Effect of chitosan and vanillin-modified chitosan on wheat resistance to spot blotch

The effect of chitosan- and vanillin-based immune modulators on the development of the phytopathogen Cochliobolus sativus (S. Ito & Kurib.) Drechsler ex Dastur, which induces dark-brown blotch (helminthosporiosis) in wheat, has been studied. It was shown that treatment with these substances led to a decreased injured area in leaves and an increase in the biotrophic period of pathogen development. It was found that vanillin-modified chitosan effectively provided wheat resistance to hemibiotrophic pathogen C. sativus. Changes in leaf peroxidase activity correlated with the manifestation of disease symptoms.     

Bioengineering of the Plant Culture of <Emphasis Type="Italic">Capsicum frutescens</Emphasis> with Vanillin Synthase Gene for the Production of Vanillin

Production of vanillin by bioengineering has gained popularity due to consumer demand toward vanillin produced by biological systems. Natural vanillin from vanilla beans is very expensive to produce compared to its synthetic counterpart. Current bioengineering works mainly involve microbial biotechnology. Therefore, alternative means to the current approaches are constantly being explored. This work describes the use of vanillin synthase (VpVAN), to bioconvert ferulic acid to vanillin in a plant system. The VpVAN enzyme had been shown to directly convert ferulic acid and its glucoside into vanillin and its glucoside, respectively. As the ferulic acid precursor and vanillin were found to be the intermediates in the phenylpropanoid biosynthetic pathway of Capsicum species, this work serves as a proof-of-concept for vanillin production using Capsicum frutescens (C. frutescens or hot chili pepper). The cells of C. frutescens were genetically transformed with a codon optimized VpVAN gene via biolistics. Transformed explants were selected and regenerated into callus. Successful integration of the gene cassette into the plant genome was confirmed by polymerase chain reaction. High-performance liquid chromatography was used to quantify the phenolic compounds detected in the callus tissues. The vanillin content of transformed calli was 0.057% compared to 0.0003% in untransformed calli.     

Global insight into the distribution of velvet-like B protein in <Emphasis Type="Italic">Cochliobolus</Emphasis> species and implication in pathogenicity and fungicide resistance

The Cochliobolus genus consist of over 55 species among which the 5 most devastating are Cochliobolus carbonum, Cochliobolus heterostrophus, Cochliobolus miyabeanus, Crocus sativus and Cochliobolus lunatus causing damages in sorghum, wheat, rice, maize, cassava and soybean estimated at over 10 billion USD per annum worldwide. The dynamic pathogenicity of Cochliobolus species and the plethora of infected hosts is determined by the evolution of virulence determinants such as the velvet-like B protein (VelB). Nonetheless, the knowledge on the distribution of Cochliobolus VelB and its implication in pathogenicity and fungicide resistance are often lacking. By scanning through the annotated genomes of C. lunatus, C. heterostrophus, C. carbonum, C. victoriae, C. sativus and C. miyabeanus, it is revealed that the numbers of ortholog VelB and cognates vary. By using the phylogenetic approach, it is established that the diversification rates among velvet-domain-containing proteins for phytopathogenic Cochliobolus species could impact differently on their oxidant and fungicide resistance potentials, ability to form appressoria-like structures and infection pegs during infection. This study provides new insights into the pathogenicity evolution of Cochliobolus species at the VelB locus which is relevant for designing effective strategies for durable management of Cochliobolus diseases.     

Inhibition of lipase activity by low-molecular-weight chitosan

Inhibition of enzymatic activity of lipase (EC 3.1.1.3) from the fungus Candida rugosa and wheat (Triticum aestivum L.) germ by low-molecular-weight chitosan with an average molecular weight of 5.7 kDa in reactions of p-nitrophenyl palmitate cleavage was studied. Preincubation of lipases with chitosan, prior to addition of the substrate to solution, showed that equilibrium during the lipase-inhibitor complex formation was reached within 30 min. The inhibition constants for C. rugosa lipase and wheat germ lipase were 1.4 and 0.9 mM, respectively. The contribution of electrostatic interactions to the complex formation between chitosan and lipases is insignificant.     

Inhibition of lipase activity by low-molecular-weight chitosan

Inhibition of enzymatic activity of lipase (EC 3.1.1.3) from the fungus Candida rugosa and wheat (Triticum aestivum L.) germ by low-molecular-weight chitosan with an average molecular weight of 5.7 kDa in reactions of p-nitrophenyl palmitate cleavage was studied. Preincubation of lipases with chitosan, prior to addition of the substrate to solution, showed that equilibrium during the lipase-inhibitor complex formation was reached within 30 min. The inhibition constants for C. rugosa lipase and wheat germ lipase were 1.4 and 0.9 mM, respectively. The contribution of electrostatic interactions to the complex formation between chitosan and lipases is insignificant.     

