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Background. Downy mildew (DM) caused by the fungus Plasmopara halstedii (Farl) Berl. & De Toni) is one of the most harmful diseases of sunflower (Helianthus annuus L.). Due to the pathogen’s attacks, annual seed harvest losses range between 30% and 70%. Lines resistant to new races of the pathogen should be obtained for the development of commercial sunflower hybrids.Materials and methods. Downy mildew resistance of 323 lines and 10 cultivars from the sunflower collection was assessed in the field at the Kuban Experiment Station of VIR in 2017–2018. Line VIR 845, susceptible during all years of observations, was used as a control. Resistance genes were identified by means of the molecular analysis using diagnostic markers of the Plarg, Pl6 and Pl8 genes that confer resistance to many known P. halstedii races.Results. The founder varieties of VIR’s lines were susceptible to a varying extent. Thirty-nine lines were resistant in 2016 and 2018; among those, 36 lines were susceptible in 2017. Presumably, a more virulent P. halstedii race became widespread in 2017, compared to the races that prevailed in 2016 and 2018, so the genes that determined resistance in 2016 and 2018 turned out to be ineffective. Lines ТА 716-18, VIR 768, and VIR 800, having originated from interspecific hybrids, exhibited absence of pathogenic damage during 3 years of the trials. Molecular markers of the Plarg, Pl6 and Pl8 genes were detected in most lines that demonstrated resistance in 2016 and 2018. There were no markers in lines VIR 768 and VIR 800, whereas in ТА 716-18 the markers of Plarg and Pl8 were present.Conclusion. As a result of the long-term studies, a trait-specific genetic collection was established for sunflower. It comprises genotyped lines with various effective DM resistance genes. Lines ТА 716-18, VIR 768 and VIR 800 appeared highly resistant to the pathogen and probably possess new resistance genes/alleles introgressed from wild species.

Background Grapevine (Vitis vinifera) productivity has been severely affected by various bacterial, viral and fungal diseases worldwide. When a plant is infected with the pathogen, various defense mechanisms are subsequently activated in plants at various molecular levels. Thus, for substantiating the disease control in an eco-friendly way, it is essential to understand the molecular mechanisms governing pathogen resistance in grapes. Results In our study, we performed genome-wide identification of various defensive genes expressed during powdery mildew (PM) and downy mildew (DM) infections in grapevine. Consequently, we identified 6, 21, 2, 5, 3 and 48 genes of Enhanced Disease Susceptibility 1 (EDS1), Non-Race-specific Disease Resistance (NDR1), Phytoalexin deficient 4 (PAD4), Nonexpressor of PR Gene (NPR), Required for Mla-specified resistance (RAR) and Pathogenesis Related (PR), respectively, in the grapevine genome. The phylogenetic study revealed that V. vinifera defensive genes are evolutionarily related to Arabidopsis thaliana. Differential expression analysis resulted in identification of 2, 4, 7, 2, 4, 1 and 7 differentially expressed Nucleotide-binding leucine rich repeat receptor (NLR), EDS1, NDR1, PAD4, NPR, RAR1 and PR respectively against PM infections and 28, 2, 5, 4, 1 and 19 differentially expressed NLR, EDS1, NDR1, NPR, RAR1 and PR respectively against DM infections in V. vinifera. The co-expression study showed the occurrence of closely correlated defensive genes that were expressed during PM and DM stress conditions. Conclusion The PM and DM responsive defensive genes found in this study can be characterized in future for impelling studies relaying fungal and oomycete resistance in plants, and the functionally validated genes would then be available for conducting in-planta transgenic gene expression studies for grapes.

Bananas are among the world’s most important cash and staple crops but are threatened by various devastating pathogens. The phytohormone salicylic acid (SA) plays a key role in the regulation of plant immune response. Tracking the expression of SA-responsive marker genes under pathogen infection is important in pathogenesis elucidation. However, the common SA-responsive marker genes are not consistently induced in different banana cultivars or different organs. Here, we conducted transcriptome analysis for SA response of a banana cultivar, ‘Pei-Chiao’ (Cavendish, AAA genome), and identified three genes, MaWRKY40, MaWRKY70, and Downy Mildew Resistant 6 (DMR6)-Like Oxygenase 1 (MaDLO1) that are robustly induced upon SA treatment in both the leaves and roots. Consistent induction of these three genes by SA treatment was also detected in both the leaves and roots of bananas belonging to different genome types such as ‘Tai-Chiao No. 7’ (Cavendish, AAA genome), ‘Pisang Awak’ (ABB genome), and ‘Lady Finger’ (AA genome). Furthermore, the biotrophic pathogen cucumber mosaic virus elicited the expression of MaWRKY40 and MaDLO1 in infected leaves of susceptible cultivars. The hemibiotrophic fungal pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) also consistently induced the expression of MaWRKY40 and MaDLO1 in the infected roots of the F. oxysporum f. sp. cubense TR4-resistant cultivar. These results indicate that MaWRKY40 and MaDLO1 can be used as reliable SA-responsive marker genes for the study of plant immunity in banana. Revealing SA-responsive marker genes provides a stepping stone for further studies in banana resistance to pathogens.

