Identification of resistance source in wheat germplasm against spot blotch disease caused by Bipolaris sorokiniana

Four hundred and twenty-two spring wheat germplasm (Triticum aestivum L.) lines belonging to Indian, CIMMYT and Chinese wheat programme were evaluated for their tolerance against natural epiphytotic conditions of spot blotch caused by Bipolaris sorokiniana at the hot spot location, Pusa, Bihar, India. Of the 422 entries screened, none of the genotype showed immunity to the disease, whereas 52 were resistant, 180 moderately susceptible, 171 susceptible and 19 highly susceptible. Indian germplasm lines tended to be more susceptible than lines originated from CIMMYT and China. Chirya 3, Chirya 7 and Mayoor from CIMMYT showed high degree of resistance to the disease both under field and polyhouse conditions. On the basis of the disease severity under field conditions, 20 promising resistant genotypes and 10 highly susceptible lines were isolated for further testing under artificial epiphytotic conditions in polyhouse for genetic analysis and their potential for spot blotch resistance breeding.     

Sources and donors of resistance to wheat spot blotch caused by Bipolaris sorokiniana Shoem. (Cochliobolus sativus Drechs. ex Dastur)

The resistance of wheat lines and cultivars from the Institute of Crop Breeding (Harbin, China) and synthetic, hexaploid wheat lines derived from T. durum and T. tauschii (CIMMYT) were screened for resistance to spot blotch Bipolaris sorokiniana Shoem. using field and laboratory tests. The highly and moderately resistant wheat samples were determined. The satisfactory coincidence of data obtained from evaluation of type reaction of seedlings and disease severity in adult plant stage was demonstrated. The genetics of resistance in Chinese lines Long 98-4554, Long 98-4546, Long mai 24, Long mai 23 and Canadian line 181-5 was studied using hybridological analysis. The resistance in these lines was inherited as quantitative traits and was conditioned by a few (one or two) genes. The absence of susceptible plants in F₂ in crosses of resistant lines Long 98-4554, Long 98-4546, Long mai 24 and 181-5 can testify to the presence of a common gene of resistance. Our data reveals the poor genetic diversity for spot blotch resistance in studying wheat genotypes.     

URP‐based DNA Fingerprinting of Bipolaris sorokiniana Isolates Causing Spot Blotch of Wheat

Spot blotch, caused by the pathogen Bipolaris sorokiniana is an important disease of wheat and is responsible for large economic losses world wide. In this study, molecular variability in B. sorokiniana isolates collected from different regions of India was investigated using URP‐PCR technique. All the 40 isolates used in the study were pathogenic when tested on susceptible host, Agra local, although they varied in pathogenicity. Isolate BS‐49 was least virulent showing 4.5 infection index while BS‐75 was the most virulent with 63.4 infection index. The universal rice primers (URPs’) are primers which have been derived from DNA repeat sequences in the rice genome. Out of the 12 URP markers used in the study, 10 markers were effective in producing polymorphic fingerprint patterns from DNA of B. sorokiniana isolates. The analysis of entire fingerprint profile using unweighted pair group method with arithmetic averages (UPGMA) differentiated B. sorokiniana isolates obtained from different geographic regions. One isolate BS‐53 from northern hill zone was different from rest of the isolates showing less than 50% similarity. Broadly, three major clusters were obtained using UPGMA method. One cluster consisted of isolates from North western plain zone; second cluster having isolates from North eastern plain zone and third cluster consisted of isolates from Peninsular zone showing more than 75% genetic similarity among them. One of the markers, URP‐2F (5′GTGTGCGATCAGTTGCTGGG3′) amplified three monomorphic bands of 0.60, 0.80 and 0.90 kb size which could be used as specific markers for identification of B. sorokiniana. Further, based on URP‐PCR analysis, the grouping of the isolates according to the geographic origin was possible. This analysis also provided important information on the degree of genetic variability and relationship between the isolates of B. sorokiniana.     