Induction of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Resistance to Pathogenic Bacteria by Lipopolysaccharide and Salicylic Acid

The effects of combined treatment with an elicitor (lipopolysaccharide) and a signaling molecule (salicylic acid) on the disease resistance of wild-type (Col-0) and mutant Arabidopsis thaliana L. plants have been compared. The mutant lines used were jin1 (with impaired jasmonate signaling), npr1 (lacking expression of pathogen-dependent PR genes), and NahG (expressing an active bacterial salicylate hydroxylase transgene). The lipopolysaccharide was isolated from a saprophytic strain (8614) of Pseudomonas aeruginosa bacteria. Treatment of A. thaliana seeds with a composite preparation (lipopolysaccharide and salicylic acid–SA) increased the resistance of seedlings to a subsequent infection by the pathogenic 9096 strain of P. aeruginosa bacteria. The protective effect was more pronounced in jin1 mutant seedlings, which was indicative of the possible compensation of jasmonate signaling impairment due to activation of the SA-dependent signaling pathway. We concluded that a preparation composed of an elicitor and a signaling molecule could affect regulatory mechanism functioning in a plant cell and, in particular, compensate for the absence of a certain signaling pathway by activating another.     

Effect of benzothiadiazole,an inducer of systemic acquired resistance,on the pathogenesis of wheat brown rust

We studied the effects of systemic acquired resistance inducer, benzothiadiazole (BT, benzo-(1,2,3-thiadiazole-7-carbothionic acid S-methyl ester, commercial name BION), on the development of brown rust caused by the fungus Puccinia triticina Erikss. in nearly isogenic lines of common wheat (Triticum aestivum L, cv. Thatcher) carrying the resistance genes Lr19 and Lr24. A dependence of BT efficiency on the time between treatment with the inducer and inoculation with urediniospores was observed. A difference in BT action on pathogenesis during wheat line infection with virulent and avirulent P. triticina clones was detected. In compatible combinations, the morphogenesis of virulent clone mycelium was suppressed without the hypersensitive response. The inhibition of the development of avirulent clone colonies was accompanied by the intense hypersensitive response. Treatment with cycloheximide enhanced avirulent clone development during 3 days after inoculation; however, later colony growth ceased.     

Root characteristics of grafted peppers and their resistance to <Emphasis Type="Italic">Fusarium solani</Emphasis>

Root rot caused by Fusarium solani, is one of the most severe diseases in pepper (Capsicum annuum L.). Grafting has been attempted as an effective means to control the disease, but little is known about the disease resistance mechanism in grafted pepper. Therefore, we investigated the changes of biomass, cell structure, and the secondary metabolism in roots of control (non-grafted pepper) and grafted peppers using cvs. Weishi and Buyeding as rootstocks and the cv. Xinfeng 2 as a scion. After a manual inoculation, less F. solani invaded grafted pepper roots and consequently less serious injury to the root cell ultra-structure compared with the control was found. The roots of grafted pepper infected with F. solani exhibited greater biomass production and root activity than the roots of infected controls. Grafting led to an increased content of salicylic acid, benzoic acid, vanillin, lignin, and polyamines, as well as activities of phenylalanine ammonia lyase, polyphenoloxidase, and peroxidase. These results suggest that grafting improved the resistance of peppers to root rot.     

Identification of 50 K Illumina-chip SNPs associated with resistance to spot blotch in barley

Background

Spot blotch, caused by Cochliobolus sativus, is one of the most widespread and harmful diseases in barley. Identification of genetic loci associated with resistance to C. sativus is of importance for future marker-assisted selection. The goal of the current study was to identify loci conferring seedling resistance to two different pathotypes of C. sativus in the Siberian spring barley core collection.

