Genetic analysis of leaf rust resistance genes and associated markers in the durable resistant wheat cultivar Sinvalocho MA

In the cross of the durable leaf rust resistant wheat Sinvalocho MA and the susceptible line Gama6, four specific genes were identified: the seedling resistance gene Lr3, the adult plant resistance (APR) genes LrSV1 and LrSV2 coming from Sinvalocho MA, and the seedling resistance gene LrG6 coming from Gama6. Lr3 was previously mapped on 6BL in the same cross. LrSV1 was mapped on chromosome 2DS where resistance genes Lr22a and Lr22b have been reported. Results from rust reaction have shown that LrSV1 from Sinvalocho is not the same allele as Lr22b and an allelism test with Lr22a showed that they could be alleles or closely linked genes. LrSV1 was mapped in an 8.5-cM interval delimited by markers gwm296 distal and gwm261 proximal. Adult gene LrSV2 was mapped on chromosome 3BS, cosegregating with gwm533 in a 7.2-cM interval encompassed by markers gwm389 and gwm493, where other disease resistance genes are located, such as seedling gene Lr27 for leaf rust, Sr2 for stem rust, QTL Qfhs.ndsu-3BS for resistance to Fusarium gramineum and wheat powdery mildew resistance. The gene LrG6 was mapped on chromosome 2BL, with the closest marker gwm382 at 0.6 cM. Lines carrying LrSV1, LrSV2 and LrG6 tested under field natural infection conditions, showed low disease infection type and severity, suggesting that this kind of resistance can be explained by additive effects of APR and seedling resistance genes. The identification of new sources of resistance from South American land races and old varieties, supported by modern DNA technology, contributes to sustainability of agriculture through plant breeding.     

Molecular characterization of a wheat -<Emphasis Type="Italic">Thinopyrum ponticum</Emphasis> partial amphiploid and its derived substitution line for resistance to stripe rust

Stripe rust (caused by Puccinia striiformis) occurs annually in most wheat-growing areas of the world. Thinopyrum ponticum has provided novel rust resistance genes to protect wheat from this fungal disease. Wheat – Th. ponticum partial amphiploid line 7430 and a substitution line X005 developed from crosses between wheat and 7430 were resistant to stripe rust isolates from China. Genomic in situ hybridization (GISH) analysis using Pseudoroegneria spicata genomic DNA as a probe demonstrated that the partial amphiploid line 7430 contained ten J^s and six J genome chromosomes, and line X005 had a pair of J^s-chromosomes. Giemsa-C banding further revealed that both lines 7430 and X005 were absent of wheat chromosomes 6B. The EST based PCR confirmed that the introduced J^s chromosomes belonging to linkage group 6, indicating that line X005 was a 6J^s/6B substitution line. Both resistance observation and sequence characterized amplified region (SCAR) markers displayed that the introduced chromosomes 6J^s were responsible for the stripe rust resistances. Therefore, lines 7430 and X005 can be used as a donor in wheat breeding for stripe rust resistance.     

Distribution,population dynamics and damage of Hessian fly,Mayetiola destructor (Diptera: Cecidomyiidae) in North Tunisia

A three years survey and monitoring studies (2013–2014–2015) were carried out through 4 regions of north Tunisia in order to follow the evolution of the distribution, the frequency of occurrence and damage caused by the Hessian fly Mayetiola destructor (Say) to bread wheat (Triticum aestivum L.) and durum wheat (Triticum durum Desf). Moreover, the effectiveness of resistance genes H3, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H22, H23, H25 and H26 to protect wheat from Hessian fly attack was assessed in natural field and under controlled laboratory conditions at INRAT-Kef Station. Results showed that Hessian fly was detected in 60.33% and 51.5% of all sampled durum and bread wheat fields, respectively. This pest was more frequent with a higher percentage of infestation in semi-arid regions. Indeed, during 2013, infestation rate attained 12.39% in Kef region against 0.9% registered in Bizerte region. In order to update information about the annual number of generations, we surveyed the population dynamic of Hessian fly in Kef region. Three generations of the fly were counted annually on wheat, with two complete and one incomplete generation. This insect affects host plant growth at different developmental stages. Plant height was the most affected parameter followed by shoot dry weight and tiller number. Field investigations on host resistance revealed that among the 16 tested resistance genes, and only three were strictly effective (H22, H25 and H26). The resistance genes H5, H9, H13 and H9H13 have also conferred high levels of protection against Hessian fly. This work indicated that H22, H25 and H26 genes could be incorporated into Tunisian wheat varieties and released to farmers to manage the threat due to Hessian fly attacks.     