Downy mildew is one of the most serious diseases of Papaver somniferum. Endophytes isolated from different parts of P. somniferum were screened for their ability to enhance resistance against downy mildew caused by the obligate biotrophic oomycete Peronospora meconopsidis. Two endophytes (SMR1 and SMR2) reduced the downy mildew on three P. somniferum genotypes (Sampada, J-16, and I-14). SMR1 (Microbacterium sp.) also enhanced the resistance of P. somniferum against downy mildew under field conditions. The biochemical markers of plant susceptibility under biotic stresses (proline and malondialdehyde) were found to be reduced in P. somniferum upon SMR1 treatment. To understand the mechanisms underlying the enhanced resistance to downy mildew in SMR1 endophyte-treated P. somniferum genotype J-16, we compared the expression profiles using the next-generation RNA sequencing approach between P. somniferum pretreated with SMR1 and untreated endophyte-free control plants following exposure to downy mildew pathogen. Comparative transcriptome analysis revealed differential expression of transcripts belonging to broad classes of signal transduction, protein modification, disease/defense proteins, transcription factors, and phytohormones in SMR1-primed P. somniferum after infection with downy mildew pathogen. Furthermore, enhanced salicylic acid content was observed in SMR1-primed P. somniferum after exposure to downy mildew pathogen. This study sheds light on molecular mechanisms underlying enhanced resistance to downy mildew in SMR1-primed P. somniferum. Finally, we propose that the SA-dependent defense pathway, the hallmark of systemic acquired resistance, is activated in SMR1-primed P. somniferum, triggering the endophyte-induced resistance.

Modern sunflower breeding is significantly diversified due to the different needs of agricultural production. The breeding of sunflower varieties and hybrids is carried out at V.S. Pustovoit All-Russian Research Institute of Oil Crops (VNIIMK) in all areas in demand on the market and is based on fundamental biological research. In the field of breeding for faster maturing, the following commercial cultivars were obtained: very early maturing, cv. Skormas and the three-way hybrid Achilles, early maturing cvs. Varyag and Uspekh, medium maturing cvs. Amelie, Aris and Aurus. Within the framework of breeding for immunity, eight hybrids and one variety have been produced. So at the Don experimental station (Rostov region), productive hybrids were bred, resistant to the virulent broomrape of the G race due to the presence of the Or7 gene: 'Gorstar', 'Gorfild', 'Grant', 'Status', 'Fogor' and the three-way hybrid Nika. On the central experimental base, the following were obtained: the mid-early hybrid Typhoon and the earlymaturing variety Platonych with resistance to common races of downy mildew and a high oil content of achenes (up to 53 %) as well as the mid-early hybrid Tayzar, which is simultaneously resistant to virulent races of broomrape and to the causative agent of downy mildew. The early maturing large-fruited sunflower variety Belochka was included in the "Russian State Register of Selection Achievements…", and the large-fruited varieties Karavan, Konditer and Kalibr are currently undergoing state tests. The breeding use of germplasm with genes for herbicide resistance was accompanied by their extensive genetic study. A practical recommendation for all three alleles of the ALS gene (Imr, CLHA-Plus, Sur) was the need to create homozygous hybrids for their reliable use in appropriate production systems. For Clearfield technology, the hybrids Imidzh, Arimi and Immi have been developed; for Clearfield Plus, the hybrid Klip; and for Express Sun (or SUMO), the hybrid Surus. Klip and Surus are mid-oleic. All newly developed fertile ornamental sunflower varieties - Aurelia, Fizalia, Zhemchuzhny, Rumyanets, Agat and Mazhor - were transferred for practical use to a sterile CMS RIG basis. Thus, new achievements have been attained across the entire spectrum of modern trends in sunflower breeding.Современная селекция подсолнечника сильно диверсифицирована различными потребностями сельскохозяйственного производства. Во Всероссийском научно-исследовательском институте масличных культур (ВНИИМК) селекция сортов и гибридов подсолнечника ведется по всем востребованным на рынке направлениям и основывается на фундаментальных биологических исследованиях. В направлении селекции на сокращение вегетационного периода растений получены: скороспелые сорт Скормас и трехлинейный гибрид Ахиллес, раннеспелые сорта Варяг и Успех, среднеспелые гибриды Амели, Арис и Аурус. При селекции на устойчивость к болезням созданы восемь гибридов и один сорт. Так, на Донской опытной станции выведены продуктивные гибриды, устойчивые к вирулентной заразихе расы G за счет наличия гена Or7: Горстар, Горфилд, Грант, Статус, Фогор и трехлинейный гибрид Ника. На центральной экспериментальной базе получены среднеранний гибрид Тайфун и раннеспелый сорт Платоныч с устойчивостью к распространенным расам ложной мучнистой росы и с высокой масличностью семянок (до 53 %), а также среднеранний гибрид подсолнечника Тайзар, который обладает устойчивостью к вирулентным расам заразихи и к возбудителю ложной мучнистой росы. В «Государственный реестр селекционных достижений…» внесенскороспелый крупноплодный сорт подсолнечника Белочка; проходят госсортоиспытание крупноплодные сорта Караван, Кондитер и Калибр. Результатом генетического изучения генов гербицидоустойчивости стала практическая рекомендация для применения в селекции подсолнечника трех генов устойчивости к ингибиторам ацетолактатсинтетазы (Imr, CLHA-Plus, Sur), которая заключается в необходимости создания гомозиготных гибридов для их надежного использования в соответствующих производственных системах. Для технологий Clearfield получены гибриды Имидж, Арими и Имми, для Clearfield Plus – гибрид Клип, а для Express Sun (или SUMO) – гибрид Сурус. Гибриды Клип и Сурус являются среднеолеиновыми по качеству масла. Все созданные фертильные декоративные сорта подсолнечника Аурелия, Физалия, Жемчужный, Агат, Мажор и гибрид Румянец переведены для практического использования на стерильную основу ЦМС RIG. Таким образом, по всему спектру современных направлений селекции подсолнечника во ВНИИМК имеются новые достижения.