Potentiality of organotin(IV) compounds in the control of foliar blight disease of wheat (Triticum aestivum) caused by Bipolaris sorokiniana

Three different types of eight organotin(IV) compounds (of which four were newly synthesised) were screened against Bipolaris sorokiniana. The new compounds were characterised by elemental, IR, ¹H and ¹³C NMR spectral analyses. The experiments were carried out in the field and laboratory between the months of November 2006 to March 2007 and November 2007 to March 2008 at Uttar Banga Krishi Viswavidyalaya, Cooch Bihar, W.B., India. All of the organotin(IV) compounds were tested for the toxicity assay against Indian wheat (Triticum aestivum L.), cv. Sonalika. The spore germination and growth of B. sorokiniana and the biochemical changes associated with the induction of resistance by these chemicals were also tested. The influence of the organic groups (attached to tin atom) of the three different types of compounds studied on the fungicidal activity is remarkably distinct. Some of the compounds tested are more active in controlling the fungus than a commonly used commercial product.     

小麦根腐病菌索氏平脐蠕孢SYBR Green I实时荧光定量PCR检测技术研究

由索氏平脐蠕孢Bipolaris sorokiniana引起的小麦根腐病,常和其他土传真菌病害混合发生,传统的症状鉴别方法很难区分,导致病害防控难度增加。为建立病菌实时荧光定量检测体系,根据ITS序列设计引物,筛选出1对特异性引物BS‐F/R,扩增片段大小为280bp。以菌丝DNA为标准品构建实时荧光定量标准曲线,并对其灵敏度、特异性、可重复性进行评价。结果表明,建立的实时荧光定量PCR检测方法速度快,灵敏度高,特异性强,重复性好。构建的荧光定量PCR标准曲线循环阈值与模板浓度呈良好的线性关系,溶解曲线的吸收峰单一,扩增效率良好。利用该定量检测体系,可以检测出田间小麦样品中52.8fg/μL的病菌DNA。     

A novel effector,CsSp1, from Bipolaris sorokiniana,is essential for colonization in wheat and is also involved in triggering host immunity

The hemibiotrophic pathogen Bipolaris sorokiniana causes root rot, leaf blotching, and black embryos in wheat and barley worldwide, resulting in significant yield and quality reductions. However, the mechanism underlying the host–pathogen interactions between B. sorokiniana and wheat or barley remains unknown. The B. sorokiniana genome encodes a large number of uncharacterized putative effector proteins. In this study, we identified a putative secreted protein, CsSp1, with a classic N-terminal signal peptide, that is induced during early infection. A split-marker approach was used to knock out CsSP1 in the Lankao 9-3 strain. Compared with the wild type, the deletion mutant ∆Cssp1 displayed less radial growth on potato dextrose agar plates and produced fewer spores, and complementary transformation completely restored the phenotype of the deletion mutant to that of the wild type. The pathogenicity of the deletion mutant in wheat was attenuated even though appressoria still penetrated the host. Additionally, the infectious hyphae in the deletion mutant became swollen and exhibited reduced growth in plant cells. The signal peptide of CsSp1 was functionally verified through a yeast YTK12 secretion system. Transient expression of CsSp1 in Nicotiana benthamiana inhibited lesion formation caused by Phytophthora capsici. Moreover, CsSp1 localized in the nucleus and cytoplasm of plant cells. In B. sorokiniana-infected wheat leaves, the salicylic acid-regulated genes TaPAL, TaPR1, and TaPR2 were down-regulated in the ∆Cssp1 strain compared with the wild-type strain under the same conditions. Therefore, CsSp1 is a virulence effector and is involved in triggering host immunity.     

Differential expression of phenylalanine ammonia-lyase genes in barley induced by fungal infection or elicitors

Gene-specific probes were used to assess the expression patterns of four different phenylalanine ammonia-lyase ( pal ) genes in infected or elicitor-treated leaves and suspension-cultured cells of barley. Genes corresponding to hpal2 , hpal3 , hpal4 , and hpal6 were all induced by mercuric chloride and fungal infection by Bipolaris sorokiniana Sacc. (Shoem.) in barley ( Hordeum vulgare L. cv. Pokko) leaves, but with considerable variation in their expression level and timing. The expression patterns of hpal2 and hpal6 were similar, both showing a rapid, strong induction after treatment with mercuric chloride and a slower induction after fungal inoculation, whereas the more divergent hpal3 was induced at a later time and at a lower level after both treatments. Hpal4 was expressed with timing like that of hpal2 and hpal6 in infected or mercuric chloride-treated leaves but its expression was much weaker. Hpal2 and hpal4 were induced in elicitor-treated, suspension-cultured barley cells, whereas the expression of hpal3 was nearly undetectable, and hpal6 was strongly and constitutively present. All pal genes except hpal4 were developmentally regulated, but differentially expressed in various barley tissues. The results suggest that the four pal genes studied here might be responsible for the activation of different branches in the phenylpropanoid biosynthesis of barley.     