Tissue‐specific and pathogen‐inducible expression of a fusion protein containing a Fusarium‐specific antibody and a fungal chitinase protects wheat against Fusarium pathogens and mycotoxins

Fusarium head blight (FHB) in wheat and other small grain cereals is a globally devastating disease caused by toxigenic Fusarium pathogens. Controlling FHB is a challenge because germplasm that is naturally resistant against these pathogens is inadequate. Current control measures rely on fungicides. Here, an antibody fusion comprised of the Fusarium spp.‐specific recombinant antibody gene CWP2 derived from chicken, and the endochitinase gene Ech42 from the biocontrol fungus Trichoderma atroviride was introduced into the elite wheat cultivar Zhengmai9023 by particle bombardment. Expression of this fusion gene was regulated by the lemma/palea‐specific promoter Lem2 derived from barley; its expression was confirmed as lemma/palea‐specific in transgenic wheat. Single‐floret inoculation of independent transgenic wheat lines of the T₃ to T₆ generations revealed significant resistance (type II) to fungal spreading, and natural infection assays in the field showed significant resistance (type I) to initial infection. Gas chromatography–mass spectrometry analysis revealed marked reduction of mycotoxins in the grains of the transgenic wheat lines. Progenies of crosses between the transgenic lines and the FHB‐susceptible cultivar Huamai13 also showed significantly enhanced FHB resistance. Quantitative real‐time PCR analysis revealed that the tissue‐specific expression of the antibody fusion was induced by salicylic acid drenching and induced to a greater extent by F. graminearum infection. Histochemical analysis showed substantial restriction of mycelial growth in the lemma tissues of the transgenic plants. Thus, the combined tissue‐specific and pathogen‐inducible expression of this Fusarium‐specific antibody fusion can effectively protect wheat against Fusarium pathogens and reduce mycotoxin content in grain.     

Novel DNA variations to characterize low molecular weight glutenin Glu-D3 genes and develop STS markers in common wheat

Low-molecular-weight glutenin subunits (LMW-GS) play an important role in bread and noodle processing quality by influencing the viscoelasticity and extensibility of dough. The objectives of this study were to characterize Glu-D3 subunit coding genes and to develop molecular markers for identifying Glu-D3 gene haplotypes. Gene specific primer sets were designed to amplify eight wheat cultivars containing Glu-D3a, b, c, d and e alleles, defined traditionally by protein electrophoretic mobility. Three novel Glu-D3 DNA sequences, designated as GluD3-4, GluD3-5 and GluD3-6, were amplified from the eight wheat cultivars. GluD3-4 showed three allelic variants or haplotypes at the DNA level in the eight cultivars, which were designated as GluD3-41, GluD3-42 and GluD3-43. Compared with GluD3-42, a single nucleotide polymorphism (SNP) was detected for GluD3-43 in the coding region, resulting in a pseudo-gene with a nonsense mutation at the 119th position of deduced peptide, and a 3-bp insertion was found in the coding region of GluD3-41, leading to a glutamine insertion at the 249th position of its deduced protein. The coding regions for GluD3-5 and GluD3-6 showed no allelic variation in the eight cultivars tested, indicating that they were relatively conservative in common wheat. Based on the 12 allelic variants of three Glu-D3 genes identified in this study and three detected previously, seven STS markers were established to amplify the corresponding gene sequences in wheat cultivars containing five Glu-D3 alleles (a, b, c, d and e). The seven primer sets M2F12/M2R12, M2F2/M2R2, M2F3/M2R3, M3F1/M3R1, M3F2/M3R2, M4F1/M4R1 and M4F3/M4R3 were specific to the allelic variants GluD3-21/22, GluD3-22, GluD3-23, GluD3-31, GluD3-32, GluD3-41 and GluD3-43, respectively, which were validated by amplifying 20 Chinese wheat cultivars containing alleles a, b, c and f based on protein electrophoretic mobility. These markers will be useful to identify the Glu-D3 gene haplotypes in wheat breeding programs. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

The endophytic fungi from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

In order to study the species composition of endophytes from wheat healthy plants in Buenos Aires Province (Argentina) and to determine their infection frequencies from leaves, stems, glumes and grains, wheat plants were collected from five cultivars at five growth stages from crop emergence to harvest. A total of 1,750 plant segments (leaves, stems, glumes and grains) were processed from the five wheat cultivars at five growth stages, and 722 isolates of endophytic fungi recovered were identified as 30 fungal genera. Alternaria alternata, Cladosporium herbarum, Epicoccum nigrum, Cryptococcus sp., Rhodotorula rubra, Penicillium sp. and Fusarium graminearum were the fungi that showed the highest colonization frequency (CF%) in all the tissues and organs analysed. The number of taxa isolated was greater in the leaves than those in the other organs analysed.     