Downy mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most economically significant grapevine diseases worldwide. Current strategies to cope with this threat rely on the massive use of chemical compounds during each cultivation season. The economic costs and negative environmental impact associated with these applications increased the urge to search for sustainable strategies of disease control. Improved knowledge of plant mechanisms to counteract pathogen infection may allow the development of alternative strategies for plant protection. Epigenetic regulation, in particular DNA methylation, is emerging as a key factor in the context of plant-pathogen interactions associated with the expression modulation of defence genes. To improve our understanding of the genetic and epigenetic mechanisms underpinning grapevine response to P. viticola, we studied the modulation of both 5-mC methylation and gene expression at 6 and 24 h post-infection (hpi). Leaves of two table grape genotypes (Vitis vinifera), selected by breeding activities for their contrasting level of susceptibility to the pathogen, were analysed. Following pathogen infection, we found variations in the 5-mC methylation level and the gene expression profile. The results indicate a genotype-specific response to pathogen infection. The tolerant genotype (N23/018) at 6 hpi exhibits a lower methylation level compared to the susceptible one (N20/020), and it shows an early modulation (at 6 hpi) of defence and epigenetic-related genes during P. viticola infection. These data suggest that the timing of response is an important mechanism to efficiently counteract the pathogen attack.

Relevance. F1 hybrids breeding programs of onion are based on the use of nuclear cytoplasmic male sterility (NCMS). Breeding and seed production based on NCMS suggest a development of a female sterile maternal line, a maintainer line and a paternal component. The development of an isogenic pair of sterile line – maintainer line is one of the most labor-, time-consuming and intellectually-intensive stages of a breeding program, the implementation of which becomes more complicated with an increase in the number of traits / genes for which selection is carried out.Methods and results. This paper presents a genetic scheme for the development of a Downy mildewresistant (pathogen P. destructor) maintainer line of onion using marker-assisted selection (MAS). F2 populations were obtained from the cross of the female maintainer inbred line, Bn1- (13) (genotype – cytN msms pdpd) and male inbred line resistant to Downy mildew, No. 136 (genotype – cytT MsMs PdPd). DNA marker DMR1 were used for Downy mildew resistance gene Pd, jnurf13 – for maintainer gene ms and marker system 5`cob: orfA501 – for N, S and T cytoplasms. It was shown that the MAS application allow to develop a stable Downy mildew resistant maintainer line cytN msms PdPd in two generations.

