymorphism (RFLP) markers, which was identified seven QTLs (chromosomes 2, 3, 6, and 9) for three DM strains. The major QTL, located on chromosome 2, consists of 12.95% of phenotypic variation explained (PVE) and a logarithm of odds (LOD) score of 14.12. Sixty-two candidate genes for P. sorghi, P. maydis, and S. macrospora resistance were obtained between the flanked markers in the QTL regions. The relative expression level of candidate genes was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) using resistant (CML228, Ki3, and Ki11) and susceptible (B73 and CML270) genotypes. For the 62 candidate genes, 15 genes were upregulated in resistant genotypes. Among these, three (GRMZM2G028643, GRMZM2G128315, and GRMZM2G330907) and AC210003.2_FG004 were annotated as leucine-rich repeat (LRR) and peroxidase (POX) genes, respectively. These candidate genes in the QTL regions provide valuable information for further studies related to P. sorghi, P. maydis, and S. macrospora resistance.

Root treatment with oxathiapiprolin, benthiavalicarb or their mixture provides prolonged systemic protection against oomycete foliar pathogens.

Oxathiapiprolin is a fungicide effective against downy mildews of cucumber (Pseudoperonospora cubensis) and basil (Peronospora belbahrii) and late blight of tomato (Phytophthora infestans). To avoid fungicide resistance, it is recommended to apply oxathiapiprolin as a mixture with a partner fungicide that have a different mode of action. Here it is shown that a single application of oxathiapiprolin, benthiavalicarb, or their mixture (3+7, w/w) to the root of nursery plants grown in multi-cell trays provided prolonged systemic protection against late blight and downy mildews in growth chambers and in field tests. Soil application of 1mg active ingredient per plant provided durable protection of up to four weeks in tomato against late blight, cucumber against downy mildew and basil against downy mildew. Not only did the mixture of oxathiapiprolin and benthiavalicarb provide excellent systemic control of these diseases but also mutual protection against resistance towards both oxathiapiprolin and benthiavalicarb.

Small RNA inhibits infection by downy mildew pathogen Hyaloperonospora arabidopsidis.

Gene silencing exists in eukaryotic organisms as a conserved regulation of the gene expression mechanism. In general, small RNAs (sRNAs) are produced within the eukaryotic cells and incorporated into an RNA-induced silencing complex (RISC) within cells. However, exogenous sRNAs, once delivered into cells, can also silence target genes via the same RISC. Here, we explored this concept by targeting the Cellulose synthase A3 (CesA3) gene of Hyaloperonospora arabidopsidis (Hpa), the downy mildew pathogen of Arabidopsis thaliana. Hpa spore suspensions were mixed with sense or antisense sRNAs and inoculated onto susceptible Arabidopsis seedlings. While sense sRNAs had no obvious effect on Hpa pathogenicity, antisense sRNAs inhibited spore germination and hence infection. Such inhibition of infection was not race-specific, but dependent on the length and capping of sRNAs. Inhibition of infection by double stranded sRNA was more efficient than that observed with antisense sRNA. Thus, exogenous sRNA targeting conserved CesA3 could suppress Hpa infection in Arabidopsis, indicating the potential of this simple and efficient sRNA-based approach for deciphering gene functions in obligate biotrophic pathogens as well as for R-gene independent control of diseases in plants.

Early stage metabolic events associated with the establishment of Vitis vinifera - Plasmopara viticola compatible interaction.

Grapevine (Vitis vinifera L.) is the most widely cultivated and economically important fruit crop in the world, with 7.5 million of production area in 2017. The domesticated varieties of grapevine are highly susceptible to many fungal infections, of which downy mildew, caused by the biotrophic oomycete Plasmopara viticola (Berk. et Curt.) Berl. et de Toni is one of the most threatening. In V. vinifera, several studies have shown that a weak and transient activation of a defense mechanism occurs, but it is easily overcome by the pathogen leading to the establishment of a compatible interaction. Major transcript, protein and physiologic changes were shown to occur at later infection time-points, but comprehensive data on the first hours of interaction is scarce. In the present work, we investigated the major physiologic and metabolic changes that occur in the first 24 h of interaction between V. vinifera cultivar Trincadeira and P. viticola. Our results show that there was a negative modulation of several metabolic classes associated to pathogen defense such as flavonoids or phenylpropanoids as well as an alteration of carbohydrate content after inoculation with the pathogen. We also found an accumulation of hydrogen peroxide and increase of lipid peroxidation but to a low extent, that seems to be insufficient to restrain pathogen growth during the initial biotrophic phase of the interaction.

Timing of Grape Downy Mildew Onset in Bordeaux Vineyards.

