Brachypodium distachyon exhibits compatible interactions with Oculimacula spp. and Ramularia collo‐cygni,providing the first pathosystem model to study eyespot and ramularia leaf spot diseases

Brachypodium distachyon (Bd) has established itself as an essential tool for comparative genomic studies in cereals and increasing attention is being paid to its potential as a model pathosystem. Eyespot and ramularia leaf spot (RLS) are important diseases of wheat, barley and other small‐grain cereals for which very little is known about the mechanisms of host resistance despite urgent requirements for plant breeders to develop resistant varieties. This work aimed to test the compatibility of interaction of two Bd accessions with the cereal pathogens Oculimacula spp. and Ramularia collo‐cygni, the causal agents of eyespot and RLS diseases, respectively. Results showed that both Bd accessions developed symptoms similar to those on the natural host for all pathogen species tested. Microscopy images demonstrated that R. collo‐cygni produced secondary conidia and both Oculimacula spp. formed characteristic infection structures on successive tissue layers. Visual disease assessment revealed that quantitative differences in disease severity exist between the two Bd accessions. The results presented here provide the first evidence that Bd is compatible with the main causal agents of eyespot and RLS diseases, and suggest that future functional genetic studies can be undertaken to investigate the mechanisms of eyespot and RLS disease resistance using Bd.     

Mining the Watkins collection of wheat landraces for novel sources of eyespot resistance

Eyespot is an economically important stem base disease of wheat caused by the soilborne fungal pathogens Oculimacula yallundae and Oculimacula acuformis. The most effective method of controlling the disease is host resistance. However, there are only three genetically characterized resistances in wheat varieties and further sources of resistance are required. Previous studies have identified resistances in wild relatives, but use of these resistances has been limited by linkage drag with deleterious traits exacerbated by low rates of recombination. Therefore, the identification of novel resistances in hexaploid wheat germplasm is desirable. The Watkins collection currently consists of 1056 hexaploid wheat landraces that represent global wheat diversity at the time of its collection in the 1920s and 1930s. As such, it may contain beneficial agronomic traits such as eyespot resistance. The Watkins collection was screened for resistance to O. yallundae based on a glasshouse test of all 1056 accessions and a polytunnel test of 44 accessions selected from a previous field trial. Resistant lines identified in these tests were retested against both O. yallundae and O. acuformis. This identified 17 accessions with resistance to one or both of the pathogen species. From these, two accessions (1190094.1 and 1190736.3) provided a high level of resistance to both pathogen species. An F₄ population derived from accession 1190736.3 indicated that the resistance to O. acuformis in this accession is conferred by a single gene and therefore would be suitable for introgression into elite wheat varieties to provide an alternative source of eyespot resistance.     

Development of Oculimacula yallundae and O. acuformis (eyespot) lesions on stems of winter wheat in relation to thermal time in the UK

Relationships between development of eyespot, caused by Oculimacula yallundae (OY) or O. acuformis (OA) on stems of winter wheat (cv. Avalon), and thermal time (°C days after sowing) were investigated in field experiments in 1985/86, 1986/87 and 1987/88 (two experiments). In all experiments, the incidence and severity of stem eyespot (uninoculated plots, OY- and OA-inoculated plots) were linearly related to accumulated thermal time after sowing. There were ca. 600–800°C days from the time of the first sample when eyespot lesions were recorded on stems to the time when maximum eyespot incidence or severity was recorded. Relationships between stem eyespot incidence or severity and thermal time differed between seasons, with more severe eyespot in 1986/87 and 1987/88 than 1985/86. Both the severity and volume of stem lesions were initially greater in OY-inoculated plots than OA-inoculated plots in spring but differences were less by harvest in all seasons. The percentage of plants with stems colonized by OY or OA over all plots (including uninoculated) showed a consistent pattern in 1986/87 and 1987/88 (two experiments), with the percentage colonized by OY greater initially and the percentage colonized by OA gradually increasing with time towards harvest.     