Grapevine (Vitis vinifera L.) is prone to fungal and oomycete diseases. Downy and powdery mildews and grey mold, are caused by Plasmopara viticola, Erisiphe necator and Botrytis cinerea, respectively. P. viticola and E. necator are obligatory biotrophs whereas B. cinerea is a necrotroph. In tolerant grapevine cultivars, plant-pathogen interaction induces defence responses, including metabolite and protein accumulation and hypersensitive reaction. Lipid and lipid-derived molecules may have a key role in the activation of defence mechanisms. Previous results suggest that V. vinifera cv Regent tolerance to P. viticola may be mediated in the first hours post inoculation by fatty acid (FA) associated signalling. In the present study we characterized FA modulation in V. vinifera cv Regent leaves upon inoculation with P. viticola, E. necator and B. cinerea and correlated FA modulation with the expression profiles of genes encoding the FA desaturases FAD6 and FAD8. In all the interactions, a progressive desaturation of stearic acid to α-linolenic acid, precursor of jasmonic acid, occurred, which was observed for a longer period against B. cinerea. Our results provide evidence of a distinct FA meditated signalling pattern in grapevine interaction with biotrophs and necrotrophs. While the interaction with the biotrophs may trigger a higher synthesis of polyunsaturated FA (PUFA) at early time-points with a tendency to return to basal levels, the interaction with B. cinerea may trigger a later and more durable induction of PUFA synthesis. In all interactions, membrane fluidity modulation occurred, which may be crucial to maintain cellular function during infection.

This is the first identification of QTLs underlying resistance to Pseudoperonospora cubensis in Cucumis melo using a genetically characterized isolate. Pseudoperonospora cubensis, causal organism of cucurbit downy mildew (CDM), is one of the largest threats to cucurbit production in the eastern USA. Currently, no Cucumis melo (melon) cultivars have significant levels of resistance. Additionally, little is understood about the genetic basis of resistance in C. melo. Recombinant inbred lines (RILs; N = 169) generated from a cross between the resistant melon breeding line MR-1 and susceptible cultivar Ananas Yok’neam were phenotyped for CDM resistance in both greenhouse and growth chamber studies. A high-density genetic linkage map with 5,663 binned SNPs created from the RIL population was utilized for QTL mapping. Nine QTLs, including two major QTLs, were associated with CDM resistance. Of the major QTLs, qPcub-10.1 was stable across growth chamber and greenhouse tests, whereas qPcub-8.2 was detected only in growth chamber tests. qPcub-10.1 co-located with an MLO-like protein coding gene, which has been shown to confer resistance to powdery mildew and Phytophthora in other plants. This is the first screening of C. melo germplasm with a genetically characterized P. cubensis isolate.

Plantago princeps var. princeps is an endangered native Hawaiian plant, and part of the recovery plan includes repopulation using plants grown in a nursery. However, disease pressure from downy mil...

Radish downy mildew (DM) caused by the oomycete Hyaloperonospora brassicae f. sp. raphani is a serious problem in radish crop, an edible root vegetable of the Brassicaceae family. The objective of this research was to assess radish germplasm for DM resistance and to evaluate the response of different radish organs to the disease under controlled conditions. Forty-four radish accessions were inoculated at cotyledons and true-leaves with H. brassicae isolate R10, collected in cotyledons of field plants. The roots were tested with isolates R10 and R6, this last one collected in roots of field radish. DM symptoms varied with the radish genotype and plant organ analysed. Twenty-seven resistant and partially resistant accessions were identified in all plant stages and are promising sources of resistance to DM, namely 16 commercial varieties, 10 breeding lines, and one landrace. A significant correlation was observed between cotyledon and leaf (1st and 2nd leaves) DM resistance, but low and no correlation was found between the resistance of true-leaves or cotyledons and roots, respectively. Cotyledon and leaf evaluation cannot be used to predict root resistance response in radish. However, cotyledon resistance has its own value because non-infected cotyledons will act as a barrier to slow disease progression to true-leaves and roots. Interesting sources of DM resistance were identified that can be used in radish breeding programs.

Grapevine, one of the most important fruit crops worldwide is host to various pathogens, some of which have been spread intercontinentally in the early history of viticulture. One of the most problematic diseases is downy mildew, caused by the oomycete Plasmopara viticola (Berk. & Curt.) Berl. & de Toni which was introduced to Europe from North America in the 19th century. In addition to the establishment of chemical plant protection measures, extensive resistance breeding activities have succeeded in establishing varieties that are able to limit the propagation of this pathogen Recent studies have indicated an important role of subtilisin-like serine proteases (subtilases) in association with this increased disease tolerance. Gene expression analysis of two candidate subtilases was performed in seven cultivars with different levels of susceptibility to P. viticola. Two P. viticola isolates with different degrees of virulence were tested. Our results show that subtilases display different patterns of expression: Cucumisin exhibits peaks of expression, while VviSBT5.3a presents a bimodal behaviour. Nevertheless, both subtilases have a more pronounced response to the more virulent P. viticola isolate, which supports the hypothesis that the two subtilases are involved in the early events of the defense mechanisms.

