甘蔗鞭黑粉菌侵染甘蔗的早期可视化观察

[目的]甘蔗鞭黑粉菌(Sporisorium scitamineum)引起的甘蔗黑穗病是我国甘蔗生产重要的病害.示踪甘蔗鞭黑粉菌侵染甘蔗的过程将有助于揭示其致病性和甘蔗抗黑穗病机制,为抗病品种的选育以及黑穗病的防治奠定基础.[方法]利用农杆菌介导的遗传转化技术对甘蔗鞭黑粉菌进行黄色荧光标记,对转化子进行配合及致病力检测...     

甘蔗品种黑穗病抗性评价体系的建立

为了建立甘蔗品种黑穗病抗性评价体系,选用9个引进品种,设计一个包括6个对照品种在内的田间试验。首次采用混合小种进行人工浸渍接种,通过整个新植蔗生长季病害进展曲线下的面积、茎感染率和株感染率3个病情指数,以及病害流行学参数潜伏侵染期和持续发病期的分析,对其扰痛性进行评价。在分析以上参数相关程度的基础上,引进了系统聚类分析法进行进一步评价。结果显示;9个引进甘蔗品种中,ROC26属于感病品种,其余属于抗病或高抗品种;3个病情指数和持续发病期的两两相关均为显著正相关,潜伏侵染期与这些参数的相关为负相关,但未达到显著水平;在类间距离大于1．0的条件下,可将15个品种聚为6类,进一步明确了备品种抗黑穗痛性的相似程度;抗性鉴定标准对照种NC0310、F134、NC0376和Ya71—374的应用,明确了接种源为小种1和小种2,通过一次接种试验,明确了供试品种对2个小种的抗性水平,标准对照种的抗性表现,还说明了本生长季发病条件基本是适宜的。本文建立的甘蔗品种抗黑穗病评价体系是切实可行的。     

Relationships between phenolics-conjugated polyamines and sensitivity of sugarcane to smut (Ustilago scitaminea)

Infection of sugarcane buds (var. Barbados 42231) with teliospores of Ustilago scitaminea changes the pattern of polyamine conjugation in several organs of 2-month-old plants. Stalks of infected plants contain SH-spermidine that does not occur in the healthy organ. Similar results have been obtained for SH-spermine in the first expanded leaf and in the stem. The amount of SH-cadaverine in the first expanded leaf, roots and stem of infected plants is always higher than that found for healthy plants. Some phenolics are also associated with different polyamine fractions. So, the amount of p-hydroxybenzoic acid in both SH and PH fractions of polyamines extracted from the root increases after infection. Syringic acid is the main phenol associated with the PH fraction in the first expanded leaf of infected plants, whereas this phenol is mainly associated with both SH and PH fractions isolated from the stem and the whip. Infection enhances conjugation of p-courmaric acid to PH polyamines, whereas caffeic acid appears in the SH fraction in leaf, root and stem. However, ferulic acid seems to be the main hydroxycinnamic acid derivative in the whip. Chlorogenic acid is associated with the SH fraction from the stem of healthy plants although this changes to free phenolics after infection.Key words:Saccharum officinarum, Ustilago sciaminea, phenolics, polyamines.      

A role for sugarcane glycoproteins in the resistance of sugarcane to Ustilago scitaminea

Smut is a major disease of sugarcane caused by Ustilago scitaminea. Germination of fungal teliospores is achieved on the internode surface of plants, and it is followed by the formation of appressoria. A primary response of sugarcane plants to the infection seems to be the production of several glycoproteins, defined as mid-molecular mass (MMMG) or high molecular mass (HMMG) macromolecules. Teliospore germination in the presence of both MMMG and HMMG decreased about 50% following 5 h of teliospore contact with glycoproteins. This may be related to the ability of glycoproteins to produce cytoagglutination. Binding of fluorescein-labelled glycoproteins was studied by fluorescence microscopy, showing that staining of cells was not uniform, but mainly in the contact zone between two individual teliospores when aggregated. HMMG was composed of only one fraction that was completely retained by smut teliospores, whereas three of the five different glycoproteins occurring in the MMMG fraction were retained by teliospore cell walls. Moreover, a unique application of salicylic acid, naturally produced by sugarcane stalks after experimental fungal infection, enhanced the production of both glycoprotein pools. A hypothesis about the role of both HMMG and MMMG as defence glycoproteins is discussed.     

