Control of Mn status and infection rate by genotype of both host and pathogen in the wheat take-all interaction

Take-all is a world-wide root-rotting disease of cereals. The causal organism of take-all of wheat is the soil-borne fungus Gaeumannomyces graminis var tritici (Ggt). No resistance to take-all, worthy of inclusion in a plant breeding programme, has been discovered in wheat but the severity of take-all is increased in host plants whose tissues are deficient for manganese (Mn). Take-all of wheat will be decreased by all techniques which lift Mn concentrations in shoots and roots of Mn-deficient hosts to adequate levels. Wheat seedlings were grown in a Mn-deficient calcareous sand in small pots and inoculated with four field isolates of Ggt. Infection by three virulent isolates was increased under conditions which were Mn deficient for the wheat host but infection by a weakly virulent isolate, already low, was further decreased. Only the three virulent isolates caused visible oxidation of Mn in vitro. The sensitivity of Ggt isolates to manganous ions in vitro did not explain the extent of infection they caused on wheat hosts. In a similar experiment four Australian wheat genotypes were grown in the same Mn-deficient calcareous sand and inoculated with one virulent isolate of Ggt. Two genotypes were inefficient at taking up manganese and were very susceptible to take-all, one was very efficient at taking up manganese and was resistant to take-all, and the fourth genotype was intermediate for both characters. All genotypes were equally resistant under Mn-adequate conditions.     

Take-all in spring-sown cereals under continuous cultivation: disease progress and decline in relation to crop succession and nitrogen

Incidence and severity of the take-all disease in spring wheat and spring barley caused by Gaeumannomyces graminis (syn. Ophiobolus graminis) were studied during seven years of monoculture. The fungus apparently survived for much longer periods in the soil under non-susceptible break-crops than previously recorded. The incidence and severity of infection increased progressively with each successive cereal crop from initially low levels to a maximum within 3–7 years, which was followed by a progressive but limited decline in the disease. Spring wheat was more susceptible to take-all than spring barley and the development of take-all decline (TAD) was recorded earlier in the sequences of wheat than of barley crops. Nitrogen did not influence the disease until the point of maximum incidence and severity, when it caused a reduction in disease levels in addition to that associated with TAD. Factors influencing the time of onset and the rate of development of take-all and of TAD are discussed and possible explanations for TAD are suggested.     

Effect of Ophiobolus graminis infection on the growth of wheat and barley

Glasshouse experiments are reported, in which the development of young wheat and barley plants was examined following inoculation with Ophiobolus graminis (Sacc.) Sacc. The dry weight, leaf area, tiller number and water content of the shoot were reduced by infection. Reductions were equally severe in wheat and barley. The seminal root system of both was severely attacked and its growth retarded. Inoculated plants, however, translocated a greater proportion of their total assimilates to the root system and produced more adventitious roots than healthy plants. As a result there was an increase in both the number and proportion of healthy roots on these plants following the initial infection of their root systems. This effect was more pronounced in barley than in wheat. It is suggested that this may in part account for the reported relative tolerance of barley to take-all attack under field conditions.     

The invasion of root tips of cereals by the cereal cyst nematode Heterodera avenue

Newly germinated seedlings of susceptible cultivars of oats, wheat, barley and rye were inoculated with second-stage juveniles of Heterodera avenae in pots of sand. Subsequent examination showed oat root tips to be more commonly invaded, and by a greater range of nematode numbers than the other cereals. A comparison of oats and barley showed that lower nematode numbers in barley were not due to a higher emigration from barley; invasion, establishment and emigration by nematodes all being greater in oats. Second-stage juveniles were more likely to migrate prior to establishment in barley than in oats.     