Resistance to quinclorac and ALS-inhibitor herbicides in Galium spurium is conferred by two distinct genes

Classical Mendelian experiments were conducted to determine the genetics and inheritance of quinclorac and acetolactate synthase (ALS)‐inhibitor resistance in a biotype of Galium spurium. Plants were screened with the formulated product of either quinclorac or the ALS‐inhibitor, thifensulfuron, at the field dose of 125 or 6 g active ingredient (a.i.) ha^?1 respectively. Segregation in the F₂ generation indicated that quinclorac resistance was a single, recessive nuclear trait, based on a 1 : 3 segregation ratio [resistant : susceptible (R : S)]. Resistance to ALS inhibitors was due to a single, dominant nuclear trait, segregating in the F₂ generation in a 3 : 1 ratio (R : S). The genetic models were confirmed by herbicide screens of F₁ and backcrosses between the F₁ and the S parent. F₂ plants that survived quinclorac treatment set seed and the resulting F₃ progeny were screened with either herbicide. Quinclorac‐treated F₃ plants segregated in a 1 : 0 ratio (R : S), hence F₂ progenitors were homozygous for quinclorac resistance. In contrast, F₃ progeny segregated into three ratios: 1 : 0, 3 : 1 and 0 : 1 (R : S) in response to ALS‐inhibitor treatment. This segregation pattern indicates that their F₂ parents were either homozygous or heterozygous for ALS‐inhibitor resistance. Therefore, there were clearly two distinct resistance mechanisms encoded by two genes that were not tightly linked as demonstrated by segregation patterns of the F₃.     

The impact of resistant and susceptible wheat cultivars on the multiplication of Heterodera filipjevi and H. avenae in parasite‐infested soil

Cereal cyst nematode (CCN) severely threatens wheat production in many regions of China. Cultivars susceptible to CCN are the main reason for its spread. This study was initiated to determine whether wheat cultivars conferring different resistance levels are the dominant determinants of CCN populations in the rhizospheric soil. Field experiments were conducted at two locations in Henan province, China, where high populations of Heterodera filipjevi or H. avenae have occurred, during the growing seasons of 2010/11 and 2011/12. Conventional enumeration of white female nematodes on the plant roots, reproductive factor (Rf) and a molecular diagnostic approach, PreDicta B test, a soil testing service based on a sensitive quantitative PCR technique, were used to determine the nematode populations in the rhizospheric soils of seven common wheat and durum wheat cultivars with different reactions to CCN. The resistant responses to CCN conferred by durum wheat Wascana and Wakooma and common wheat Madsen were effective against both H. filipjevi and H. avenae and resulted in significantly fewer nematodes (<5 females) on the roots, and lower Rf. Those cultivars were effective in limiting nematode propagation, resulting in fewer CCN eggs in their rhizospheric soils. Taikong 6 conferred moderate resistance (5–10 females) to both Heterodera species. Tianmin 668 only showed resistance to H. avenae. Aikang 58 and Wenmai 19 were susceptible to both CCN species, which facilitated increases in the nematode populations. These results demonstrate that the reactions to CCN of wheat genotypes have obvious impact on the propagation of nematodes.     

Effects of blast on components of wheat physiology and grain yield as influenced by fungicide treatment and host resistance

Two field experiments (Exp. 1 and Exp. 2) were carried out to assess the physiological performance and grain yield of wheat cultivars BR‐18 (moderately resistant) and Guamirim (susceptible) inoculated with Pyricularia oryzae in plots treated or untreated with Ópera (fungicide 13.3% epoxiconazole + 5% pyraclostrobin). Results from regression analyses indicated that spike and leaf blast severity at 10–14 days after inoculation (dai) were associated with greater yield losses (highest negative slope) than severity at 18–22 dai. Relative to untreated Guamirim, there were 0.3% and 16% increases in Exp. 1 and 2, respectively, for untreated BR‐18 (resistance alone). For fungicide treatment alone, the mean yield of Guamirim increased by 20% and 61% in Exp. 1 and 2, respectively, relative to the untreated fungicide control, whereas for the fungicide treated BR‐18, the mean yield increased by 26% and 83% in Exp. 1 and 2, respectively. Fungicide application and cultivar resistance resulted in higher measures of leaf health and photosynthetic performance in both spikes and leaves than in the untreated susceptible reference treatment. The results from this study may be useful in future efforts to develop crop loss models and management guidelines for wheat blast.     