Changes in cinnamyl alcohol dehydrogenase activities from sugarcane cultivars inoculated with Sporisorium scitamineum sporidia

This study describes a method for determining cinnamyl alcohol dehydrogenase activity in sugarcane stems using reverse phase (RP) high-performance liquid chromatography to elucidate their possible lignin origin. Activity is assayed using the reverse mode, the oxidation of hydroxycinnamyl alcohols into hydroxycinnamyl aldehydes. Appearance of the reaction products, coniferaldehyde and sinapaldehyde is determined by measuring absorbance at 340 and 345 nm, respectively. Disappearance of substrates, coniferyl alcohol and sinapyl alcohol is measured at 263 and 273 nm, respectively. Isocratic elution with acetonitrile:acetic acid through an RP Mediterranea sea C18 column is performed. As case examples, we have examined two different cultivars of sugarcane; My 5514 is resistant to smut, whereas B 42231 is susceptible to the pathogen. Inoculation of sugarcane stems elicits lignification and produces significant increases of coniferyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD). Production of lignin increases about 29% in the resistant cultivar and only 13% in the susceptible cultivar after inoculation compared to uninoculated plants. Our results show that the resistance of My 5514 to smut is likely derived, at least in part, to a marked increase of lignin concentration by the activation of CAD and SAD.     

Development of a loop‐mediated isothermal amplification assay for rapid and sensitive detection of Sporisorium scitamineum in sugarcane

Smut disease caused by Sporisorium scitamineum is one of the most destructive sugarcane diseases worldwide. The pathogen spreads primarily through infected sugarcane setts, and hence, the use of disease‐free planting materials is essential for preventing disease development in the field. In this study, a species‐specific loop‐mediated isothermal amplification (LAMP) assay was developed for rapid and accurate detection of S. scitamineum. Based on the differences in internal transcribed spacer (ITS) sequences of S. scitamineum, a set of four species‐specific primers, F3, B3, FIP and BIP, were designed by using a panel of fungal and bacterial species as controls. After optimization of the reaction conditions, the detection limit of LAMP assay was about 2 fg of the S. scitamineum genomic DNA in 25 µL reaction solution, 100‐fold lower than that of conventional polymerase chain reaction. The assay showed high specificity to discriminate all S. scitamineum isolates from nine other fungal and bacterial pathogens. The LAMP assay also detected smut infection from young sugarcane leaves with no visible smut‐disease symptoms. The findings from this study provide a simple, highly sensitive, rapid and reliable technique for early detection of S. scitamineum, which may be useful for sugarcane quarantine and production of smut‐free seedcanes. This is the first report of LAMP‐based assay for the detection of S. scitamineum in sugarcane.     

Glycoproteins from sugarcane plants regulate cell polarity of Ustilago scitaminea teliospores

Saccharum officinarum, cv. Mayarí, is a variety of sugarcane resistant to smut disease caused by Ustilago scitaminea. Sugarcane naturally produces glycoproteins that accumulate in the parenchymatous cells of stalks. These glycoproteins contain a heterofructan as polysaccharide moiety. The concentration of these glycoproteins clearly increases after inoculation of sugarcane plants with smut teliospores, although major symptoms of disease are not observed. These glycoproteins induce homotypic adhesion and inhibit teliospore germination. When glycoproteins from healthy, non-inoculated plants are fractionated, they inhibit actin capping, which occurs before teliospore germination. However, inoculation of smut teliospores induce glycoprotein fractions that promote teliospore polarity and are different from those obtained from healthy plants. These fractions exhibit arginase activity, which is strongly enhanced in inoculated plants. Arginase from healthy plants binds to cell wall teliospores and it is completely desorpted by sucrose, but only 50% of arginase activity from inoculated plants is desorpted by the disaccharide. The data presented herein are consistent with a model of excess arginase entry into teliospores. Arginase synthesized by sugarcane plants as a response to the experimental infection would increase the synthesis of putrescine, which impedes polarization at concentration values higher than 0.05 mM. However, smut teliospores seem to be able to change the pattern of glycoprotein production by sugarcane, thereby promoting the synthesis of different glycoproteins that activate polarization after binding to their cell wall ligand.     

Molecular cloning and characterization of two pathogenesis-related β-1,3-glucanase genes ScGluA1 and ScGluD1 from sugarcane infected by Sporisorium scitamineum

Key message

Two β-1,3-glucanase genes from sugarcane were cloned and characterized. They were all located in apoplast and involves in different expression patterns in biotic and abiotic stress.

