Model Wheat Genotypes as Tools to Uncover Effective Defense Mechanisms Against the Hemibiotrophic Fungus Bipolaris sorokiniana

ABSTRACT We investigated the interaction of several differentially resistant wheatwith the hemibiotrophic phytopathogenic fungus Bipolaris sorokiniana (teleomorph Cochliobolus sativus). Wheat genotypes Yangmai, M 3 (W7976), Shanghai 4, and Chirya 7 showed higher levels of resistancewith cv. Sonalika, used as a susceptible control. In amicroscopic inspection, we found that fungal penetration intoepidermal layer failed mostly through a cell wall-associated defense. In cases where the fungus successfully overcame epidermal, its spread within the mesophyll tissue (necrotrophic phase) wasin the more resistant genotypes. Epidermal cell wall-associated, spreading as well as the extent of electrolyte leakage of infected, correlated well with field resistance. We propose that cellular hostsuch as formation of cell wall appositions as well as the degreeearly mesophyll spreading of fungal hyphae are indicative of thepotential of the respective host genotype and, therefore, could befor the characterization of new spot blotch resistance traits in cereals.     

Complex interplay of future climate levels of CO2, ozone and temperature on susceptibility to fungal diseases in barley

Barley (Hordeum vulgare) was grown in different climatic environments with elevated [CO₂] (700 vs 385 ppm), [O₃] (60/90 vs 20 ppb) and temperature (24/19 vs 19/12°C day/night) as single factors and in combinations, to evaluate the impact of these climatic factors on photosynthesis and susceptibility to powdery mildew and spot blotch disease. No significant increase in net CO₂ assimilation rate was observed in barley grown under elevated [CO₂] at ambient temperature. However, this rate was positively stimulated under elevated temperature together with a slightly higher potential quantum efficiency of PSII, both at ambient and elevated [CO₂], suggesting that photosynthesis was not limited by [CO₂] at ambient temperature. When growing under elevated temperature or [O₃], infection by the biotrophic powdery mildew fungus decreased, whereas disease symptoms and growth of the toxin‐secreting hemibiotrophic spot blotch fungus increased compared to ambient conditions, implying that climate‐induced changes in disease severity could be linked to the trophic lifestyle of the pathogens. Elevated [CO₂] decreased powdery mildew infection but had no effect on spot blotch disease compared to ambient condition. However, the effect of elevated [CO₂], [O₃] and temperature did not act in an additive manner when combined. This led to a surprising disease development in the combination treatments, where powdery mildew infection increased despite the individual reducing effect of the climatic factors, and spot blotch disease decreased despite the individual promoting effect of temperature and ozone, emphasizing the importance of conducting multifactorial experiments when evaluating the potential effects of climate change.     

Differential effects of phenylalanine ammonia lyase, cinnamyl alcohol dehydrogenase, and energetic metabolism inhibition on resistance of appropriate host and nonhost cereal-rust interactions

ABSTRACT Effects of phenylpropanoid and energetic metabolism inhibition on resistance were studied during appropriate host and nonhost cereal-rust interactions. In the appropriate barley-Puccinia hordei interaction, phenylalanine ammonia lyase (PAL) and cinnamyl alcohol dehydrogenase (CAD) inhibition reduced penetration resistance in two genotypes, suggesting a role for phenolics and lignins in resistance. Interestingly, penetration resistance of the barley genotype 17.5.16 was not affected by phenylpropanoid biosynthesis but penetration resistance was almost completely inhibited by D-mannose, which reduces the energy available in plant host cells. This suggests a parallel in the cellular basis of penetration resistance between 17.5.16 rust and mlo barleys powdery mildew interaction. Results revealed differing patterns of programmed cell death (PCD) in appropriate versus nonhost rust interactions. PAL and CAD inhibitors reduced PCD (hypersensitivity) in appropriate interactions. Conversely, they had no effect in PCD of wheat to P. hordei; whereas D-mannose dramatically reduced nonhost resistance and allowed colony establishment. The differential effects of inhibitors in the expression of the different resistances and the commonalities with the cereal-powdery mildew interaction is analyzed and discussed.     