Results

A total of 96 spring barley cultivars and lines were phenotyped at the seedling stage with two C. sativus isolates (Kr2 and Ch3). According to the Fetch-Steffenson rating scale 16%/17% of genotypes were resistant and 26%/30% were moderate-resistant to the Kr2/Ch3 isolates respectively. A total of 94 genotypes were analyzed with the barley 50 K Illumina Infinium iSELECT assay. From 44,040 SNPs, 40,703 were scorable, from which 39,140 were polymorphic. 27,319 SNPs passed filtering threshold and were used for association mapping. Data analysis by GLM revealed 48 and 41 SNPs for Kr2 and Ch3 isolates, respectively. After application of 5% Bonferroni multiple test correction, only 3 and 27 SNPs were identified, respectively. A total of three genomic regions were associated with the resistance. The region on chromosome 3H associated with Ch3-resistance was expanded between markers SCRI_RS_97417 and JHI-Hv50k-2016-158003 and included 11 SNPs, from which JHI-Hv50k-2016-157070, JHI-Hv50k-2016-156842 had the lowest p-values. These two SNPs were also significant in case of Kr2 isolate. The region on chromosome 2H included 16 loci (7 of them with the lowest p-values were tightly linked to BOPA2_12_11504). Three loci corresponding to this region had suggestive p-values in case of Kr2 tests, so the locus on chromosome 2H may also contribute to resistance to Kr2 isolate. The third region with significant p-value in case of Kr2 tests was identified on chromosome 1H at the locus JHI-Hv50k-2016-33568.

Conclusions

Three genomic regions associated with the resistance to one or both isolates of C. sativus were identified via screening of the Siberian spring barley core collection. Comparison of their location with QTLs revealed previously either with biparental mapping populations studies or with GWAS of distinct germplasm and other isolates, demonstrated that resistance to isolates Kr2 and Ch3 is conferred by known spot blotch resistance loci. Information on SNPs related can be used further for development of DNA-markers convenient for diagnostics of resistance-associated alleles in barley breeding programs.

     

<Emphasis Type="Italic">Sr33</Emphasis> and <Emphasis Type="Italic">Sr35</Emphasis> gene homolog identification in genomes of cereals related to <Emphasis Type="Italic">Aegilops tauschii</Emphasis> and <Emphasis Type="Italic">Triticum monococcum</Emphasis>

Using bioinformatics analysis, the homologs of genes Sr33 and Sr35 were identified in the genomes of Triticum aestivum, Hordeum vulgare, and Triticum urartu. It is known that these genes confer resistance to highly virulent wheat stem rust races (Ug99). To identify amino acid sites important for this resistance, the found homologs were compared with the Sr33 and Sr35 protein sequences. It was found that sequences S5DMA6 and E9P785 are the closest homologs of protein RGAle, a Sr33 gene product, and sequences M7YFA9 (CNL-C) and F2E9R2 are homologs of protein CNL9, a Sr35 gene product. It is assumed that the homologs of genes Sr33 and Sr35, which were obtained from the wild relatives of wheat and barley, can confer resistance to various forms of stem rust and can be used in the future breeding programs aimed at improvement of national wheat varieties.     

<Emphasis Type="Italic">VpUR9</Emphasis>, a novel RING-type ubiquitin ligase gene from <Emphasis Type="Italic">Vitis pseudoreticulata</Emphasis>, is involved in powdery mildew response in transgenic <Emphasis Type="Italic">V. vinifera</Emphasis> plants

Ubiquitination plays important roles in disease resistance in plants. We report the identification and functional characterization of the RING-type ubiquitin ligase gene VpUR9 from Chinese wild Vitis pseudoreticulata accession Baihe-35-1. VpUR9, encodes 164 amino acids and possesses a RING conserved motif. It is homologously cloned from the cDNA library of the high powdery mildew (Erysiphe necator [Schw.] Burr) resistant V. pseudoreticulata accession Baihe-35-1 inoculated with E. necator. The gene is induced in response to powdery mildew and salicylic acid. VpUR9 fused with FLAG-tag controlled by 35S promoter was transformed into 15 regenerated V. vinifera L. cv. Red Globe lines via Agrobacterium tumefaciens-mediated transformation. Twelve of these lines were confirmed by Western blot of FLAG-tag. As a result, the powdery mildew-resistance of Red Globe transformed with VpUR9 was repressed. Furthermore, the expression of some disease-resistant related genes (NPR1, PR1, PR10 and PAL) of the transgenic Red Globe declined compared with wild type grapes when inoculated with powdery mildew or salicylic acid. When treated with jasmonic acid methyl ester, its PR1 gene expression decreased, while the expressions of NPR1, PR10 and PAL all increased, contrasting with the wild type grape.     