Grapevine downy mildew (GDM) is a severe disease of grapevines. Because of the lack of reliable information about the dates of GDM symptom onset, many vine growers begin fungicide treatments early in the season. We evaluate the extent to which such preventive treatments are justified. Observational data for 266 untreated sites for the years between 2010 and 2017 were used to estimate the timing of GDM onset on vines and bunches of grapes in South West France (Bordeaux region) through survival analyses. The onset of GDM was not apparent on vines and bunches before early to mid-May, and the rate of GDM symptom appearance was highly variable across years. Depending on the year, 50% of the plots displayed symptoms between mid-May and late June for vines. For several years, our statistical analysis revealed that the proportion of plots with no symptoms was high in early August on vines (27.5 and 43.7% in 2013 and 2016) and on bunches (between 23 and 79% in 2011, 2013, and 2016). We found a significant effect of the amount of rainfall in spring on the date of symptom appearance. These results indicate that preventive fungicide application is unjustified in many vineyards, and that regional disease surveys should be used to adjust fungicide treatment dates according to local characteristics, in particular according to rainfall conditions in spring.

Comparative genomics of downy mildews reveals potential adaptations to biotrophy.

BACKGROUND: Spinach downy mildew caused by the oomycete Peronospora effusa is a significant burden on the expanding spinach production industry, especially for organic farms where synthetic fungicides cannot be deployed to control the pathogen. P. effusa is highly variable and 15 new races have been recognized in the past 30 years. RESULTS: We virulence phenotyped, sequenced, and assembled two isolates of P. effusa from the Salinas Valley, California, U.S.A. that were identified as race 13 and 14. These assemblies are high quality in comparison to assemblies of other downy mildews having low total scaffold count (784 & 880), high contig N50s (48 kb & 52 kb), high BUSCO completion and low BUSCO duplication scores and share many syntenic blocks with Phytophthora species. Comparative analysis of four downy mildew and three Phytophthora species revealed parallel absences of genes encoding conserved domains linked to transporters, pathogenesis, and carbohydrate activity in the biotrophic species. Downy mildews surveyed that have lost the ability to produce zoospores have a common loss of flagella/motor and calcium domain encoding genes. Our phylogenomic data support multiple origins of downy mildews from hemibiotrophic progenitors and suggest that common gene losses in these downy mildews may be of genes involved in the necrotrophic stages of Phytophthora spp. CONCLUSIONS: We present a high-quality draft genome of Peronospora effusa that will serve as a reference for Peronospora spp. We identified several Pfam domains as under-represented in the downy mildews consistent with the loss of zoosporegenesis and necrotrophy. Phylogenomics provides further support for a polyphyletic origin of downy mildews.

Fine mapping and candidate gene screening of the downy mildew resistance gene RPF1 in Spinach.

KEY MESSAGE: A SLAF-BSA approach was used to locate the RPF1 locus. The three most likely candidate genes were identified which provide a basic for cloning the resistance gene at the RPF1 locus. Spinach downy mildew is a globally devastating oomycete disease. The use of downy mildew resistance genes constitutes the most effective approach for disease management. Hence, the objective of the present study was to fine map the first-reported resistance locus RPF1. The resistance allele at this resistance locus was effective against races 1-7, 9, 11, 13, and 15 of Peronospora farinosa f. sp. spinaciae (P. effusa). The approach fine mapped RPF1 using specific-locus amplified fragment sequencing (SLAF-Seq) technology combined with bulked segregant analysis. A 1.72 Mb region localized on chromosome 3 was found to contain RPF1 based on association analysis. After screening recombinants with the SLAF markers within the region, the region was narrowed down to 0.89 Mb. Within this region, 14 R genes were identified based on the annotation information. To identify the genes involved in resistance, resequencing of two resistant inbred lines (12S2 and 12S3) and three susceptible inbred lines (12S1, 12S4, and 10S2) was performed. The three most likely candidate genes were identified via amino acid sequence analysis and conserved domain analysis between resistant and susceptible inbred lines. These included Spo12729, encoding a receptor-like protein, and Spo12784 and Spo12903, encoding a nucleotide-binding site and leucine-rich repeat domains. Additionally, based on the sequence variation in the three genes between the resistant and susceptible lines, molecular markers were developed for marker-assisted selection. The results could be valuable in cloning the RPF1 alleles and improving our understanding of the interaction between the host and pathogen.

Unique resistance traits against downy mildew from the center of origin of grapevine (Vitis vinifera).