Associations between fungal and abiotic leaf spotting and the presence of mlo alleles in barley

The hypothesis that the increased use of the powdery mildew-resistance gene mlo has caused the increase in spotting diseases of barley over the past 20 years was tested in field trials. Near-isogenic lines with alleles of the Mlo gene for susceptibility or resistance to mildew in two parental backgrounds were trialled at four sites in Scotland and two in Ireland that were prone to spotting diseases, over 3 consecutive years. Mildew was controlled by sprays with quinoxyfen. Disease levels were low in the trials, the two most important diseases being scald caused by Rhynchosporium secalis and ramularia leaf spot caused by Ramularia collo-cygni . There were high levels of abiotic spotting. Lines with mutant mlo alleles consistently developed less Rh. secalis and Ra. collo-cygni , but more abiotic spots. This study indicates that the mlo mildew-resistance gene has not alone been responsible for the rise in spotting diseases over the past 20 years. Possible reasons for the rise are discussed, including the interaction of the mlo gene with the environment.     

Interaction between powdery mildew and barley with mlo5 mildew resistance

Powdery mildew infection of barley with the mlo5 barley powdery mildew resistance gene was examined, using near-isogenic barley lines, with and without mlo5 resistance, and two near-isogenic powdery mildew isolates, HL3/5 and GE3 with high (virulent) or low (avirulent) penetration efficiency on the resistant barley line. In all isolate–host combinations (except GE3 on the resistant barley line), frequency of haustorium formation increased significantly from zero at 11 h after inoculation to a maximum by 13 h, and there was no subsequent increase up to 24 h. In the susceptible barley line, 27% of appressoria from both isolates formed haustoria. Although this was significantly higher than the frequency of haustorium formation (18%) of HL3/5 on the resistant barley line, HL3/5 was much more successful than GE3 (frequency of haustorium formation less than 1%). The fact that HL3/5 did not possess a generally higher ability to penetrate successfully to form haustoria on the susceptible barley line, indicates that HL3/5 did not overcome the mlo5 resistance by being generally more vigorous. In the resistant barley line, papillae were larger than in the susceptible line; however, both isolates were associated with papillae of the same diameter at the time of penetration. We suggest that the mlo5 resistant barley line confers two different forms of resistance: isolate-specific and isolate-nonspecific.     

大麦种质对叶斑病的抗性鉴定与评价

 由麦根腐平脐蠕胞菌引起的叶斑病在世界各大麦种植区均有发生,严重影响大麦的产量和品质。选育和应用抗性品种是防控该病害最有效的策略,然而可利用的抗源非常有限。在本研究中对中国233份具有代表性的大麦种质资源进行成株期抗叶斑病田间人工接种鉴定,发现只有垦啤麦5号等10份材料对3个供试菌株都表现抗病,仅占供试材料的4.3%。另外对37份国内外重要的叶斑病抗源材料进行苗期及成株期抗叶斑病鉴定,结果显示成株期抗叶斑病材料所占比例为41%~46%,苗期抗性材料所占比例为50%~64%,其中ND17293等11份材料在苗期和成株期对3个菌株均表现为抗病,可作为抗源继续加以利用;基于上述鉴定结果,进一步分析发现供试大麦苗期对三个菌株的抗病比例均高于成株期抗病比例,说明大麦在不同生育期对叶斑病的抗性存在较大差异。另外发现大麦对B. sorokiniana不同致病类型的抗性也存在明显的专化性。     

Temporary partial breakdown of mlo-resistance in spring barley by sudden relief of soil water-stress under field conditions: the effects of genetic background and mlo allele