Studies on virulence of the take-all fungus, Gaeumannomyces graminis, with reference to methology

Current methods for take-all assessment in laboratory experiment were examined; it was shown that the extent of vascular discoloration may not reflect virulence of a fungal isolate or host resistance to the pathogen under some experimental conditions. A new assessment method for take-all is described, based on the ability of transport eosin past infection sites. It enables hosts or isolates to be compared by ET₅₀ values, the times from inoculation when 50% of plants fail in eosin-uptake through the three oldest seminal roots. Use of this technique suggested that barley roots were less affected than were wheat roots by Gaeumannomyces graminis var. tritici. Further experimental results showed that an isolate of G. graminis that had lost part of its virulence in culture yielded some single-conidium progeny more virulent than itself. When single-condium isolates or a mycelial isolate and its single-conidium progeny were jointly inoculated on wheat, the amount of disease was less than that caused by the more virulent isolate alone.     

Effects of shading and powdery mildew infection on senescence of the root cortex and coleoptile of wheat and barley seedlings,and implications for root- and foot-rot fungi

Summary Nuclear and cytoplasmic staining methods were used to study natural senescence of the root cortex and coleoptile of wheat and barley seedlings grown in glasshouse conditions. Coleoptiles of barley senesced more slowly than those of wheat, paralleling the known difference in rates of root cortex senescence in these cereals. The coleoptiles and root cortices of both cereals senesced more slowly in shaded than in unshaded conditions, but infection of the shoots of barley byErysiphe graminis had little effect on root cortex senescence. The results are discussed in relation to infection by root- and foot-rot fungi. Previous reports on the effects of illumination on take-all infection (Gaeumannomyces graminis) are explained. It is suggested that natural senescence of the coleoptile might affect establishment of infection by the eyespot fungus,Pseudocercosporella herpotrichoides, either directly or through the activities of competing microorganisms.     

Genetic mapping of Dn7, a rye gene conferring resistance to the Russian wheat aphid in wheat

The Russian wheat aphid is a significant pest problem in wheat and barley in North America. Genetic resistance in wheat is the most effective and economical means to control the damage caused by the aphid. Dn7 is a rye gene located on chromosome 1RS that confers resistance to the Russian wheat aphid. The gene was previously transferred from rye into a wheat background via a 1RS/1BL translocation. This study was conducted to genetically map Dn7 and to characterize the type of resistance the gene confers. The resistant line '94M370' was crossed with a susceptible wheat cultivar that also contains a pair of 1RS/1BL translocation chromosomes. The F₂ progeny from this cross segregated for resistance in a ratio of 3 resistant: 1 susceptible, indicating a single dominant gene. One-hundred and eleven RFLP markers previously mapped on wheat chromosomes 1A, 1B and 1D, barley chromosome 1H and rye chromosome 1R, were used to screen the parents for polymorphism. A genetic map containing six markers linked to Dn7, encompassing 28.2 cM, was constructed. The markers flanking Dn7 were Xbcd1434 and XksuD14, which mapped 1.4 cM and 7.4 cM from Dn7, respectively. Dn7 confers antixenosis, and provides a higher level of resistance than that provided by Dn4. The applications of Dn7 and the linked markers in wheat breeding are discussed.Communicated by J. Dvorak     

Pathogenic Variation of Fusarium Isolates Associated with Head Blight of Wheat in Australia

This paper examines the level of pathogenic diversity in Australian Fusarium pseudograminearum and Fusarium graminearum isolates for head blight from the assessment of 51 wheat germplasm lines, barley, triticale, rye, maize and sorghum plants. A set of nine putative wheat differentials were selected and assessed with 10 F. graminearum and 12 F. pseudograminearum isolates. Isolates of both species were pathogenic on all the wheat germplasm lines, barley triticale and rye. The isolates differed largely in a quantitative way with only small differential effects and were statistically demarcated into three pathogenicity groups: low, intermediate and high. Such distribution patterns suggest that wheat germplasm lines employ different resistance mechanisms to each group of isolates and the three pathogenicity groups may have different mechanisms controlling pathogenicity. The aggressiveness of F. graminearum and F. pseudograminearum isolates on the wheat germplasm lines were marginally correlated (r = 0.40). Durum wheats were ranked as the most susceptible while Sumai 3, Ituo Komugi, Sotome A, Sotome and Nobeokabouzu komugi were consistently grouped as resistant by both species. These findings reiterate the need to consider pathogen variability in the screening, selection and improvement of resistance to head blight in wheat.     