Cucurbit downy mildew (DM), caused by the obligate biotrophic oomycete Pseudoperonospora cubensis, is a major foliar disease of cucumber. Cucumber accession PI197088 was previously shown to be one of the most promising donors for DM resistance. The resistance in PI197088 is controlled by multiple quantitative trait loci (QTL), each with a relatively small effect. We developed a cucumber line with a 12 Mb introgression from PI197088 in a susceptible background. This introgression line was partially resistant to DM. Subsequently, we combined fine mapping data with RNAseq and whole genome resequencing to identify candidate genes for the resistance conferred by this introgression. Interestingly, fine-mapping data suggested that the partial resistance is caused by a combination of multiple genetically linked genes, leading to three subQTL, each with a different effect on the disease phenotype. In one of these subQTL we identified several Receptor Like Kinase genes (RLK). A novel RLK gene appeared to be present in resistant genotypes. In susceptible genotypes, including the reference genotype “Chinese Long 9930”, this novel gene has a 551 base pair deletion, and was therefore not correctly predicted during the annotation of the cucumber genome. In another subQTL, we found a loss-of-function mutation in the novel susceptibility gene (S-gene) CsAAP2A, encoding an amino acid transporter. In the resistant genotype a non-functional allele of this gene is present due to integration of a transposable element in its coding sequence.

Opium poppy, belonging to the family Papaveraceae, is grown for its alkaloid compounds of pharmaceutical value. Downy mildew caused by Peronospora somniferi and Pe. meconopsidis, substantially impacts crop production. The present study was conducted to identify the host range of Pe. somniferi and Pe. meconopsidis within selected weed and ornamental members of the Papaveraceae family. Nine Papaver spp., Meconopsis cambrica and a nonhost control (tomato, Solanum lycopersicum) were challenged with both pathogens under controlled glasshouse or laboratory conditions using infested soil or foliar applied sporangia as inocula. Peronospora somniferi and Pe. meconopsidis induced disease symptoms, including sporulation, in at least one trial for all tested species except for Pa. atlanticum and tomato. Species‐specific PCR testing of foliage of challenged plants confirmed infections by both pathogen species of symptomatic plants, identifying these as hosts. Positive PCR tests were also obtained from Pa. atlanticum plants for both pathogens. However, in the absence of pathogen sporulation structures as further evidence of infection, the host status of Pa. atlanticum remains inconclusive. Testing of seeds collected from Pe. somniferi and Pe. meconopsidis infected plants of Pa. somniferum, Pa. dubium, Pa. rhoeas and Pa. nudicaule showed presence of both pathogens, indicating likely ability for seed transmission in these species. We identified new hosts of these pathogens and discuss potential implications of these alternative hosts in pathogen survival, dissemination and epidemic initiation.

The viticulture industry needs advanced grape cultivars with genes that enhance disease resistance and environmental stress tolerance to meet the challenges of a changing climate. To discover beneficial allelic variants of grape genes, we established an F1 mapping population from a cross between two North American grapevines, Vitis rupestris Scheele and Vitis riparia Michx. We generated genotyping-by-sequencing (GBS) markers and constructed parental linkage maps consisting of 1177 and 1115 GBS markers, respectively (LOD threshold ≥ 14), which were validated by mapping the sex-determining locus to chromosome 2. Taking advantage of loci heterozygous in both parents, we also constructed an integrated map containing 2583 markers. We mapped a major quantitative trait locus (QTL) for downy mildew (Plasmopara viticola) resistance to chromosome 10 of V. rupestris using both greenhouse- and in vitro-generated leaf resistance data. This QTL explains 66.5% of the phenotypic variance under greenhouse conditions, and its 2-LOD confidence interval corresponds to region 2,470,297 to 3,024,940 bp on chromosome 10 in the Vitis vinifera L. PN40024 reference genome sequence (assembly 12X.v2). We provide PN40024-projected positions of the GBS markers, which can be used as anchors to develop additional markers for the introgression of this V. rupestris haplotype into cultivated grape varieties.