Genetic analysis and molecular mapping of resistance to Puccinia striiformis f. sp. pseudo‐hordei in common wheat

This is the first genetic study reporting on the interaction and molecular mapping of resistance to the barley grass stripe rust pathogen (Puccinia striiformis f. sp. pseudo‐hordei, Psph) in common wheat. Seedlings of 638 wheat accessions were tested and it was determined that wheat is a near‐nonhost to Psph based on rare susceptibility observed in <2% of commercial cultivars and <5% of wheat landraces. As previously observed for P. striiformis f. sp. tritici (Pst), the Australian cultivar Teal was highly susceptible to Psph. In contrast, a selection of cv. Avocet carrying complementary resistance genes Yr73 and Yr74 (Avocet R; AvR) was resistant. The Teal × AvR (T/A) doubled haploid (DH) population was used to map resistance in AvR to Psph. Infection types on the T/A DH lines inoculated with Psph and Pst indicated that all DH lines carrying both Yr73 and Yr74 were also resistant to Psph; however, fewer DH lines were susceptible to Psph than expected, suggesting the resistance was more complex. QTL analysis using 9053 DArT‐Seq markers determined that resistance to Psph was polygenically inherited and mapped to chromosomes 3A, 3D, 4A and 5B. The 3DL and 5BL markers co‐located with Yr73 and Yr74, suggesting an overlap between host and non‐host resistance mechanisms.     

Absence of detectable yield penalty associated with insensitivity to Pleosporales necrotrophic effectors in wheat grown in the West Australian wheat belt

Genetic disease resistance is widely assumed, and occasionally proven, to cause host yield or fitness penalties due to inappropriate activation of defence response mechanisms or diversion of resources to surplus preformed defences. The study of resistance gene trade‐offs has so far been restricted to biotrophic pathogens. In some Pleosporales necrotrophic interactions, quantitative resistance is positively associated with insensitivity to effectors. Host lines that differ in sensitivity can easily be identified amongst current cultivars and advanced breeding lines. Large wheat cultivar trials were used to test whether lines sensitive or insensitive to three necrotrophic effectors from Pyrenophora tritici‐repentis and Parastagonospora nodorum differed in yield when subjected to natural disease and stress pressures in the West Australian wheat belt. There was no significant yield penalty associated with insensitivity to the fungal effectors ToxA, SnTox1 and SnTox3. Some yield gains were associated with insensitivity and some of these gains could be attributed to increased disease resistance. It is concluded that insensitivity to these effectors does not render such plants more vulnerable to any relevant biotic or abiotic stress present in these trials. These results suggest that the elimination of sensitivity alleles for necrotrophic effectors is a safe and facile strategy for improving disease resistance whilst maintaining or improving other desirable traits.     

Role of co‐infection by Pectobacterium and Verticillium dahliae in the development of early dying and aerial stem rot of Russet Burbank potato

Potato early dying (PED) is a disease complex primarily caused by the fungus Verticillium dahliae. Pectolytic bacteria in the genus Pectobacterium can also cause PED symptoms as well as aerial stem rot (ASR) of potato. Both pathogens can be present in potato production settings, but it is not entirely clear if additive or synergistic interactions occur during co‐infection of potato. The objective of this study was to determine if co‐infection by V. dahliae and Pectobacterium results in greater PED or ASR severity using a greenhouse assay and quantitative real‐time PCR to quantify pathogen levels in planta. PED symptoms caused by Pectobacterium carotovorum subsp. carotovorum isolate Ec101 or V. dahliae isolate 653 alone included wilt, chlorosis and senescence and were nearly indistinguishable. Pectobacterium wasabiae isolate PwO405 caused ASR symptoms including water‐soaked lesions and necrosis. Greater Pectobacterium levels were detected in plants inoculated with PwO405 compared to Ec101, suggesting that ASR can result in high Pectobacterium populations in potato stems. Significant additive or synergistic effects were not observed following co‐inoculation with these strains of V. dahliae and Pectobacterium. However, infection coefficients of V. dahliae and Ec101 were higher and premature senescence was greater in plants co‐inoculated with both pathogens compared to either pathogen alone in both trials, and V. dahliae levels were greater in basal stems of plants co‐inoculated with either Pectobacterium isolate. Overall, these results indicate that although co‐infection by Pectobacterium and V. dahliae does not always result in significant additive or synergistic interactions in potato, co‐infection can increase PED severity.     

