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Background Powdery mildew is a major disease that causes great losses in soybean yield and seed quality. Disease-resistant varieties, which are generated by reducing the impact of susceptibility genes through mutation in host plants, would be an effective approach to protect crops from this disease. The Mildew Locus O (MLO) genes are well-known susceptibility genes for powdery mildew in plant. In this study, we utilized the CRISPR/Cas9 system to induce targeted mutations in the soybean GmMLO genes to improve powdery mildew resistance. Results A dual-sgRNA CRISPR/Cas9 construct was designed and successfully transferred into the Vietnamese soybean cultivar DT26 through Agrobacterium tumefaciens -mediated transformation. Various mutant forms of the GmMLO genes including biallelic, chimeric and homozygous were found at the T0 generation. The inheritance and segregation of CRISPR/Cas9-induced mutations were confirmed and validated at the T1 and T2 generations. Out of six GmMLO genes in the soybean genome, we obtained the Gmmlo02/Gmmlo19/Gmmlo23 triple and Gmmlo02/Gmmlo19/Gmmlo20/Gmmlo23 quadruple knockout mutants at the T2 generation. When challenged with Erysiphe diffusa , a fungus that causes soybean powdery mildew, all mutant plants showed enhanced resistance to the pathogen, especially the quadruple mutant. The powdery mildew severity in the mutant soybeans was reduced by up to 36.4% compared to wild-type plants. In addition, no pleiotropic effect on soybean growth and development under net-house conditions was observed in the CRISPR/Cas9 mutants. Conclusions Our results indicate the involvement of GmMLO02 , GmMLO19 , GmMLO20 and GmMLO23 genes in powdery mildew susceptibility in soybean. Further research should be conducted to investigate the roles of individual tested genes and the involvement of other GmMLO genes in this disease infection mechanism. Importantly, utilizing the CRISPR/Cas9 system successfully created the Gmmlo transgene-free homozygous mutant lines with enhanced resistance to powdery mildew, which could be potential materials for soybean breeding programs.

Grapevines ( Vitis vinifera L.) form the basis of viticulture, and are susceptible to diseases such as downy mildew ( Plasmopara viticola ) and powdery mildew ( Erysiphe necator ). Therefore, successful viticulture programs require the use of pesticides. Breeding for resistance is the only eco-friendly solution. Marker-assisted selection is currently widely used for grapevine breeding. Consequently, traits of interest must be tagged with molecular markers linked to quantitative trait loci (QTL). We herein present our findings regarding genetic mapping and QTL analysis of resistance to downy and powdery mildew diseases in the progenies of the GF.GA-47-42 (‘Bacchus’ × ‘Seyval’) × ‘Villard blanc’ cross. Simple sequence repeats and single nucleotide polymorphisms of 151 individuals were analyzed. A map consisting of 543 loci was screened for QTL analyses based on phenotypic variations observed in plants grown in the field or under controlled conditions. A major QTL for downy mildew resistance was detected on chromosome 18. For powdery mildew resistance, a QTL was identified on chromosome 15. This QTL was replaced by a novel QTL on chromosome 18 in 2003 (abnormally high temperatures) and 2004. Subsequently, both QTLs functioned together. Additionally, variations in the timing of the onset of veraison, which is a crucial step during grape ripening, were studied to identify genomic regions affecting this trait. A major QTL was detected on linkage group 16, which was supplemented by a minor QTL on linkage group 18. This study provides useful information regarding novel QTL-linked markers relevant for the breeding of disease-resistant grapevines adapted to current climatic conditions.

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.