Effect of Host Plant Resistance on Haustorium Formation in Cereal Rust Fungi

Haustoria of Puccinia triticina (wheat leaf rust fungus) and P. hordei (barley leaf rust fungus) were isolated from susceptible and partially resistant wheat lines, and susceptible, hypersensitive and partially resistant barley lines. Haustoria were counted and measured. The size of haustoria was similar in the partially resistant and susceptible genotypes but haustoria were smaller in the hypersensitive barley line L94+Pa7. The number of haustoria was reduced in both partially and hypersensitive lines when compared with susceptible ones. Therefore it seems that the reduction in the number of haustoria is a consequence of the resistance that can be attributable either to early abortion of infection units or reduced colony growth. The reduction of the number of haustoria was more pronounced in the adult plant stage.     

Effect of different potting systems; inoculation time; nematode density and sources of cereal cyst nematode (Heterodera filipjevi) on juvenile penetration into wheat root system

Investigations were designed to optimize testing systems for screening wheat breeding lines for resistance to Heterodera filipjevi. The effects of: 1) plant potting systems 2) inoculum level and time of inoculation 3) and type of inoculum of H. filipjevi on detection accuracy were examined in growth chamber experiments in Turkey. The rate of nematode penetration in the highly susceptible variety Bezostaya was used as the base measurement of efficacy. The results showed that the highest level of penetration coupled with high level of germination was obtained in plastic tubes (13 cm long x 3 cm in diam.) when compared to both small flower pots (400 cm3) and smaller plastic tubes (10.2 cm long x 0.8 cm in diam.). The highest rate of nematode penetration into wheat root system was obtained by inoculating the seedlings with 1000 J2 per plant. However, inoculation with 200 J2 at sowing or 200 J2 at sowing plus an additional 200 J2 after germination improved percent penetration when compared to inoculation with 600 or 1000 J2/plant at sowing. The test on the optimum form of inoculum showed that inoculating the seedling with J2's gave the highest rate of nematode penetration over inoculum with eggs or cysts. The results of these experiments demonstrated that screening wheat for resistance can be optimized by raising the seedlings in plastic tubes and inoculating them with 400 J2.     

Wheat Resistance to Spot Blotch Potentiated by Silicon

Spot blotch, caused by the fungus Bipolaris sorokiniana, is one of the most important diseases on wheat. The effects of silicon (Si) on this wheat disease were studied. Plants of wheat cultivars BR‐18 and BRS‐208 were grown in plastic pots containing Si‐deficient soil amended with either calcium silicate (+Si) or calcium carbonate (?Si). The content of Si in leaf tissue was significantly increased by 90.5% for the +Si treatment. There was no significant difference between Si treatments for calcium content, so variations in Si accounted for differences in the level of resistance to spot blotch. The incubation period was significantly increased by 40% for the +Si treatment. The area under spot blotch progress curve, number of lesions per cm² of leaf area, and real disease severity significantly decreased by 62, 36 and 43.5% in +Si treatment. There was no significant effect of Si on lesion size. The role played by total soluble phenolics in the increased resistance to spot blotch of plants from both cultivars supplied with Si was not clear. Plants from cultivar BR‐18 supplied with Si showed the highest values for concentration of lignin‐thioglycolic acid derivatives during the most advanced stages of fungus infection. Chitinase activity was high at the most advanced stages of fungus infection on leaves from both cultivars supplied with Si and may have had an effect on fungus growth based on the reduction of the components of resistance evaluated. Peroxidase activity was found to be high only at 96 h after inoculation of both cultivars supplied with Si. Polyphenoloxidase activity had no apparent effect on resistance regardless of Si treatments. Results revealed that supplying Si to wheat plants can increase resistance against spot blotch.     