This study, carried out under field conditions, assessed the extent to which temporary breakdown of mlo- resistance, following relief of water-stress, was determined by genetic background and mlo allele. Commercial barley cultivars expressing the mlo gene for resistance to powdery mildew ( Erysiphe graminis ( Blumeria graminis ) f.sp. hordei ) were tested as well as doubled haploid progeny from spring barley genotypes, a proportion of which were sown in the field in two successive years. Plants were protected from natural rain by a mobile rain shelter and either watered by trickle-irrigation or allowed to dry. Percentage mildew infection resulting from natural inoculum was recorded and the doubled haploid genotypes were classified as resistant, intermediate or susceptible on the basis of their control (watered) treatment scores. In each of the three designated classes, particular genotypes developed infection levels, following relief of water-stress, that were higher than those observed on the well-watered controls. This was found not to be related to the mlo allele as there was no significant difference between the increases observed on resistant plants carrying mlo9 and resistant plants carrying mlo11 . Differences in the degree of breakdown of resistance were attributed to genetic background rather than to the specific mlo allele.     

Calcium ion promotes successful penetration of powdery mildew fungi into barley cells

To determine whether Ca²⁺ promotes powdery mildew penetration, Ca²⁺-treated barley coleoptiles were inoculated with conidia of pathogenic and nonpathogenic fungi. Penetration efficiency of the pathogenic powdery mildew Blumeria graminis was enhanced by Ca²⁺ treatment, but that of the necrotrophic pathogen Helminthosporium sp. remained unaffected. Similarly, when actin-dependent penetration resistance is suppressed with cytochalasin A, Ca²⁺ treatment specifically enhanced penetration of the nonpathogenic powdery mildew Erysiphe pisi but not that of other nonpathogens. Calmodulin inhibitors suppressed the promotive effect of Ca²⁺ on B. graminis penetration. These results suggest that barley powdery mildew specifically requires Ca²⁺ and calmodulin for penetration.     

Effects of phenylpropanoid and energetic metabolism inhibition on faba bean resistance mechanisms to rust

ABSTRACT Effects on penetration and hypersensitive resistance of the cinnamyl acid dehydrogenase (CAD) suicide inhibitor ([(2-hydroxyphenyl) amino] sulphinyl) acetic acid, 1.1 dimethyl ester, which suppresses phenylpro-panoid biosynthesis, and of D-mannose, which sequesters phosphate and reduces energy available in host cells, were studied in faba bean (Vicia faba) genotypes with differing resistance mechanisms to faba bean rust (Uromyces viciae-fabae). Inhibition of CAD reduced penetration resistance in lines 2N-34, 2N-52, V-1271, and V-1272, revealing an important role for phenylpropanoid biosynthesis in the resistance of these lines. Inhibition of CAD also inhibited hypersensitive cell death in these lines. D-mannose had little or no effect on resistance. By contrast, CAD inhibition did not affect penetration resistance of line BPL-261, which has a high degree of penetration resistance not associated with hypersensitive cell death. In BPL-261, D-mannose inhibited penetration resistance. The parallelism between the faba bean genotype responses to rust observed here and the response of barley genotypes with differing resistance mechanisms to powdery mildew after similar inhibitor treatments is analyzed and discussed.     

Ectopic expression of barley constitutively activated ROPs supports susceptibility to powdery mildew and bacterial wildfire in tobacco

ROPs (also called RACs) are RHO-like monomeric G-proteins of plants, well-known as molecular switches in plant signal transduction processes, which are involved in plant development and a variety of biotic and abiotic stress responses. The barley (Hordeum vulgare) ROPs RACB, RAC1 and RAC3 have been shown to be involved in cellular growth, polarity and in susceptibility to the biotrophic barley powdery mildew fungus Blumeria graminis f.sp. hordei. We produced transgenic tobacco (Nicotiana tabacum) plants expressing constitutively activated (CA) mutants of the barley ROPs RACB and RAC3 to monitor the impact of heterologous ROP expression on cell polarity and disease susceptibility of tobacco. CA HvROPs influenced leaf texture, disturbed root hair polarity and induced cell expansion in tobacco. Both barley ROPs induced super-susceptibility to the compatible powdery mildew fungus Golovinomyces cichoracearum but only CA HvRAC3 induced super-susceptibility to the bacterial leaf pathogen Pseudomonas syringae pv. tabaci. Data suggest involvements of ROPs in tobacco cell expansion, polar growth and in response to bacterial and fungal leaf pathogens.     