S-Nitrosylation belongs to principal signalling pathways of nitric oxide in plant development and stress responses. Protein S-nitrosylation is regulated by S-nitrosoglutathione reductase (GSNOR) as a key catabolic enzyme of S-nitrosoglutathione (GSNO), the major intracellular S-nitrosothiol. GSNOR expression, level and activity were studied in leaves of selected genotypes of lettuce (Lactuca sativa) and wild Lactuca spp. during interactions with biotrophic mildews, Bremia lactucae (lettuce downy mildew), Golovinomyces cichoracearum (lettuce powdery mildew) and non-pathogen Pseudoidium neolycopersici (tomato powdery mildew) during 168 h post inoculation (hpi). GSNOR expression was increased in all genotypes both in the early phase at 6 hpi and later phase at 72 hpi, with a high increase observed in L. sativa UCDM2 responses to all three pathogens. GSNOR protein also showed two-phase increase, with highest changes in L. virosa–B. lactucae and L. sativa cv. UCDM2–G. cichoracearum pathosystems, whereas P. neolycopersici induced GSNOR protein at 72 hpi in all genotypes. Similarly, a general pattern of modulated GSNOR activities in response to biotrophic mildews involves a twophase increase at 6 and 72 hpi. Lettuce downy mildew infection caused GSNOR activity slightly increased only in resistant L. saligna and L. virosa genotypes; however, all genotypes showed increased GSNOR activity both at 6 and 72 hpi by lettuce powdery mildew. We observed GSNOR-mediated decrease of S-nitrosothiols as a general feature of Lactuca spp. response to mildew infection, which was also confirmed by immunohistochemical detection of GSNOR and GSNO in infected plant tissues. Our results demonstrate that GSNOR is differentially modulated in interactions of susceptible and resistant Lactuca spp. genotypes with fungal mildews and uncover the role of S-nitrosylation in molecular mechanisms of plant responses to biotrophic pathogens.

• Loss of barley Mildew Resistance Locus O (MLO) is known to confer durable and robust resistance to powdery mildew (Blumeria graminis), a biotrophic fungal leaf pathogen. Based on the increased expression of MLO in mycorrhizal roots and its presence in a clade of the MLO family that is specific to mycorrhizal-host species, we investigated the potential role of MLO in arbuscular mycorrhizal interactions. • Using mutants from barley (Hordeum vulgare), wheat (Triticum aestivum), and Medicago truncatula, we demonstrate a role for MLO in colonization by the arbuscular mycorrhizal fungus Rhizophagus irregularis. • Early mycorrhizal colonization was reduced in mlo mutants of barley, wheat, and M. truncatula, and this was accompanied by a pronounced decrease in the expression of many of the key genes required for intracellular accommodation of arbuscular mycorrhizal fungi. • These findings show that clade IV MLOs are involved in the establishment of symbiotic associations with beneficial fungi, a role that has been appropriated by powdery mildew.

Tobacco mildew is a common postharvest problem caused by fungal growth. It can directly decrease product quality and cause serious economic loss in the tobacco industry. However, the fungal community characteristics of mildewed tobacco leaves and the related influencing factors remain unknown. Here, next-generation sequencing was used to characterize the fungal communities present in mildewed and healthy tobacco leaves stored under three different climatic conditions. Mildewed leaves showed a higher pH and total nitrogen content as well as a lower carbon nitrogen ratio than healthy leaves. Fungal diversity and richness were significantly lower in the mildewed tobacco leaves than in healthy tobacco leaves, with saprophytic fungi such as Xeromyces , Aspergillus , and Wallemia being the dominant molds. Network analysis showed that the complexity, connectivity, and stability of the fungal network were significantly poorer in heavy mildew tobacco leaves than in healthy leaves. NMDS and PERMANOVA analysis showed that the distribution of fungal communities in warehoused tobacco leaves differed significantly across different regions, and temperature and humidity were the key factors affecting these differences. Mildew-causing fungi were significantly enriched in tobacco leaf samples collected in the period between the completion of flue-curing and the start of pre-re-curing. This study demonstrated that mildew is an irreversible process that destroys the balance of the tobacco ecosystem, and that environmental factors play important roles in shaping fungal communities in tobacco leaves. Key points • The diversity and composition of the fungal communities in mildewed tobacco leaves were significantly different from those in healthy tobacco leaves. • Climatic factors may play an important role in shaping fungal communities in tobacco leaves. • Tobacco leaves were most vulnerable to mold contamination between the post-flue-curing and pre-re-curing period. Graphical abstract