Generation of PCR-based markers for the detection of rye chromatin in a wheat background

Oligonucleotide primers were developed to detect the presence of four rye sequences using a PCR assay. These assays give a rye-specific signal from wheat DNA template which contains various rye chromosomes or chromosome segments. The sequences identified were associated with the nucleolar organiser region, the 5S-Rrna-R1 locus, the telomere, and a widely dispersed, rye-specific repetitive element Ris-1. The primers amplified from the well-established loci Nor-R1 and 5S-Rrna-R1 on rye chromosome arm 1RS, and also located a 5s-Rrna locus on chromosome 3R. The telomere-associated sequence was present on every rye chromosome, and was also present, at a low copy number, in both wheat and barley. These assays will be particularly useful for introgression programmes aimed at reducing the rye content of the 1BL.1RS wheat-rye translocation. When multiplexed, the primers will enable a rapid, simultaneous assay for a number of distinct rye loci, which can be derived from a small portion of mature endosperm tissue.     

Fermentation, purification, formulation, and pharmacological evaluation of a prolyl endopeptidase from Myxococcus xanthus: implications for Celiac Sprue therapy

Celiac Sprue is a multi-factorial disease characterized by an inflammatory response to ingested wheat gluten and similar proteins in rye and barley. Proline-rich gluten peptides from wheat, rye, and barley are relatively resistant to gastrointestinal digestion, and therefore persist in the intestinal lumen to elicit immunopathology in genetically susceptible individuals. In this study, we characterize the in vitro gluten detoxifying properties of a therapeutically promising prolyl endopeptidase from Myxococcus xanthus (MX PEP), and describe the development of a prototypical enteric-coated capsule containing a pharmacologically useful dose of this enzyme. A high-cell density fed-batch fermentation process was developed for overproduction of recombinant MX PEP in E. coli, yielding 0.25-0.4 g/L purified protein. A simple, scalable purification and lyophilization procedure was established that yields >95% pure, highly active and stable enzyme as a dry powder. The dry powder was blended with excipients and encapsulated in a hard gelatin capsule. The resulting capsule was enteric coated using Eudragit L30-D55 polymer coat, which provided sufficient resistance to gastric conditions (> 1 h in 0.01 M HCl, pH 2 with pepsin) and rapid release under duodenal conditions (15-30 min release in pH 6.0 in the presence of trypsin and chymotrypsin). In conjunction with pancreatic enzymes, MX PEP breaks down whole gluten into a product mixture that is virtually indistinguishable from that generated by the Flavobacterium meningosepticum (FM) PEP as judged by chromatographic assays. Competitive studies involving selected immunogenic peptides mixed with whole gluten reveal that both PEPs have a wide range of substrate specificity. Our results support further in vitro and in vivo evaluation of the MX PEP capsule as an oral therapeutic agent for Celiac Sprue patients.     

Investigations on Adult Plant Resistance of Barley Against Erysiphe graminis f.sp. hordei

Spring barley cultivars and lines were tested for 3 years in field studies for adult plant resistance against Erysiphe graminis f.sp. hordei. The cultivars Osiris and Asse were selected for further detailed cytological studies and compared with the susceptible cultivar Peruvian. Under controlled greenhouse conditions, the percentage of conidia that had formed a functional haustorium and secondary hyphae (infection efficiency) was reduced in fifth leaves of the adult plant resistant cultivars. On fifth and flag leaves of adult plant resistant cultivars, papillae were formed more frequently under primary germ tubes and appressoria, and fungal penetration was prevented more often than on the susceptible cultivar Peruvian. In ultrastructural studies various types of papillae were observed, but could not be strictly correlated with penetration success or failure of the fungus.     