Antioxidant,ethylene and membrane leakage responses to powdery mildew infection of near-isogenic barley lines with various types of resistance

Leaves of powdery mildew-susceptible barley (Hordeum vulgare cv. Ingrid) and related near-isogenic lines bearing various resistance genes (Mla12, Mlg or mlo5) were inoculated with Blumeria graminis f. sp. hordei race A6. Fungal attack induced several-fold increases in ethylene emission and electrolyte leakage in leaves of susceptible Ingrid beginning 3 days after inoculation. Activities of peroxidase, superoxide dismutase, glutathione S-transferase, ascorbate peroxidase and glutathione reductase enzymes were induced markedly in susceptible leaves 5–7 days after inoculation. Similar, but less pronounced pathogen-induced changes were detected in inoculated leaves of Mla-type resistant plants that show hypersensitive cell death upon inoculation, and, to an even lesser extent, in the Mlg and mlo lines, where no visible symptoms accompanied the incompatible interaction. Glutathione content increased only in susceptible barley 7 days after inoculation. Catalase activity, total ascorbate content and redox state were not influenced by inoculation in any of the genotypes. The activity of dehydroascorbate reductase was significantly reduced 3–5 days after inoculation in the susceptible parental plants and after 5 days in Mla and Mlg lines, while it was stable in the mlo barley. Slightly elevated levels of H₂O₂ were observed in the inoculated resistant plants. In contrast, H₂O₂ content decreased in the susceptible line 7 days after pathogen attack. These data indicate that high levels of antioxidants are involved in the compatible interaction of susceptible barley and powdery mildew by protecting the pathogen from oxidative damage.     

The importance of non‐penetrated papillae formation in the resistance response of triticale to powdery mildew (Blumeria graminis)

Triticale is the intergeneric hybrid between wheat and rye. With the expansion of the triticale growing area, powdery mildew has emerged and become a significant disease on this new host. Recent research demonstrated that this ‘new’ powdery mildew on triticale has emerged through a host range expansion of powdery mildew of wheat. Moreover, isolates sampled from triticale still infect their previous host, wheat, but isolates sampled from wheat hardly infect triticale. Race‐specific and adult‐plant resistance have been identified in triticale cultivars. The main objective of this study was to characterize the cellular basis of powdery mildew resistance in triticale. Commonalities with resistance responses in other cereals such as wheat, barley and oat are discussed. A detailed comparative histological study of various resistance responses during cross‐inoculation of either virulent or avirulent wheat and triticale isolates on both hosts was carried out. The present data provide evidence that for incompatible interactions, the formation of non‐penetrated papillae is the predominant resistance response, while the hypersensitive response (HR) acts as a second line of defence, to cut the fungus off from nutrients, if penetration resistance fails. It is not clear yet what causes the slower growth and reduced colony size of triticale isolates when inoculated on wheat. Possibly, post‐penetration resistance mechanisms, other than HR, are switched on during these (semi‐) compatible interactions. Molecular studies on gene expression and gene function of defence‐related genes might reveal further insights into the genetic basis of these resistance responses.     

What types of powdery mildew can infect wheat-barley introgression lines?

This work is a detailed study of the infection of fungal biotrophic pathogens causing powdery mildew diseases on introgression lines originating from the intergeneric hybridisation between wheat and barley (Triticum aestivum L. × Hordeum vulgare L.). Powdery mildew fungi are among the most widespread biotrophic pathogens of plants also and infect dicot and monocot species. Most powdery mildew species are strictly host specific. They colonize only a narrow range of species or one particular host species. The intergeneric hybridisation between wheat and barley could result in expansions of host ranges of the barley powdery mildew. Our experiments covered natural infections in the field and artificial infections under greenhouse conditions. Formae speciales of powdery mildew were identified on the basis of the sequencing results of ribosomal internal transcribed spacer sequences (rDNA-ITS). We identified Blumeria graminis f.sp. tritici isolate 14 (HM484334) on the wheat parent and all wheat-barley introgression lines and B. g. f. sp. hordei isolate MUMH1723 (AB 273556) on the barley parent, respectively. The wheat-barley introgression lines were inoculated with barley powdery mildew under greenhouse conditions. According to our results the added barley chromosomes (or segments) do not cause host range expansion of barley powdery mildew.     