Silencing of copine genes confers common wheat enhanced resistance to powdery mildew

Powdery mildew, caused by the biotrophic fungal pathogen Blumeria graminis f. sp. tritici (Bgt), is a major threat to the production of wheat (Triticum aestivum). It is of great importance to identify new resistance genes for the generation of Bgt‐resistant or Bgt‐tolerant wheat varieties. Here, we show that the wheat copine genes TaBON1 and TaBON3 negatively regulate wheat disease resistance to Bgt. Two copies of TaBON1 and three copies of TaBON3, located on chromosomes 6AS, 6BL, 1AL, 1BL and 1DL, respectively, were identified from the current common wheat genome sequences. The expression of TaBON1 and TaBON3 is responsive to both pathogen infection and temperature changes. Knocking down of TaBON1 or TaBON3 by virus‐induced gene silencing (VIGS) induces the up‐regulation of defence responses in wheat. These TaBON1‐ or TaBON3‐silenced plants exhibit enhanced wheat disease resistance to Bgt, accompanied by greater accumulation of hydrogen peroxide and heightened cell death. In addition, high temperature has little effect on the up‐regulation of defence response genes conferred by the silencing of TaBON1 or TaBON3. Our study shows a conserved function of plant copine genes in plant immunity and provides new genetic resources for the improvement of resistance to powdery mildew in wheat.     

Identification of RAPD markers for 11 Hessian fly resistance genes in wheat

 The pyramiding of genes that confer race- or biotype-specific resistance has become increasingly attractive as a breeding strategy now that DNA-based marker-assisted selection is feasible. Our objective here was to identify DNA markers closely linked to genes in wheat (Triticum aestivum L.) that condition resistance to Hessian fly [Mayetiola destructor (Say)]. We used a set of near-isogenic wheat lines, each carrying a resistance gene at 1 of 11 loci (H3, H5, H6, H9, H10, H11, H12, H13, H14, H16 or H17) and developed by backcrossing to the Hessian fly-susceptible wheat cultivar ‘Newton’. Using genomic DNA of these 11 lines and ‘Newton’, we have identified 18 randomly amplified polymorphic DNA (RAPD) markers linked to the 11 resistance genes. Seven of these markers were identified by denaturing gradient gel electrophoresis and the others by agarose gel electrophoresis. We confirmed linkage to the Hessian fly resistance loci by cosegregation analysis in F₂ populations of 50–120 plants for each different gene. Several of the DNA markers were used to determine the presence/absence of specific Hessian fly resistance genes in resistant wheat lines that have 1 or possibly multiple genes for resistance. The use of RAPD markers presents a valuable strategy for selection of single and combined Hessian fly resistance genes in wheat improvement. Received: 20 March 1996 / Accepted: 6 September 1996     

Influence of plant resistance at the third trophic level: interactions between parasitoids and entomopathogenic fungi of cereal aphids

Host-plant resistance can affect herbivorous insects and their natural enemies such as parasitoids and entomopathogenic fungi. This tritrophic effect acts on interspecific interactions between the two groups of natural enemies distantly related in phylogenetic terms. The intra- and extra-host aspects of the interaction between the cereal aphid parasitoid Aphidius rhopalosiphi and the entomopathogenic fungus Erynia neoaphidis developing on the grain aphid, Sitobion avenae, on resistant and susceptible wheat (Triticum aestivum) cultivars, were studied. The competitive outcome of the intra-host interaction depended on the timing of parasitoid oviposition and fungal infection and was affected by wheat resistance. In particular, survival of the parasitoid was lower on the resistant wheat cultivar than the susceptible wheat cultivar, when the competitive outcome of the interaction was favourable for either parasitoid or fungal development. Before and after this period the influence of plant resistance was not significant. Furthermore, the extra-host interaction was not affected by the wheat cultivar, although an increase in fungal infection of S. avenae was observed when parasitoids foraged in the experimental arena with sporulating aphid cadavers compared with foraging in the absence of sporulating cadavers. Our results showed that the host plant may affect interspecific interactions between parasitoids and fungi and that these interactions depended on the timing of parasitoid oviposition and fungal infection. Received: 16 March 1998 / Accepted: 24 August 1998     

The role of reactive oxygen species in the virulence of wheat leaf rust fungus Puccinia triticina