Virulence and toxin synthesis of an azole insensitive Fusarium culmorum strain in wheat cultivars with different levels of resistance to fusarium head blight

Fusarium culmorum causes head blight, produces toxins and reduces yield and quality of cereals. To prevent damage caused by fusarium head blight (FHB), azole fungicides are mainly applied. The occurrence of insensitivity to azoles is a major problem in agriculture. The present study shows that a tebuconazole insensitive strain of F. culmorum can be readily produced in the laboratory, but that the resulting strain of the fungus is of lower fitness in vitro. Insensitivity was confirmed microscopically and by cell viability and metabolic activity. The tebuconazole insensitive strain shows cross insensitivity to nine important azoles. In addition, plants inoculated with the insensitive F. culmorum strain showed no reduction of FHB symptoms and deoxynivalenol (DON) content after tebuconazole treatment, compared to an inoculation with the sensitive strain. Use of wheat cultivars carrying a high resistance level (i.e. cv. Toras) was the most effective method for reducing symptoms and decreasing DON content, independent from the level of fungicide insensitivity of the F. culmorum strain. In conclusion, resistant cultivars and a fungicide mixture which combines different mechanisms of action in fungal metabolism should be applied to avoid fungicide insensitivity of Fusarium spp. in future.     

Mapping of Aegilops umbellulata‐derived leaf rust and stripe rust resistance loci in wheat

Aegilops umbellulata, a non‐progenitor diploid species, is an excellent source of resistance to various wheat diseases. Leaf rust and stripe rust resistance genes from A. umbellulata were transferred to the susceptible wheat cultivar WL711 through induced homoeologous pairing. A doubly resistant introgression line IL 393‐4 was crossed with wheat cultivar PBW343 to develop a mapping population. Tests on BC₂F₇ RILs indicated monogenic inheritance of seedling leaf rust and stripe rust resistance in IL 393‐4 and the respective co‐segregating genes were tentatively named LrUmb and YrUmb. Bulked segregant analysis placed LrUmb and YrUmb in chromosome 5DS, 7.6 cM distal to gwm190. Aegilops geniculata‐derived and completely linked leaf rust and stripe rust resistance genes Lr57 and Yr40 were previously located in chromosome 5DS. STS marker Lr57/Yr40MAS‐CAPS16 (Lr57/Yr40‐CAPS16), linked with Lr57/Yr40 (T756) also co‐segregated with LrUmb/YrUmb. Seedling infection types differentiated LrUmb from Lr57. Absence of leaf rust‐susceptible segregants among F₃ families of the intercross (IL 393‐4/T756) indicated repulsion linkage between LrUmb and Lr57. YrUmb expressed a consistently low seedling response under greenhouse conditions, whereas Yr40 expressed a higher seedling response. Based on the origin of LrUmb/YrUmb from the U genome and Lr57/Yr40 from the M genome, as well as phenotypic differences, LrUmb and YrUmb were formally named Lr76 and Yr70, respectively. These genes have been transferred to Indian wheat cultivars PBW343 and PBW550, and advanced breeding lines are being tested in state and national trials.     

Histopathological assessment of wheat seedling tissues infected by Fusarium pseudograminearum

Histopathological assessment of infection by the crown rot pathogen Fusarium pseudograminearum in wheat seedling tissues was performed using fluorescence microscopy. The coleoptiles and leaf sheaths of four host cultivars of differing susceptibility were examined. Leaf sheaths were most frequently penetrated via stomata, indicated by initial lesions forming at the guard cells. Internally, cell wall penetration was facilitated by penetration structures which appeared as hyphal swellings or septate foot‐shaped appressoria. Colonization of leaf sheaths resulted in the re‐emergence of hyphae from stomata on both surfaces of the sheath. These hyphae are hypothesized to have two major roles; first as exploratory hyphae for colonization of new tissues, and secondly as sites of profuse conidial production. The formation of conidia on the leaf sheath surface was only recorded on the most susceptible bread wheat genotype. No other major differences in host–pathogen interactions were observed among these cultivars. Almost all cell types in the leaf sheath tissues were extensively colonized, except for the vascular bundles and silica cells. This investigation provides the first comprehensive assessment of F. pseudograminearum infection structures and growth patterns during the infection of wheat seedlings.     