61份大麦种质资源对蠕孢菌叶斑病的抗性鉴定

 麦根腐平脐蠕孢菌[Bipolaris sorokiniana(Sacc.)Shoemaker]引起的叶斑病是大麦上重要的病害叶部之一,在世界各大麦主要种植区均能造成严重的经济损失。本研究利用3个致病力不同的蠕孢叶斑病菌株Z 12010、Z 12014和Z 13010分别对来源于中国及国外引进的61份大麦种质资源进行苗期抗蠕孢菌叶斑病鉴定,田间接种Z 12014菌株进行成株期抗病性鉴定。在所鉴定的大麦种质中未发现在苗期或成株期对叶斑病免疫的材料。苗期抗叶斑病分析发现,品种之间对3个菌株的抗性存在明显差异,Varunda、Legacy、Tradition、09GW-01、09GW-07、08PJ-36、09PJ-39、垦啤麦7号、垦啤麦10号、驻大麦3号、S-4和中饲麦1号等12份材料对3个菌株表现为中抗至高抗反应型。7份材料成株期抗Z 12014菌株,其中Legacy、蒙啤麦3号、09PJ-39、垦啤麦9号、垦啤麦11号和龙啤麦3号等6份材料具有全生育期抗性特点,品种10-3苗期对菌株Z 12014感病,而成株期抗该菌株。     

Victoria Blight,defense turned upside down

Genes that confer disease resistance to biotrophic pathogens typically encode nucleotide-binding, leucine-rich-repeat proteins (NB-LRRs). These proteins confer resistance by detecting the presence of virulence effectors secreted by biotrophic pathogens. Recognition triggers NB-LRR activation and subsequently, the defense response which often includes localized host cell death. The fungus, Cochliobolus victoriae, is a necrotrophic pathogen that causes a disease called Victoria Blight. Virulence of this fungus is dependent on its production of a peptide called “victorin” that has been traditionally described as a toxin. Only plants that respond to victorin are susceptible to Cochliobolus victoriae whereas those that do not are resistant to the fungus. Genetic and molecular analyses have revealed that victorin functions like a biotrophic effector recognized by a NB-LRR resistance protein in Arabidopsis. Further, numerous plant species express victorin sensitivity suggesting there are numerous NB-LRRs that recognize victorin. Thus, through expression of victorin, C. victoriae is able to exploit plant defense to cause disease and is capable of evoking this response in an array of different plants.     

61份大麦种质资源对蠕孢菌叶斑病的抗性鉴定

 麦根腐平脐蠕孢菌[Bipolaris sorokiniana(Sacc.)Shoemaker]引起的叶斑病是大麦上重要的病害叶部之一,在世界各大麦主要种植区均能造成严重的经济损失。本研究利用3个致病力不同的蠕孢叶斑病菌株Z 12010、Z 12014和Z 13010分别对来源于中国及国外引进的61份大麦种质资源进行苗期抗蠕孢菌叶斑病鉴定,田间接种Z 12014菌株进行成株期抗病性鉴定。在所鉴定的大麦种质中未发现在苗期或成株期对叶斑病免疫的材料。苗期抗叶斑病分析发现,品种之间对3个菌株的抗性存在明显差异,Varunda、Legacy、Tradition、09GW-01、09GW-07、08PJ-36、09PJ-39、垦啤麦7号、垦啤麦10号、驻大麦3号、S-4和中饲麦1号等12份材料对3个菌株表现为中抗至高抗反应型。7份材料成株期抗Z 12014菌株,其中Legacy、蒙啤麦3号、09PJ-39、垦啤麦9号、垦啤麦11号和龙啤麦3号等6份材料具有全生育期抗性特点,品种10-3苗期对菌株Z 12014感病,而成株期抗该菌株。     