Grape downy mildew (Plasmopara viticola) is one of the most destructive diseases of grapevine worldwide. In order to better understand the cellular processes involved in resistance to downy mildew, leaves of Vitis vinifera cv. ‘Centennial Seedless’ (susceptible) and Vitis labrusca×Vitis riparia cv. ‘Beta’ (resistant) were inoculated with Plasmopara viticola, and were used in the Illumina HiSeq™2000 platform for deep transcriptome sequencing. We performed transcriptome analysis and identified a total of 1091 differentially expressed genes (DEGs) in the disease-resistant cultivar and 849 in the susceptible cultivar. To increase understanding of the DEGs, the datasets were analyzed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genome (KEGG) database. Most of them were found to be associated with plant–pathogen interaction, flavonoid synthesis, phenylpropanoid synthesis metabolism and phytohormone signal transduction. Moreover, transcription factors ERF, MYB, WRKY, and bHLH associated with disease resistance were screened. A total of 196 genes were identified as the candidate resistant genes. The expression of 18 differentially expressed genes was detected by RT-qPCR. Finally, the pattern of differentially expressed genes was consistent with the result of transcriptome sequencing. The present study identified several candidate resistance genes and signal transduction pathways that may contribute to downy mildew resistance in grapes.

Peronospora salviae‐officinalis, the causal agent of downy mildew on common sage, is an obligate biotrophic pathogen. It grows in the intercellular spaces of the leaf tissue of sage and forms intracellular haustoria to interface with host cells. Although P. salviae‐officinalis was described as a species of its own 10 years ago, the infection process remains obscure. To address this, a histological study of various infection events, from the adhesion of conidia on the leaf surface to de novo sporulation is presented here. As histological studies of oomycetes are challenging due to the lack of chitin in their cell wall, we also present an improved method for staining downy mildews for confocal laser scanning microscopy as well as evaluating the potential of autofluorescence of fixed nonstained samples. For staining, a 1:1 mixture of aniline blue and trypan blue was found most suitable and was used for staining of oomycete and plant structures, allowing discrimination between them as well as the visualization of plant immune responses. The method was also used to examine samples of Peronospora lamii on Lamium purpureum and Peronospora belbahrii on Ocimum basilicum, demonstrating the potential of the presented histological method for studying the infection processes of downy mildews in general.

A spectrophotometric method for determining the viability of sporangia and zoospores of the oomycete Plasmopara viticola (causal agent of grapevine downy mildew) is described and evaluated to overcome the limitations of currently available methods for assessing propagule viability. Sporangia produced on leaf discs in the laboratory were harvested at different days after the initiation of sporulation (DAS) to yield differences in sporangium viability. Sporangia were suspended in sterile water, the suspensions were placed in a cuvette, and sporangium germination was monitored in a spectrophotometer (λ = 600 nm) at 2‐ to 3‐min intervals for 5 hr. Absorbance started to increase after sporangia were suspended in water for ~30–60 min followed by major peak(s) for younger sporangia (1–3 DAS), whereas low to no increase in absorbance was observed for senescent sporangia (>7 DAS). Microscopic observation confirmed that the increase in absorbance corresponded to the release and active swimming of zoospores, whereas absorbance decreased when zoospores encysted and settled. A positive correlation (r = .839, p = .0365) was observed when the time to the initial increase in absorbance was plotted against the age of sporangia. The time to the absorbance peak (marking the time of maximum zoospore movement) was shortest for immature sporangia (0 DAS), longest for young sporangia (2 DAS) and decreased for mature and senescent sporangia. A similar pattern was observed for the standardized area under the absorbance curve (indicating the overall quantity of zoospores released), for which values were lowest for immature and senescent sporangia, highest for young sporangia and intermediate for mature sporangia. Consistent patterns obtained across two independent experiments suggest that the method is reproducible and may be further developed for other zoospore‐releasing pathogens.

Sorghum downy mildew (SDM), caused by Peronosclerospora sorghi is one of the important diseases in maize. Here, we illustrate the transfer of two QTLs for SDM on chromosome 3 and 6 from UMI 936 (w) into an elite maize inbred line UMI 79 through marker-assisted backcross breeding (MABC). For foreground selection, plants carrying heterozygote allele for the QTLs for SDM were selected from F1 and three back cross (BC1-BC3) generations using simple sequence repeat (SSR) markers linked to the targeted QTLs region of SDM. Consequently, foreground positive plants from BC3F1 generation selfed to produce BC3F2 and plants carrying the favorable alleles for the QTLs for SDM were selected followed by stringent phenotyping against SDM and advanced to BC3F3. As a result, two improved lines (79/936–1–7-7-7-46-46 and 79/936–1–7-7-10-80-80) possessing QTLs for SDM were obtained. Background selection using 146 SSR markers distributed evenly across the maize genome revealed 92.45% and 89.68% recovery of recurrent parent genome (RPG) in two improved lines. Phenotypic characterization of the improved lines showed more than 90% of recovery of the recurrent parents in morphological traits and a resistance to SDM. In conclusion, the newly developed SDM resistant inbred lines are a potential source for improving SDM resistance in maize breeding programs.