Fusarium species and chemotypes associated with fusarium head blight and fusarium root rot on wheat in Sardinia

Environmental conditions in Sardinia (Tyrrhenian Islands) are conducive to fusarium root rot (FRR) and fusarium head blight (FHB). A monitoring survey on wheat was carried out from 2001 to 2013, investigating relations among these diseases and their causal agents. FHB was more frequently encountered in the most recent years while FRR was constantly present throughout the monitored period. By assessing the population composition of the causal agents as well as their genetic chemotypes and EF‐1α polymorphisms, the study examined whether the two diseases could be differentially associated to a species or a population. Fusarium culmorum chemotypes caused both diseases and were detected at different abundances (88% 3‐ADON, 12% NIV). Fusarium graminearum (15‐ADON genetic chemotype) appeared only recently (2013) and in few areas as the causal agent of FHB. In F. culmorum, two haplotypes were identified based on an SNP mutation located 34 bp after the first exon of the EF‐1α partial sequence (60% adenine, 40% thymine); the two populations did not segregate with the chemotype but the A‐haplotype was significantly associated with FRR in the Sardinian data set (P = 0·001), suggesting a possible fitness advantage of the A‐haplotype in the establishment of FRR that was neither dependent on the sampling location nor the sampling year. The SNP determining the Sardinian haplotype is distributed worldwide. The question whether the A‐haplotype segregates with characters facilitating FRR establishment will require further validation on a specifically sampled international data set.     

Resistance to Xanthomonas perforans race T4 causing bacterial spot in tomato breeding lines

Tomato (Solanum lycopersicum) is the second most important vegetable crop in the world. Bacterial spot (BS) of tomato, caused by four species of Xanthomonas: X. euvesicatoria, X. vesicatoria, X. perforans and X. gardneri, results in severe loss in yield and quality due to defoliation and formation of lesions on fruits, respectively. Currently management practices do not offer effective control under conditions of high disease pressure. Thus, developing BS resistance is a critical priority for tomato growers in order to minimize crop losses. Sixty‐three advanced tomato breeding lines, heirlooms and wild tomato lines with diverse genetic backgrounds were screened under greenhouse and field conditions for BS resistance using X. perforans race T4, which was found to be a prevalent race in North Carolina. Race T4 isolate 9 was used to inoculate the plants by spraying, and disease severity was measured using the Horsfall–Barratt scale. Tomato lines 74L‐1W(2008), NC2CELBR, 081‐12‐1X‐gsms, NC22L‐1 (2008) and 52LB‐1 showed resistance to BS in the field and/or greenhouse trials. These lines were derived from S. pimpinellifolium L3707. Screening L3707 followed by development of a mapping population and mapping resistance genes might be useful for breeding resistance against BS in future breeding programmes.     

Involvement of FgMad2 and FgBub1 in regulating fungal development and carbendazim resistance in Fusarium graminearum

Resistance to carbendazim of Fusarium graminearum is conferred by point mutation in the β₂‐tubulin gene that plays an important role in spindle assembly. The spindle assembly checkpoint is a cellular surveillance system that is critical for maintaining genomic stability. Predicted protein Mad2‐ and Bub1‐encoding genes in F. graminearum (FgMad2 and FgBub1) were isolated and characterized. There was no difference in FgMad2 and FgBub1 expression levels between carbendazim‐sensitive and ‐resistant strains; however, after carbendazim treatment FgMad2 expression increased while FgBub1 expression stayed the same. Both the FgMad2 and FgBub1 deletion mutants became more sensitive to carbendazim. The FgMad2 deletion mutants grew more slowly, produced fewer conidia and both hyphae and conidia were malformed. Conversely, deletion of FgBub1 had no effect on fungal development other than a reduction in conidia production. FgMad2 deletion mutants exhibited a severe decrease in perithecia production and pathogenicity along with a down‐regulation of trichothecene production, whereas FgBub1 deletion mutants exhibited only a slight reduction in perithecia production and was accompanied by a twofold increase in trichothecene production. Overall, the results indicate that both FgMad2 and FgBub1 are involved in carbendazim resistance and trichothecene biosynthesis, and FgMad2 plays an important role in fungal development in F. graminearum.     