牛筋草离孺孢生物学特性及代谢产物活性测定

对牛筋草离孺孢属(Bipolaris sorokiniana)NJ-08菌株的生物学特性及代谢产物除草活性测定结果表明：此菌株的菌丝体生长速度慢, 但产孢量大, 致病性强。菌丝体生长最适温度25°C, 致死温度是55°C/10 min, 最适pH值为8, 碳源以葡萄糖最佳, 供试氮源则不利于菌丝体生长。产生分生孢子最适温度25°C, 最适pH值为8, 碳源以葡萄糖最好, 氮源以硝酸钠产孢量最高。分生孢子萌发温度在20°C~35°C之间没有差异, 适宜pH值为5~7, 碳源以D-木糖的孢子萌发率最高, 氮源以蛋白胨的孢子萌发率最高, 不同处理间存在明显差异。NJ-08菌株产生的代谢产物作用于牛筋草, 可使叶片黄化或枯死, 代谢产物对指示植物及杂草如柱花草、地桃花、猪屎豆和辣椒的胚根胚芽生长都有明显的抑制作用, 抑制率均超过了90%, 显示出很好的除草活性。     

A new technique for monoconidial culture of the most aggressive isolate in a given population of Bipolaris sorokiniana, cause of foliar spot blotch in wheat and barley

We developed a new technique for monoconidial culture of the most aggressive isolate in a given population of Bipolaris sorokiniana, to facilitate the evaluation of spot blotch resistance in wheat and barley. Blotched portions of infected barley leaves were placed on a glass slide in a moist chamber for production of conidia by associated fungal hyphae. Conidia were collected separately and grown on water agar discs. Individual water agar discs having conidium growth were inoculated on barley leaves. The conidium producing the earliest symptom with the largest lesion was considered most aggressive. This lesion was incubated in a moist chamber and the conidial offspring were tested for pathogenicity. When a uniform infection was observed, a small piece of the lesion was cut using a sterilized scalpel, surface sterilized with NaOCl, and inoculated in the centre of Petri dishes containing potato dextrose agar medium. The inoculated Petri dishes were incubated at 25 ± 1 °C to yield monoconidial cultures of the most aggressive isolate. Variability in symptom expression caused by the most aggressive isolate of a given population was much less than variability in symptom expression caused by all isolates collectively. The techniques will be useful for plant pathologists and breeders in screening for spot blotch resistance in wheat and barley.     

Interactive signal transfer between host and pathogen during successful infection of barley leaves by Blumeria graminis and Bipolaris sorokiniana

Using ion-selective microprobes, interactive signalling between barley and Blumeria graminis or Bipolaris sorokiniana has been investigated. The question was raised whether a biotrophically growing fungus manipulates the electrical driving forces (membrane potential, transmembrane pH), required for H+ cotransport of energy-rich compounds. Electrodes were positioned in the substomatal cavity of open stomata or on the leaf surface, and pH was measured continuously up to several days during fungal development. We demonstrate that surface and apoplastic fluids are electrically coupled and respond in a similar manner to stimuli. Apoplastic pH, monitored from the moment of inoculation with conidia, reveals several phases: 2-4h after inoculation of the barley leaf with either fungus, the host displays rapid transient responses after its first contact with the fungal cell wall; apoplastic pH and pCa increases, cytoplasmic pH and pCa decreases. About 1 day after inoculation, the apoplastic pH increases by up to 2 pH units, which is thought to reflect a resistance response against the intruder. Whereas barley leaf cells possess a membrane potential of -152+/-5 mV, hyphae of B. graminis yield -251+/-8 mV, indicative of a substantial driving force advantage for the fungus. Although the resting membrane potential of barley remains constant during the first days after inoculation, leaves infected with B. sorokiniana get confronted with an energy problem, indicated by a retarded repolarization following a "light-off" stimulus. Five days after inoculation, apoplastic pH has increased to 5.97+/-0.47 (n=11) and does no longer respond to "light-off" when measured within lesions. In contrast, it stays at near normal values outside the lesions and responds to "light-off". It is concluded that biotrophically growing fungi do not manipulate the cotransport driving forces since (i) any change in apoplastic pH would be experienced by both partners; (ii) the resting membrane potential is not changed. It is suggested that measured pH changes reflect defence responses of the host against the fungus rather than fungal action to increase compatibility.     