The Eurasian grapevine (Vitis vinifera), an Old World species now cultivated worldwide for high-quality wine production, is extremely susceptible to the agent of downy mildew, Plasmopara viticola. The cultivation of resistant V. vinifera varieties would be a sustainable way to reduce the damage caused by the pathogen and the impact of disease management, which involves the economic, health and environmental costs of frequent fungicide application. We report the finding of unique downy mildew resistance traits in a winemaking cultivar from the domestication center of V. vinifera, and characterize the expression of a range of genes associated with the resistance mechanism. Based on comparative experimental inoculations, confocal microscopy and transcriptomics analyses, our study shows that V. vinifera cv. Mgaloblishvili, native to Georgia (South Caucasus), exhibits unique resistance traits against P. viticola. Its defense response, leading to a limitation of P. viticola growth and sporulation, is determined by the overexpression of genes related to pathogen recognition, the ethylene signaling pathway, synthesis of antimicrobial compounds and enzymes, and the development of structural barriers. The unique resistant traits found in Mgaloblishvili highlight the presence of a rare defense system in V. vinifera against P. viticola which promises fresh opportunities for grapevine genetic improvement.

Chitosan nanoparticles having higher degree of acetylation induce resistance against pearl millet downy mildew through nitric oxide generation.

Downy mildew of pearl millet caused by the biotrophic oomycete Sclerospora graminicola is the most devastating disease which impairs pearl millet production causing huge yield and monetary losses. Chitosan nanoparticles (CNP) were synthesized from low molecular weight chitosan having higher degree of acetylation was evaluated for their efficacy against downy mildew disease of pearl millet caused by Sclerospora graminicola. Laboratory studies showed that CNP seed treatment significantly enhanced pearl millet seed germination percentage and seedling vigor compared to the control. Seed treatment with CNP induced systemic and durable resistance and showed significant downy mildew protection under greenhouse conditions in comparison to the untreated control. Seed treatment with CNP showed changes in gene expression profiles wherein expression of genes of phenylalanine ammonia lyase, peroxidase, polyphenoloxidase, catalase and superoxide dismutase were highly upregulated. CNP treatment resulted in earlier and higher expression of the pathogenesis related proteins PR1 and PR5. Downy mildew protective effect offered by CNP was found to be modulated by nitric oxide and treatment with CNP along with NO inhibitors cPTIO completely abolished the gene expression of defense enzymes and PR proteins. Further, comparative analysis of CNP with Chitosan revealed that the very small dosage of CNP performed at par with recommended dose of Chitosan for downy mildew management.

New Races and Novel Strains of the Spinach Downy Mildew Pathogen Peronospora effusa.

Downy mildew disease, caused by Peronospora effusa (=P. farinosa f. sp. spinaciae [Pfs]), is the most economically important disease of spinach. Current high-density fresh-market spinach production provides conducive conditions for disease development, and downy mildew frequently forces growers to harvest early owing to disease development, to cull symptomatic leaves prior to harvest, or to abandon the field if the disease is too severe. The use of resistant cultivars to manage downy mildew, particularly on increasing acreages of organic spinach production, applies strong selection pressure on the pathogen, and many new races of Pfs have been identified in recent years in spinach production areas worldwide. To monitor the virulence diversity in the Pfs population, downy mildew samples were collected from spinach production areas and tested for race identification based on the disease reactions of a standard set of international spinach differentials. Two new races (designated races 15 and 16) and eight novel strains were identified between 2013 and 2017. The disease reaction of Pfs 15 was similar to race 4, except race 4 could not overcome the resistance imparted by the RPF9 locus. Several resistance loci (RPF1, 2, 4, and 6) were effective in preventing disease caused by Pfs 15. The race Pfs 16 could overcome several resistance loci (RPF2, 4, 5, 9, and 10) but not others (RPF1, 3, 6, and 7). One novel strain (UA1014) could overcome the resistance of spinach resistant loci RPF1 to RPF7 but only infected the cotyledons and not the true leaves of certain cultivars. A new set of near-isogenic lines has been developed and evaluated for disease reactions to the new races and novel strains as differentials. None of the 360 U.S. Department of Agriculture spinach germplasm accessions tested were resistant to Pfs 16 or UA1014. A survey of isolates over several years highlighted the dynamic nature of the virulence diversity of the Pfs population. Identification of virulence diversity and evaluation of the genetics of resistance to Pfs will continue to allow for a more effective disease management strategy through resistance gene deployment.

Phytohormone and genome variations in Vitis amurensis resistant to downy mildew.