Genome wide association studies (GWAS) of spot blotch resistance at the seedling and the adult plant stages in a collection of spring barley

Spot blotch (SB) in barley is caused by the fungal pathogen Cochliobolus sativus and considered one of the major constraints to successful barley production. Resistance to C. sativus was evaluated, using a barley collection of 336 genotypes (AM-2014), at the seedling and adult stages. Seedling resistance was evaluated by using a mixture of 19 virulent isolates in Morocco. Virulent isolates prevalent in Uttar Pradesh were used for phenotyping resistance at the adult stage in India. The AM-2014 panel was genotyped with 9-K single-nucleotide polymorphism (SNP) markers using iSelect Illumina Infinium. Genome wide association studies (GWAS) were carried out using SNP markers, infection responses, disease severity, and area under the disease progress curve (AUDPC). The mixed linear model was employed in TASSEL using principal component analysis (PCA) and Kinship matrix (K) as covariates. Higher SB severity, 82.3?±?13.5 (mean?±?SD), was recorded at the Banaras Hindu University (BHU) compared to 47.6?±?15.0 at the Narendra Dev University of Agriculture and Technology (NDUAT). Nine QTL, Rcs-qtl-1H-126.9, Rcs-qtl-2H-148.16, Rcs-qtl-3H-25.27, Rcs-qtl-5H-80.35, Rcs-qtl-6H-58.24, Rcs-qtl-7H-29.62, Rcs-qtl-7H-29.72, Rcs-qtl-7H-32.81, and Rcs-qtl-7H-34.74, were detected for SB resistance at the seedling stage. For SB severity at the adult stage, a QTL, Rcs-qtl-7H-32.81, was detected at BHU while seven QTL, Rcs-qtl-2H-91.09, Rcs-qtl-3H-145.64, Rcs-qtl-4H-14.43, Rcs-qtl-6H-6.49, Rcs-qtl-7H-114.43, Rcs-qtl-7H-151.66, and Rcs-qtl-7H-150.36, were found for SB severity at NDUAT. Three QTL, Rcs-qtl-4H-18.61, Rcs-qtl-4H-67.91, and Rcs-qtl-5H-110.25, were significant for AUDPC of SB at BHU. The QTLs reported in this study are important to advance marker-assisted selection and gene pyramiding of SB resistance in South Asia and North Africa in future.     

QTL mapping of Fusarium head blight resistance in three related durum wheat populations

Key message

The QTL Fhb1 was successfully introgressed and validated in three durum wheat populations. The novel germplasm and the QTL detected will support improvement of Fusarium resistance in durum wheat.

Abstract

Durum wheat (Triticum durum Desf.) is particularly susceptible to Fusarium head blight (FHB) and breeding for resistance is hampered by limited genetic variation within this species. To date, resistant sources are mainly available in a few wild relative tetraploid wheat accessions. In this study, the effect of the well-known hexaploid wheat (Triticum aestivum L.) quantitative trait locus (QTL) Fhb1 was assessed for the first time in durum wheat. Three F7-RIL mapping populations of about 100 lines were developed from crosses between the durum wheat experimental line DBC-480, which carries an Fhb1 introgression from Sumai-3, and the European T. durum cultivars Karur, Durobonus and SZD1029K. The RILs were evaluated in field experiments for FHB resistance in three seasons using spray inoculation and genotyped with SSR as well as genotyping-by-sequencing markers. QTL associated with FHB resistance were identified on chromosome arms 2BL, 3BS, 4AL, 4BS, 5AL and 6AS at which the resistant parent DBC-480 contributed the positive alleles. The QTL on 3BS was detected in all three populations centered at the Fhb1 interval. The Rht-B1 locus governing plant height was found to have a strong effect in modulating FHB severity in all populations. The negative effect of the semi-dwarf allele Rht-B1b on FHB resistance was compensated by combining with Fhb1 and additional resistance QTL. The successful deployment of Fhb1 in T. durum was further substantiated by assessing type 2 resistance in one population. The efficient introgression of Fhb1 represents a significant step forward for enhancing FHB resistance in durum wheat.

     

Study of the Protective Activity of Chitosan Hydrolyzate Against Septoria Leaf Blotch of Wheat and Brown Spot of Tobacco

Chitosan hydrolyzate containing low-molecular-weight chitosan (≤24 kDa) and its oligomers (≤1.2 kDa) has been obtained via chemical depolimerization of high-molecular-weight chitosan by nitric acid. The fractions of the obtained chitosan hydrolyzate have been characterized by high performance gel permeation chromatography and proton magnetic resonance. The test performed on detached leaves of wheat has shown that the hydrolyzate completely inhibits the development of Stagonospora nodorum, a casual agent of the septoria leaf blotch, at a concentration of 200 μg/mL. A similar test with detached tobacco leaves has shown that the hydrolyzate at a concentration of 100 μg/mL also inhibits the development of Alternaria longipes, which causes brown spot of tobacco, by 75%.