TALEN-induced mutation of all homologous copies of a gene that represses resistance to an important wheat pathogen confers a trait that has eluded plant breeders for decades. Sequence-specific nucleases have been applied to engineer targeted modifications in polyploid genomes 1 , but simultaneous modification of multiple homoeoalleles has not been reported. Here we use transcription activator–like effector nuclease (TALEN) 2 , 3 and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 (refs. 4 , 5 ) technologies in hexaploid bread wheat to introduce targeted mutations in the three homoeoalleles that encode MILDEW-RESISTANCE LOCUS (MLO) proteins 6 . Genetic redundancy has prevented evaluation of whether mutation of all three MLO alleles in bread wheat might confer resistance to powdery mildew, a trait not found in natural populations 7 . We show that TALEN-induced mutation of all three TaMLO homoeologs in the same plant confers heritable broad-spectrum resistance to powdery mildew. We further use CRISPR-Cas9 technology to generate transgenic wheat plants that carry mutations in the TaMLO-A1 allele. We also demonstrate the feasibility of engineering targeted DNA insertion in bread wheat through nonhomologous end joining of the double-strand breaks caused by TALENs. Our findings provide a methodological framework to improve polyploid crops.

Ecological intensification of agriculture calls for ecological mechanisms to replace anthropogenic inputs. Cereal/legume intercropping increases yields due to species complementarities, it produces high protein food and feed, and it reduces the need for artificial N fertilizer because legumes fix N biologically. In addition, intercropping has the potential to suppress plant diseases, but its efficacy for disease suppression in cereal/legume mixtures has not been well characterized quantitatively. Here we conducted meta-analysis to quantify the disease suppressive effect of intercropping cereals with legumes at different levels of N fertilizer. Intercropping reduced disease incidence (measured by the odds ratio of disease occurrence) by 45% on average. This reduction was significant (P < 0.01) for four out of six studied pathogens: yellow rust (Puccinia striiformis f.sp. tritici) and mildew (Blumeria graminis) in wheat (Triticum aestivum), and chocolate spot (Botrytis fabae) and Fusarium wilt (Fusarium oxysporum) in faba bean (Vicia faba). Disease reduction was marginally significant for yellow rust in barley (Puccinia striiformis f.sp. hordei) (P < 0.10) and not significant for bean rust (Uromyces fabae). The reduction in disease incidence was greatest during the early stages of epidemics. N fertilizer strongly increased the incidence of powdery mildew of wheat, but it did not affect the incidence of the other diseases and it did not affect the effectiveness of intercropping as a management strategy for disease control. While nitrogen input increased powdery mildew incidence in both sole and intercropped wheat, the incidence was lower in the intercropped than sole wheat at all levels of N input. The disease suppressive effect of intercropping on wheat powdery mildew or any other disease was not affected by the amount of nitrogen fertilizer. The results show that intercropping has a substantial and consistent effect on disease incidence in cereal/faba bean mixtures across studies, but is not sufficient to provide complete disease control. Intercropping is therefore best used as a component in an integrated approach for managing plant diseases.

Key messageA single recessive gene for complete resistance to powdery mildew and a major-effect QTL for partial resistance to downy mildew were co-localized in aCucumis hystrixintrogression line of cucumber.Downy mildew (DM) and powdery mildew (PM) are two major foliar diseases in cucumber. DM resistance (DMR) and PM resistance (PMR) may share common components; however, the genetic relationship between them remains unclear. IL52, a Cucumis hystrix introgression line of cucumber which has been reported to possess DMR, was recently identified to exhibit PMR as well. In this study, a single recessive gene pm for PMR was mapped to an approximately 468-kb region on chromosome 5 with 155 recombinant inbred lines (RILs) and 193 F2 plants derived from the cross between a susceptible line ‘changchunmici’ and IL52. Interestingly, pm was co-localized with the major-effect DMR QTL dm5.2 confirmed by combining linkage analysis and BSA-seq, which was consistent with the observed linkage of DMR and PMR in IL52. Further, phenotype–genotype correlation analysis of DMR and PMR in the RILs indicated that the co-localized locus pm/dm5.2 confers complete resistance to PM and partial resistance to DM. Seven candidate genes for DMR were identified within dm5.2 by BSA-seq analysis, of which Csa5M622800.1, Csa5M622830.1 and Csa5M623490.1 were also the same candidate genes for PMR. A single nucleotide polymorphism that is present in the 3ˊ untranslated region (3′UTR) of Csa5M622830.1 co-segregated perfectly with PMR. The GATA transcriptional factor gene Csa5M622830.1 may be a likely candidate gene for DMR and PMR. This study has provided a clear evidence for the relationship between DMR and PMR in IL52 and sheds new light on the potential value of IL52 for cucumber DMR and PMR breeding program.