Genetic architecture of adult plant resistance to leaf rust in a wheat association mapping panel

Leaf rust caused by Puccina triticina is one of the most destructive fungal diseases of wheat (Triticum aestivum). Adult plant resistance (APR) is an effective strategy to achieve long‐term protection from the disease. In this study, findings are reported from a genome‐wide association study (GWAS) using a panel of 96 wheat cultivars genotyped with 874 Diversity Arrays Technology (DArT) markers and tested for adult leaf rust response in six field trials. A total of 13 quantitative trait loci (QTL) conferring APR to leaf rust were identified on chromosome arms 1BL, 1DS, 2AS, 2BL, 2DS, 3BS, 3BL, 4AL, 6BS (two), 7DS, 5BL/7BS and 6AL/6BS. Of these, seven QTLs mapped close to known resistance genes and QTLs, while the remaining six are novel and can be used as additional sources of resistance. Accessions with a greater number of combined QTLs for APR showed lower levels of disease severity, demonstrating additive and significant pyramiding effects. All QTLs had stable main effects and they did not exhibit a significant interaction with the experiments. These findings could help to achieve adequate levels of durable resistance through marker‐assisted selection and pyramiding resistance QTLs in local germplasm.     

Quantitative trait loci associated with resistance to a potato isolate of Verticillium albo‐atrum in Medicago truncatula

Verticillium albo‐atrum is responsible for considerable yield losses in many economically important crops, among them alfalfa (Medicago sativa). Using Medicago truncatula as a model for studying resistance and susceptibility to V. albo‐atrum, previous work has identified genetic variability and major resistance quantitative trait loci (QTLs) to Verticillium. In order to study the genetic control of resistance to a non‐legume isolate of this pathogen, a population of recombinant inbred lines (RILs) from a cross between resistant line F83005.5 and susceptible line A17 was inoculated with a potato isolate of V. albo‐atrum, LPP0323. High genetic variability and transgressive segregation for resistance to LPP0323 were observed among RILs. Heritabilites were found to be 0·63 for area under the disease progress curve (AUDPC) and 0·93 for maximum symptom score (MSS). A set of four QTLs associated with resistance towards LPP0323 was detected for the parameters MSS and AUDPC. The phenotypic variance explained by each QTL (R²) was moderate, ranging from 4 to 21%. Additive gene effects showed that favourable alleles for resistance all came from the resistant parent. The four QTLs are distinct from those described for an alfalfa V. albo‐atrum isolate, confirming the existence of several resistance mechanisms in this species. None of the QTLs co‐localized with regions involved in resistance against other pathogens in M. truncatula.     

Components of quantitative resistance to leaf rust in wheat cultivars: diversity,variability and specificity

The aim of this study was to investigate the potential diversity and pathogen‐specificity of sources of quantitative resistance to leaf rust caused by Puccinia triticina in French wheat germplasm. From a set of 86 genotypes displaying a range of quantitative resistance levels during field epidemics, eight wheat genotypes were selected and challenged in a greenhouse with three isolates of the pathogen, belonging to different pathotypes. Five components of resistance were assessed: infection efficiency, for which an original methodology was developed, latent period, lesion size, spore production per lesion, and spore production per unit of sporulating tissue. High diversity and variability for all these components were expressed in the host × pathotype combinations investigated; isolate‐specificity was found for all the components. The host genotypes displayed various resistance profiles, based on both the components affected and the isolate‐specificity of the interaction. Their usefulness as sources of quantitative resistance was assessed: line LD7 probably combines diversified mechanisms of resistance, being highly resistant for all the components, but displaying isolate‐specificity for all the components; cv. Apache did not show isolate specificity for any of the components, which could be related to the durability of its quantitative resistance in the field over more than 11 years.