Allele-specific amplification of polymorphic sites for the detection of powdery mildew resistance loci in cereals

Primers for the polymerase chain reaction (PCR) were tailored to selectively amplify RFLP marker alleles associated with resistance and susceptibility for powdery mildew in cereals. The differentiation between marker alleles for susceptible and resistant genotypes is based on the discrimination of a single nucleotide by using allele-specific oligonucleotides as PCR primers. The PCR assays developed are diagnostic for RFLP alleles at the loci MWG097 in the barley genome and Whs350 in the wheat genome. The first marker locus is closely linked to MlLa resistance in barley, while the latter is linked to Pm2 resistance locus in wheat. PCR analysis of 31 barley and 30 wheat cultivars, with some exceptions, verified the presence or absence of the resistance loci investigated. These rapid PCR-based approaches are proposed as an efficient alternative to conventional procedures for selecting powdery mildew-resistant genotypes in breeding programs.     

A role for managanese in the resistance of wheat plants to take-all

Summary The hypothesis that wheat plants deficient in managenese are predisposed to infection byGaeumannomyces graminis is outlined, and a test of the hypothesis in a soil system is reported. The results supported the hypothesis: wheat plants growing in managanese-deficient soil, although not showing foliar symptoms, were markedly more susceptible to infection; plant analysis confirmed the nutrient status of the plants. A review of the literature on take-all in wheat coupled with the results of our experiments suggests a reinterpretation of the etiology of this disease, since those edaphic factors which promote infection by this organism are those which also render managese unavailable to the host. Managenese nutrition is proposed as a common factor in many of the environmental conditions which influence the host-pathogen balance.     

On the origin of cultivated rye

The origin of cultivated rye has been studied, taking into consideration evidence from various fields. Based on morphological resemblances and cytogenetic affinities, cultivated rye is included in Secale cereale L. emend. Sencer, which also includes annual wild and weedy ryes. Wild populations of 5. cereale , which have evolved from Secale montanum Guss. emend. Sencer, invaded wheat and barley fields during the early days of cultivation and gave rise to weedy ryes with varying degrees of rachis brittleness. Cultivated rye was selected from weedy ryes for non-brittle rachis and bigger caryopsis both unconsciously and consciously by man. The geographic origin of cultivated rye is postulated for the Büyük Ari Dai (Mt. Ararat) and Lake Van area in eastern Turkey. It spread from this area as a weed in wheat and barley fields towards the north, east and west and imposed itself as a secondary crop under conditions unfavourable for wheat and barley. It thus became a crop in its own right in several places independently, in addition to it being known by the people living in the Caucasus and Transcaucasus from very early agricultural times.     

Death of the cereal root cortex: its relevance to biological control of take-all

Cell death in the root cortex of cereals was assessed by an inability to detect nuclei, using acridine orangelfluorescence microscopy after fixation and mild acid hydrolysis. Seminal roots were scanned at x 100 magnification and their cortices were considered dead when nuclei were absent from all cell layers except the innermost one, adjacent to the endodermis; this cell layer remains alive long after the rest of the cortex has died. Cortical death of wheat and barley roots occurred in the absence of major pathogens. Cell death started behind the root hair zone of the main root axis, initially in the outermost cell layer of the cortex and then progressively inwards towards the endodermis; however, the cortex remained alive for a distance of c. 800 μm around emerging root laterals. The rate of cortical death was more rapid in wheat than in barley, both under field conditions and in the glasshouse at 20 °C. Thus, field-grown spring wheat (Sicca) showed 50% death of the root cortex in the top 6 cm of first seminal roots after 35 days (growth stage 1–2), whereas spring barley (Julia) showed 50% death of the root cortex after 67 days (growth stage 8). In the glasshouse, the top 9 cm of first seminal roots on 16-day plants showed 55% cortical death in wheat (Cappelle-Desprez) but only 2.5% cortical death in barley (Igri). The same rates of death were found in all subsequent seminal roots. The wheat root cortex died at the same rate in sterile and unsterile conditions, and at the same rate in the presence/absence of Phialophora radicicola Cain var. graminicola Deacon or Aureobasidium bolleyi (Sprague) von Arx. Hence, although P. radicicola and other soil microorganisms may benefit from root cortex death they do not exert biological control of take-all by enhancing or retarding the rate of this process. To study the effects of cortical death on take-all, Gaeumannomyces graminis (Sacc.) Arx & Olivier var. tritici Walker was point-inoculated at the tips and on older (5 and 15 day) regions of wheat seminal roots. After 17 days at 20 °C the fungus had grown to the same extent as runner-hyphae in all cases, but the severity of disease decreased with increasing age of the root cortex prior to inoculation; thus, G. graminis caused most extensive vascular discoloration and most intense vascular blockage in roots inoculated at their tips. Similar experiments on wheat and barley roots inoculated separately with P. radicicola and G. graminis suggest that at least three factors associated with cortical death influence infection by these fungi: (1) initially, cell death may enhance infection because nutrients are made available to the parasites and host resistance within the cortex is reduced; (2) weak parasites and soil saprophytes may colonise dead and dying cortices in competition with G. graminis and P. radicicola and thereby reduce infection by these fungi; (3) changes in the endodermis and adjacent cell layers may be associated with cortical death and may retard invasion of the stele. Future work will seek to establish the relative importance of these factors and extend this study to other cereal host-fungus combinations.     