Made available in DSpace on 2022-05-01T09:30:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-02-01 During the winter, there is a high occurrence of downy mildew on lettuce caused by Bremia lactucae. This oomycete shows variability in virulence, so understanding the genetic structure of the pathogen population becomes essential for obtaining resistant cultivars. Thus, the objective of this study was to determine sexual compatibility in Brazilian populations of B. lactucae and investigate the occurrence of sexual reproduction of the pathogen on lettuce (Lactuca sativa) and prickly lettuce (Lactuca serriola). Leaf samples were collected in 33 municipalities in seven Brazilian states. The virulence structure of the populations was monitored using the EU-C sextet code. B. lactucae populations from the states of São Paulo, Paraná, Rio de Janeiro, and Rio Grande do Sul shared six of the 15 virulence factors evaluated. Twenty-five virulence phenotypes (v-phenotypes) were found, with the sextet codes 31-00-02, 31-16-02, 31-24-02, and 31-01-02 being more frequent. The predominance of some v- phenotypes indicates that clonal reproduction is still the main form of B. lactucae propagation. The genes and resistance factors of the cultivars Argelès (Dm38), Balesta, and Bartoli are recommended as suitable sources of lettuce resistance in Brazil. Natural occurrence of oospores was detected in most sampled locations, in lettuce and prickly lettuce plants. Virulence variability of Brazilian isolates is the result of the pathogen's ability to reproduce both sexually and asexually, with a prevalence of homothallic isolates; although the majority were of the predominant B2 mating type, there was a high incidence of predominant B1 in addition to B1 = B2. Department of Agricultural Production Sciences São Paulo State University (Unesp) School of Agricultural and Veterinarian Sciences, SP Department of Plant Pathology Gulf Coast Research and Education Center University of Florida Research Department – Lettuce Breeding Agristar do Brasil, SP Department of Botany Faculty of Science Palacký University in Olomouc Department of Agricultural Production Sciences São Paulo State University (Unesp) School of Agricultural and Veterinarian Sciences, SP

Background Papain-like cysteine proteases (PLCPs), a large group of cysteine proteases, are structurally related to papain. The members belonging to PLCPs family contribute to plant immunity, senescence, and defense responses in plants. The PLCP gene family has been identified in Arabidopsis, rice, soybean, and cotton. However, no systematic analysis of PLCP genes has been undertaken in grapevine. Since Plasmopara viticola as a destructive pathogen could affect immunity of grapes in the field, we considered that the members belonged to PLCPs family could play a crucial role in defensive mechanisms or programmed cell death. We aimed to evaluate the role of PLCPs in 2 different varieties of grapevines and compared the changes of their expressions with the transcriptional data in response to P. viticola. Results In this study, 23 grapevine PLCP (VvPLCP) genes were identified by comprehensive bioinformatics analysis. Subsequently, the chromosomal localizations, gene structure, conserved domains, phylogenetic relationship, gene duplication, and cis-acting elements were analyzed. Numerous cis-acting elements related to plant development, hormone, and stress responses were identified in the promoter of the VvPLCP genes. Phylogenetic analysis grouped the VvPLCP genes into nine subgroups. The transcription of VvPLCP in different inoculation time points and varieties indicated that VvPLCP may have vital functions in grapevine defense against Plasmopara viticola. According to transcriptome data and qPCR analysis, we observed the increasing expression levels of VvRD21–1 at 72 h after inoculation in resistant variety, inferring that it was related to grape downy mildew resistance. Meanwhile, 3 genes including VvXBCP1, VvSAG12–1, and VvALP1 showed higher expression at 24 h after pathogen inoculation in the susceptible variety and might be related to the downy mildew phenotype. We nominated these four genes to function during hypersensitive response (HR) process, inferring that these genes could be associated with downy mildew resistance in grapes. Conclusions Our results provide the reference for functional studies of PLCP gene family, and highlight its functions in grapevine defense against P. viticola. The results help us to better understand the complexity of the PLCP gene family in plant immunity and provide valuable information for future functional characterization of specific genes in grapevine.