Downy mildew (DM) resistance is a highly desirable agronomic trait in grape breeding. High variation in Plasmopara viticola resistance was found in Vitis cultivars. Some accessions show high P. viticola resistance even under conditions highly conducive to DM. Here, leaf disc inoculation experiments revealed that Vitis amurensis 'Zuoshaner' exhibited DM resistance with necrotic spots, whereas the V. amurensis × V. vinifera hybrid cultivar 'Zuoyouhong' was susceptible. Changes in plant hormones accumulation profiles differed between the cultivars. To investigate the genetic mechanisms related to DM resistance, we performed genome-wide sequencing of 'Zuoshaner' and 'Zuoyouhong' and identified cultivar-specific single-nucleotide polymorphisms, insertions/deletions (indels), structural variations (SVs), and copy number variations (CNVs), identifying 5399 SVs and 191 CNVs specific for 'Zuoshaner'. Genes affected by these genetic variations were enriched in biological processes, including defense response and response to stress and stimulation, and were associated with sesquiterpenoid and triterpenoid biosynthesis, ABC transporters, and phenylalanine metabolism pathways. Additionally, indels and SVs were detected in six NBS-LRR disease resistance genes, and a CNV was mapped to the Rpv8 locus responsible for downy mildew resistance. These findings further our understanding of the genetic mechanisms underlying grape mildew resistance, and will facilitate genomic marker-assisted breeding for improved V. amurensis cultivars.

Trichogenic-selenium nanoparticles enhance disease suppressive ability of Trichoderma against downy mildew disease caused by Sclerospora graminicola in pearl millet.

Trichoderma spp. are well known biocontrol agents used against phytopathogens. In the present work Trichoderma-mediated Selenium nanoparticles (SeNPs) were synthesized and extent of downy mildew (DM) disease control in pearl millet (PM) was studied. Six species of Trichoderma namely, T. asperellum, T. harzianum, T. atroviride, T. virens, T. longibrachiatum and T. brevicompactum were evaluated in the form of culture filtrate (CF), cell lysate (CL) and crude cell wall (CW) to synthesize SeNPs. All these components produced SeNPs, but CF was significant than CL and CW. The size of SeNPs ranged from 49.5 to 312.5 nm with zeta potential of +3.3 mv to -200 mv. The nanoparticles suppressed the growth, sporulation and zoospore viability of Sclerospora graminicola and these biological activities were inversely proportional to the size of SeNPs. Under greenhouse conditions, application of SeNPs and T. asperellum together enhanced the early plant growth and suppressed DM incidence as compared to their individual application. This study demonstrated the ability of Trichogenic-SeNPs to suppress growth and proliferation of S. graminicola, the incitant of DM of PM and their activity is inversely proportional to size of nanoparticles.

Proteomics towards the understanding of elicitor induced resistance of grapevine against downy mildew.

Elicitors are known to trigger plant defenses in response to biotic stress, but do not systematically lead to effective resistance to pathogens. The reasons explaining such differences remain misunderstood. Therefore, elicitation and induced resistance (IR) were investigated through the comparison of two modified ß-1,3 glucans applied on grapevine (Vitis vinifera) leaves before and after inoculation with Plasmopara viticola, the causal agent of downy mildew. The sulfated (PS3) and the shortened (H13) forms of laminarin are both known to elicit defense responses whereas only PS3 induces resistance against downy mildew. The analysis of the 2-DE gel electrophoresis revealed that PS3 and H13 induced distinct proteomic profiles after treatment and pathogen inoculation. Our results point out that the PS3-induced resistance is associated with the activation of the primary metabolism especially on amino acids and carbohydrates pathways. In addition, few proteins, such as the 12-oxophytodienoate reductase (OPR-like) related to the OPDA pathway, and an Arsenite-resistance protein (Serrate-like protein) could be considered as useful markers of induced resistance. SIGNIFICANCE: One strategy to reduce the application of fungicides is the use of elicitors which induce plant defense responses. Nonetheless, the elicitors do not systematically lead to resistance against pathogens. The lack of correlation between plant defense activation and induced resistance (IR) requires the investigation of what makes the specificity of elicitor-IR. In this study, the two ß-glucans elicitors, sulfated (PS3) and short (H13) laminarins, were used in the grapevine/Plasmopara viticola interaction since only the first one leads to resistance against downy mildew. To disclose IR specificity, proteomic approach has been employed to compare the two treatments before and after P. viticola inoculation. The analysis of the 2-DE revealed that PS3 and H13 induced distinct proteomic profiles after treatment and pathogen inoculation. Significant increase of the number of proteins regulated by PS3, relative to both H13 and time-points, is correlated with the resistance process establishment. Our results point that the PS3-induced resistance requires the activation of the primary metabolism especially on amino acids and carbohydrates pathways. In addition, few proteins, such as the 12-oxophytodienoate reductase (OPR-like) related to the OPDA pathway, and an Arsenite-resistance protein (Serrate-like protein) could constitute useful markers of PS3 induced resistance.