Screening for Barley Yellow Dwarf Luteovirus Resistance in Barley on the Basis of Virus Movement

The movement of barley yellow dwarf luteovirus (BYDV) was evaluated in susceptible and resistant barley and bread wheat genotypes. After leaf inoculation, the virus infected the root system and the growing point of susceptible earlier than resistant, barley genotypes. No difference in virus movement occurred in resistant and susceptible wheat genotypes. It was possible to reliably differentiate susceptible from resistant genotypes when root extracts of 41 barley genotypes were tested by DAS-ELISA 3 or 4 days after inoculation at the oneleaf stage. When barley plants inoculated at the two- or three-leaf stage were assayed by tissue-blot ELISA on nitrocellulose membrane, virus was detected in the phloem vessels of the growing points of the susceptible, but not of the resistant genotype, 4–6 days after inoculation. Our results thus suggest that screening for BYDV resistance in barley could be done quickly and cheaply especially when assays are made by the tissue-blot test.     

Pathogen‐inducible Ta‐Lr34res expression in heterologous barley confers disease resistance without negative pleiotropic effects

Plant diseases are a serious threat to crop production. The informed use of naturally occurring disease resistance in plant breeding can greatly contribute to sustainably reduce yield losses caused by plant pathogens. The Ta‐Lr34res gene encodes an ABC transporter protein and confers partial, durable, and broad spectrum resistance against several fungal pathogens in wheat. Transgenic barley lines expressing Ta‐Lr34res showed enhanced resistance against powdery mildew and leaf rust of barley. While Ta‐Lr34res is only active at adult stage in wheat, Ta‐Lr34res was found to be highly expressed already at the seedling stage in transgenic barley resulting in severe negative effects on growth. Here, we expressed Ta‐Lr34res under the control of the pathogen‐inducible Hv‐Ger4c promoter in barley. Sixteen independent barley transformants showed strong resistance against leaf rust and powdery mildew. Infection assays and growth parameter measurements were performed under standard glasshouse and near‐field conditions using a convertible glasshouse. Two Hv‐Ger4c::Ta‐Lr34res transgenic events were analysed in detail. Plants of one transformation event had similar grain production compared to wild‐type under glasshouse and near‐field conditions. Our results showed that negative effects caused by constitutive high expression of Ta‐Lr34res driven by the endogenous wheat promoter in barley can be eliminated by inducible expression without compromising disease resistance. These data demonstrate that Ta‐Lr34res is agronomically useful in barley. We conclude that the generation of a large number of transformants in different barley cultivars followed by early field testing will allow identifying barley lines suitable for breeding.