Impatiens downy mildew (IDM) disease is a primary constraint on the production of Impatiens walleriana, a popular and economically important floriculture plant. IDM is caused by the biotrophic. oomycete Plasmopara destructor that emerged as a pathogen of I. walleriana in the 2000s. To enable P. destructor detection and quantification, a hydrolysis-probe-based quantitative PCR diagnostic assay was developed based on unique orientation and order of the mitochondrial cytochrome c oxidase subunit1 (cox1) and ATP synthase subunit alpha (atp1) genes in the genus Plasmopara. Nucleotide sequences and analysis of the cox1/atp1 region distinguished P. destructor and its sister-species P. obducens, consistent with prior phylogenetic analyses using cox2 and rDNA markers. Specificity for P. destructor was incorporated into a hydrolysis probe targeting the cox1 gene and flanking primers that amplified across the cox1/atp1 intergenic region. The limit of detection was 0.5 fg/μl of P. destructor DNA (∼100 plasmid copies/μl), with amplification efficiency = 0.95. The assay was validated against a panel of target and nontarget oomycetes, which showed that the primers were specific for Plasmopara spp., while the probe was specific for P. destructor infecting both I. walleriana and I. balsamina. Testing of Impatiens tissue collected from 23 locations across 13 states indicated all samples with IDM symptoms tested positive for P. destructor. Asymptomatic plants from two locations also tested positive for P. destructor.

Bacillus has been extensively studied for agricultural application as a biocontrol agent. B. altitudinis GLB197, an endophytic bacterium isolated from grape leaves, exhibits distinctive inhibition to grape downy mildew based on unknown mechanisms. To determine the genetic traits involved in the mechanism of biocontrol and host-interaction traits, the genome sequence of GLB197 was obtained and further analyzed. The genome of B. altitudinis GLB197 consisted of one plasmid and a 3,733,835-bp circular chromosome with 41.56% G + C content, containing 3,770 protein-coding genes. Phylogenetic analysis of 17 Bacillus strains using the concatenated 1,226 single-copy core genes divided into different clusters was conducted. In addition, average nucleotide identity (ANI) values indicate that the current taxonomy of some B. pumilus group strains is incorrect. Comparative analysis of B. altitudinis GLB197 proteins with other B. altitudinis strains identified 3,157 core genes. Furthermore, we found that the pan-genome of B. altitudinis is open. The genome of B. altitudinis GLB197 contains one nonribosomal peptide synthetase (NRPS) gene cluster which was annotated as lichenysin. Interestingly, the cluster in B. altitudinis has two more genes than other Bacillus strains (lgrD and lgrB). The two genes were probably obtained via horizontal gene transfer (HGT) during the evolutionary process from Brevibacillus. Taken together, these observations enable the future application of B. altitudinis GLB197 as a biocontrol agent for control of grape downy mildew and promote our understanding of the beneficial interactions between B. altitudinis GLB197 and plants.

Hyaloperonospora brassicae causes downy mildew, a major disease of Brassicaceae species. We sequenced the genomes of two H. brassicae isolates of high (Sample B) and low (Sample C) virulence. Sequencing reads were first assembled de novo with software's SOAPdenovo2, ABySS V2.1 and Velvet V1.1 and later combined to create meta-assemblies with genome sizes of 72.762 and 76.950Mb and predicted gene densities of 1628 and 1644 /Mb, respectively. We could annotate 12.255 and 13,030 genes with high proportions (91-92%) of complete BUSCOs for Sample B and C, respectively. Comparative analysis revealed conserved and varied molecular machinery underlying the physiological specialisation and infection capabilities. BLAST analysis against PHI gene database suggested a relatively higher loss of genes for virulence and pathogenicity in Sample C compared to Sample B, reflecting pathogen evolution through differential rates of mutation and selection. These studies will enable identification and monitoring of H. brassicae virulence factors prevailing in-field.

Glyoxalase I (GLYI) is part of the glyoxalase system; its major function is the detoxification of α-ketoaldehydes, including the potent and cytotoxic methylglyoxal (MG). Methylglyoxal disrupts mitochondrial respiration and increases production of reactive oxygen species (ROS), which also increase during pathogen infection of plant tissues; however, there have been few studies relating the glyoxalase system to the plant pathogen response. We used the promoter of VvGLYI-4 to screen the upstream transcription factors and report a NAC (NAM/ATAF/CUC) domain-containing transcription factor VvNAC72 in grapevine, which is localized to the nucleus. Our results show that VvNAC72 expression is induced by downy mildew, Plasmopara viticola, while the transcript level of VvGLYI-4 decreases. Further analysis revealed that VvNAC72 can bind directly to the promoter region of VvGLYI-4 via the CACGTG element, leading to inhibition of VvGLYI-4 transcription. Stable overexpression of VvNAC72 in grapevine and tobacco showed a decreased expression level of VvGLYI-4 and increased content of MG and ROS, as well as stronger resistance to pathogen stress. Taken together, these results demonstrate that grapevine VvNAC72 negatively modulates detoxification of MG through repression of VvGLYI-4, and finally enhances resistance to downy mildew, at least in part, via the modulation of MG-associated ROS homeostasis through a salicylic acid-mediated defense pathway.