The intensity of respiration and heat emission from seedlings of Festuca pratensis (Hud.) and Hordeum vulgare L. during pathogenesis caused by Bipolaris sorokiniana (Sacc.) Shoem

The presented work was conducted on seedlings of spring barley and meadow fescue which differ in the degree of sensitivity to leaf spot pathogen Bipolaris sorokiniana (Sacc.) Shoem. The seedling reaction to inoculation with mycelium and conidia was examined in glasshouse conditions on the basis of respiration intensity and heat production. The leaf respiration was measured using Clark-type electrode, while heat emission was evaluated by means of isotermic microcalorimeter. The measurements were performed after 1, 3, 6, 10, 24, 48, 72, 168 and 240 hours since the inoculation moment. Leaves of meadow fescue were characterized by the most intense respiration at the 6^th hour, while barley leaves at the 24^th and 72^nd hour after inoculation. In the case of meadow fescue the greatest heat production was noted in the period between 24 and 168 hours after inoculation. Simultaneously, at the 48^th hour the smallest rate of respiration was observed. Barley leaves emitted the greatest amount of heat only in the first 3 hours of the pathogenesis. In these hours the smallest respiration rate was noted. The observed, opposing reaction of respiration intensity and heat emission in the infected seedlings of both species may illustrate a disorder in metabolic processes in plants during pathogenesis. The plants studied differed in the time of their reaction to pathogen attack: barley responded earlier in heat production, while fescue extended respiration rate in the first hours after inoculation. This is clearly observable, when coefficients of metabolic inefficiency (heat rates per mole O₂) are compared. In the case of barley the highest rates were noticed just after inoculation, whereas in fescue at the 48^th hour. In both species attack of pathogen caused high metabolic efficiency.     

The discovery of the virulence gene ToxA in the wheat and barley pathogen Bipolaris sorokiniana

Bipolaris sorokiniana is the causal agent of multiple diseases on wheat and barley and is the primary constraint to cereal production throughout South Asia. Despite its significance, the molecular basis of disease is poorly understood. To address this, the genomes of three Australian isolates of B. sorokiniana were sequenced and screened for known pathogenicity genes. Sequence analysis revealed that the isolate BRIP10943 harboured the ToxA gene, which has been associated previously with disease in the wheat pathogens Parastagonospora nodorum and Pyrenophora tritici‐repentis. Analysis of the regions flanking ToxA within B. sorokiniana revealed that it was embedded within a 12‐kb genomic element nearly identical to the corresponding regions in P. nodorum and P. tritici‐repentis. A screen of 35 Australian B. sorokiniana isolates confirmed that ToxA was present in 12 isolates. Sequencing of the ToxA genes within these isolates revealed two haplotypes, which differed by a single non‐synonymous nucleotide substitution. Pathogenicity assays showed that a B. sorokiniana isolate harbouring ToxA was more virulent on wheat lines that contained the sensitivity gene when compared with a non‐ToxA isolate. This work demonstrates that proteins that confer host‐specific virulence can be horizontally acquired across multiple species. This acquisition can dramatically increase the virulence of pathogenic strains on susceptible cultivars, which, in an agricultural setting, can have devastating economic and social impacts.     

Elicitor-induced changes of phenylalanine ammonia-lyase activity in barley cell suspension cultures

Suspension-cultured barley cells responded to treatments with crude yeast extract and purified glucan preparation by rapidly and transiently (4 h postelicitation) inducing L-phenylalanine ammonia-lyase activity. Similarly, treatment of cell cultures with chitosan resulted in increased phenylalanine ammonia-lyase activity 2–4 h after elicitation, whereas a mycelium preparation of a fungal pathogen, Bipolaris sorokiniana, and purified chitin caused a more delayed induction of phenylalanine ammonia-lyase (8 h postelicitation). The most abundant of the plant cell wall degrading enzymes produced by Bipolaris sorokiniana, β-1,4-xylanase, had only a weak elicitor activity in barley cells suggesting that fungal cell wall components rather than the hydrolytic enzymes secreted by the fungus function as recognizable components that cause barley cells to induce defences. Treatment of the elicited cells with a phenylalanine ammonia-lyase inhibitor, α-aminooxy-β-phenylpropionic acid, resulted in the superinduction of the enzyme indicating the blocking of the feedback regulation mechanisms, whereas in the presence of 1 mM trans-cinnamic acid the elicitor-induction of phenylalanine ammonia-lyase was completely inhibited. Elicitor treatments increased the accumulation of wall-bound phenolics as evidenced by phloroglucinol-HCl staining and thioglycolic acid methods. However, α-aminooxy-β-phenylpropionic acid applied in combination with the elicitor did not prevent the accumulation of phenolics in barley cell walls. This suggested that phenylalanine ammonia-lyase might not play an important role in the synthesis wall-bound phenolic compounds in barley. However, cinnamic acid, whether applied alone or together with the elicitor, increased the amount of wall-bound phenolics in suspension-cultured barley cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Studies on the mechanism of resistance to Bipolaris sorokiniana in the barley lesion mimic mutant bst1