A microscopic study of the wheat-powdery mildew relationship after application of the systemic compounds procaine,griseofulvin and 6-azauracil

On wheat seedlings systemically protected by root application of procaine-hydrochloride, griseofulvin or 6-azauracil, germination of oidia ofErysiphe graminis f. sp.tritici and penetration of this fungus into the epidermal cell wall was as high as on control plants. Inhibition of powdery mildew development became apparent only after the penetration process had started. About 85% of the infections were halted within 24 hours after the inoculation, and did not result in the formation of a haustorium. In the other cases usually not more than one haustorium per infection court was formed, which often showed anomalies, characteristic for each compound used. Development of mycelium was scanty or absent and no sporulation occurred. Similarly, on plants of a resistant wheat variety, powdery mildew inhibition became apparent only after penetration of the host had started. There was no development of mycelium or sporulation. A severe reaction of certain epidermal cells to penetration by powdery mildew was observed on resistant as well as on treated and untreated susceptible plants. However, in relation to the total number of infections, the percentage of this type of reaction was low.     

大麦白粉菌种群毒性监测及抗性材料鉴定

2005和2006年从我国冬大麦区采集和分离大麦白粉菌单胞菌株729个,利用Pallas近等基因系进行致病型鉴定和群体毒性频率分析.同时,利用分离得到的不同致病型菌株,通过抗谱分析的方法鉴定了328份大麦品种(系)的白粉病抗性和抗病基因.结果显示:大麦白粉菌群体对抗病基因Mlal Mla(A12)、Mla3、Mla6 Mla14、Mla7 Mla(No3)、Mla7 Ml(Lg2)、Mla9 Mlk、Mla9、Mlal3 MlaRu3、Mlpl、Mlg(Cp)和mlo5的毒性频率为0;对Mla12 MlaEm2、Mla7 Mlk、Mlat Mla8、Mla10MlaDu2和Mlk1的毒性频率很低,分别为0.1%、0.4%、0.9%、2.8%和4.2%.两年共鉴定出不同的致病型21个,致病型000、001和003在两个年度皆为优势致病型.所鉴定的328份材料绝大多数感病,仅37份抗病材料,能明确推导出抗白粉病基因的品种(品系)很少,这些品种(品系)含有的抗白粉病基因为Mr(Bw)Mla8、Mlg、Mira Mla8、Mla9 Mla1、Mla Mla(A12)和mlo5.     

Comparison of resistance-inducing abilities of virulent and avirulent races of Erysiphe graminis f.sp. hordei and a race of Erysiphe graminis f.sp. tritici in barley

Resistance to powdery mildew was induced in barley by preinoculation with virulent and avirulent races of barley powdery mildew ( Erysiphe graminis f.sp. hordei ), and with a race of wheat powdery mildew ( E. graminis f.sp. tritici ). Four inducer densities were tested in 13 different induction periods between 1 and 24 h. Generally, the resistance induced by barley powdery mildew increased up to 10-12 h of induction and was maintained in longer induction periods. The inducing abilities of virulent and avirulent races could not be distinguished up to 10-12 h of induction, after which the inducing ability of avirulent races increased significantly in relation to virulent races. Wheat powdery mildew was able to induce more resistance than barley powdery mildew in induction periods up to 8 h. In a single inoculation procedure the number of haustoria developing from virulent barley powdery mildew decreased as inoculum density increased. The effect was ascribed to induction of resistance. This reduction of infection efficiency in the compatible interaction was compared to induced resistance. However, the inoculum density needed for 50% resistance induction in the double inoculation procedure was approximately 40 times higher than the inoculum density needed for 50% reduction in infection efficiency in the single inoculation procedure.