Reactive oxygen species (ROS) play an important role during host–pathogen interactions and are often an indication of induced host defence responses. In this study, we demonstrate for the first time that Puccinia triticina (Pt) generates ROS, including superoxide, H₂O₂ and hydroxyl radicals, during wheat infection. Through pharmacological inhibition, we found that ROS are critical for both Pt urediniospore germination and pathogenic development on wheat. A comparative RNA-Seq analysis of different stages of Pt infection process revealed 291 putative Pt genes associated with the oxidation–reduction process. Thirty-seven of these genes encode known proteins. The expressions of five Pt genes, including PtNoxA, PtNoxB, PtNoxR, PtCat and PtSod, were subsequently verified using RT-qPCR analysis. The results show that the expressions of PtNoxA, PtNoxB, PtNoxR, PtCat and PtSod are up-regulated during urediniospore germination. In comparison, the expressions of PtNoxA, PtNoxB, PtNoxR and PtCat are down-regulated during wheat infection from 12 to 120 h after inoculation (HAI), whereas the expression of PtSod is up-regulated with a peak of expression at 120 HAI. We conclude that ROS are critical for the full virulence of Pt and a coordinate down-regulation of PtNox genes may be important for successful infection in wheat.     

Organic farming increases richness of fungal taxa in the wheat phyllosphere

Organic farming is often advocated as an approach to mitigate biodiversity loss on agricultural land. The phyllosphere provides a habitat for diverse fungal communities that are important for plant health and productivity. However, it is still unknown how organic farming affects the diversity of phyllosphere fungi in major crops. We sampled wheat leaves from 22 organically and conventionally cultivated fields in Sweden, paired based on their geographical location and wheat cultivar. Fungal communities were described using amplicon sequencing and real‐time PCR. Species richness was higher on wheat leaves from organically managed fields, with a mean of 54 operational taxonomic units (OTUs) compared with 40 OTUs for conventionally managed fields. The main components of the fungal community were similar throughout the 350‐km‐long sampling area, and seven OTUs were present in all fields: Zymoseptoria, Dioszegia fristingensis, Cladosporium, Dioszegia hungarica, Cryptococcus, Ascochyta and Dioszegia. Fungal abundance was highly variable between fields, 10³–10⁵ internal transcribed spacer copies per ng wheat DNA, but did not differ between cropping systems. Further analyses showed that weed biomass was the strongest explanatory variable for fungal community composition and OTU richness. These findings help provide a more comprehensive understanding of the effect of organic farming on the diversity of organism groups in different habitats within the agroecosystem.     

In Silico Identification and Evolutionary Analysis of Plant <Emphasis Type="Italic">MAPKK6s</Emphasis>

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in plants. Linking upstream MAPK kinase kinase (MAPKKK) to downstream MAPK, MAPK kinase (MAPKK) plays a crucial role in MAPK cascade. MAPKK6 is one member of the MAPKK family. In this study, we have found that plant MAPKK6 genes are widely distributed in different plant species, including moss, seedless vascular plants, gymnosperms, and angiosperms. However, no MAPKK6 can be found in genomes of algae. Analysis of exon–intron organization and intron phase showed that plant MAPKK6s are highly conserved genes during plant evolution. In Physcomitrella patens, Selaginella moellendorffii, and Picea glauca, MAPKK6s exist as multicopy genes. In most high plants, however, MAPKK6s exist as single-copy. Phylogenetic analysis indicated that the occurrence of single-copy of MAPKK6s in high plants is likely because of genomic copy-number loss.     

Virulence Spectrum of Mycosphaerella graminicola Isolates on Wheat Genotypes Carrying Known Resistance Genes to Septoria tritici Blotch

The virulence spectrum of 23 monopycnidiospore isolates of Mycosphaerella graminicola was determined using wheat genotypes that carried different resistance genes (Stb1–Stb8 and Stb15). Disease severity was measured as the percentage of necrotic leaf area. The isolates used in the experiments were of diverse origin: eight from Poland, seven from Germany, and eight from other countries around the world. Analysis of variance revealed significant differences in the virulence of the isolates. Using multiple regression and Cook’s D statistic, 26 significant cultivar × isolate interactions were detected. The Israeli isolate IPO86036 showed the widest spectrum of specific reactions. It expressed specific virulence on at least four cultivars and specific avirulence on at least three. The other isolates showed specific interactions with 1–6 different cultivars. Despite the limited number of isolates that were tested, we recommend that a number of resistant lines, namely cultivars Veranopolis (Stb2), Cs/Synthetic 7D (Stb5), Arina (Stb15, Stb6 and partial resistance), and Liwilla (unknown resistance factors), could be incorporated into central European wheat breeding programmes that are aimed at developing resistance against septoria tritici blotch. In contrast, resistance gene Stb7, which is carried by cultivar Estanzuela Federal, was ineffective against most of the isolates that were used. These results on the virulence spectrum of M. graminicola isolates provide valuable information for effective wheat breeding programmes to develop resistance to the pathogen.