The Bipolaris sorokiniana tolerant 1 ( bst1 ) barley mutant is derived from fast neutron-irradiated seeds of wild-type Bowman( Rph3 ). The induced mutation was genetically localized to a position on chromosome 5HL distal to the centromere using amplified fragment length polymorphism markers. In addition, the defence responses and related gene expression in the bst1 mutant after fungal challenge were compared with those occurring in wild-type plants. Hydrogen peroxide generation, determined by 3,3-diaminobenzidine staining, revealed a clearly reduced level of bst1 , compared with the wild-type, during the entire experimental time: 8–120 h post-inoculation (hpi). At 48 hpi, the wild-type samples displayed twice as much fungal mass and three times greater H₂O₂ production than bst1 . At the same time, staining of B. sorokiniana showed less fungal growth in the spontaneous lesions of bst1 compared with the wild-type. Monitoring of defence-related genes at 48 hpi demonstrated strong expression of PR-1a , PR-2 , PR-5 and PR-10 in bst1. A gene coding for a unique oxidoreductase enzyme, designated as HCP1 , was expressed at much higher levels in inoculated leaves of the bst1 mutant than in those of the wild-type plant. Taken together, the results suggest that the defence to B. sorokiniana largely relies on salicylic acid-responsive pathogenesis-related ( PR ) genes, as well as selected reactive oxygen species and unknown HCP1 -associated factors.     

Effect of light on abscisic acid‐induced proline accumulation in leaves: comparison between barley and wheat

Light enhanced the abscisic acid‐induced accumulation of proline in barley ( Hordeum vulgare L. cv. Georgie) and wheat ( Triticum durum L. cv. Valnova). In wheat ABA is ineffective in the dark. In both barley and wheat, the accumulation of proline in the light showed the same characteristics as those of the process that occurs in barley in the dark, namely a synergistic interaction between the hormone and K(Na)Cl, an enhancing effect of Cl⁻ anion in excess over K⁺ cation in the incubation medium, and an inhibiting effect of D ‐mannose and monensine. In wheat, furthermore, light is needed during treatment with ABA if proline is to accumulate. Light was effective in both wheat and barley during the second or accumulation phase of the hormonal process, whereas the events occurring in the first (or lag) phase did not require light. The results suggest that in wheat light induces a putative factor(s) involved in the proline accumulation pathway that is lost in the dark, whereas in barley it is present in the dark.     

Seed Treatment of Barley with Idriella bolleyi causes Systemically Enhanced Defence against Root and Leaf Infection by Bipolaris sorokiniana

Inoculation of barley seed with the saprophytic fungus Idriella bolleyi caused systemically improved resistance on both leaves and roots of young plants to subsequent infection with the necrotrophic fungus Bipolaris sorokiniana . Lesion sizes were reduced by up to 50% as a result of the seed treatment. Seed and root inoculation with I. bolleyi induced biochemical defence responses in the plants as shown by slight accumulation of pathogenesis-related (PR) proteins, but the accumulation was not as great as when the roots were inoculated with B. sorokinina . Conidia of I. bolleyi applied to seed survived well during storage, with no significant reduction in colony forming units (CFUs) occurring at 15°C after 2 months. However, storage at 28°C decreased the viability of the applied conidia. The fungus colonized the seed and roots during field conditions as indicated by high numbers of CFUs. Two months after sowing, frequencies of I. bolleyi were higher on plants treated with the fungus than on control plants where colonization occurred naturally from field soil. The implications of the results are discussed in relation to earlier reports of biological control of cereal root diseases by I. bolleyi and induced resistance.