Abstract

Smut caused by Sporisorium scitamineum is a serious disease in the sugarcane industry. β-1,3-Glucanase, a typical pathogenesis-related protein, has been shown to express during plant–pathogen interaction and involves in sugarcane defense response. In this study, β-1,3-glucanase enzyme activity in the resistant variety increased faster and lasted longer than that of the susceptible one when inoculated with S. scitamineum, along with a positive correlation between the activity of the β-1,3-glucanase and smut resistance. Furthermore, two β-1,3-glucanase genes from S. scitamineum infected sugarcane, ScGluA1 (GenBank Accession No. KC848050) and ScGluD1 (GenBank Accession No. KC848051) were cloned and characterized. Phylogenetic analysis suggested that ScGluA1 and ScGluD1 clustered within subfamily A and subfamily D, respectively. Subcellular localization analysis demonstrated that both gene products were targeted to apoplast. Escherichia coli Rosetta (DE3) cells expressing ScGluA1 and ScGluD1 showed varying degrees of tolerance to NaCl, CdCl₂, PEG, CuCl₂ and ZnSO₄. Q-PCR analysis showed up-regulation of ScGluA1 and slight down-regulation of ScGluD1 in response to S. scitamineum infection. It suggested that ScGluA1 may be involved in the defense reaction of the sugarcane to the smut, while it is likely that ScGluD1 was inhibited. The gene expression patterns of ScGluA1 and ScGluD1, in response to abiotic stresses, were similar to sugarcane response against smut infection. Together, β-1,3-glucanase may function in sugarcane defense mechanism for S. scitamineum. The positive responses of ScGluA1 and the negative responses of ScGluD1 to biotic and abiotic stresses indicate they play different roles in interaction between sugarcane and biotic or abiotic stresses.     

Free and conjugated polyamines during de novo floral and vegetative bud formation in thin cell layers of tobacco

The concentrations of three classes of polyamines, trichloroacetic acid-soluble (free), TCA-soluble conjugated (to small molecules) and TCA-insoluble conjugated (to macromolecules), was examined during de novo floral and vegetative bud formation in thin cell layers of Nicotiana tabacum L. cv. Samsun. Explants (consisting of 5–6 layers of epidermal, subepidermal and parenchyma cells) were excised either from floral pedicels or from stem internodes at the unripe fruit stage and cultured on the same medium. In the former, the first de novo formed flower buds appeared on day 8 of culture, while in the latter the first vegetative domes appeared on day 10. In both cases the number of floral and vegetative buds increased up to day 12 and 15, respectively. Changes in dry weight were determined throughout the culture period. Free and conjugated putrescine titer increased 5–60 times in both types of culture and in the three classes of polyamines examined; spermidine content also increased, while spermine, when present, did not show significant changes. TCA-soluble conjugated polyamines were most abundant, being about 2-fold the TCA-insoluble conjugated ones and 10-fold the free ones. The major increment in putrescine and spermidine content occurred in stem internode explants developing vegetative buds. In pedicel explants the maximum putrescine level was reached before or on day 8 in culture (emergence of the first flower buds with calyx initials), while in stem internode explants the maximum level was reached on day 12, at the emergence of the first vegetative buds with leaf primordia. While spermidine prevailed on day 0, putrescine was the most abundant polyamine during both differentiation processes. The putrescine content rapidly increased immediately after the onset of culture. Thus conjugated polyamines, especially putrescine, and not only the free ones, seem to be involved in both the reproductive and vegetative phases of tobacco growth and development.     

Metabolic changes in cherry flower buds associated with breaking of dormancy in early and late blooming cultivars

Changes of metabolic activities during dormancy and breaking of dormancy in the cherry flower buds of early blooming (EB) cultivar ( Prunus avium L. cv. Coeur de Pigeon) and late blooming (LB) cultivar ( Prunus serrulata Lindl. cv. Kwanzan) were determined. The LB buds had higher polyamines, protein and 1-(malonylamino) cyclopropane-1-carboxylic acid (MACC) content than the EB buds. During the dormant state, the DNA, RNA, protein and polyamines in the EB buds were low but increased slowly and steadily, whereas those in the LB buds remained at a consistently higher level. The transition from dormancy to the active state in both cultivars was characterized by a sharp increase in DNA, RNA, protein, polyamines, S-adenosyl-methionine (SAM), 1-aminocyclopropane-1-carboxylic acid (ACC) and MACC. After initial swelling and development of flowers, the levels of all these components decreased. Polyamine and ethylene biosyntheses did not seem to be competing for their common substrate, SAM, during flower bud development.     

Genome sequencing of Sporisorium scitamineum provides insights into the pathogenic mechanisms of sugarcane smut

Background

Sugarcane smut can cause losses in cane yield and sugar content that range from 30% to total crop failure. Losses tend to increase with the passage of years. Sporisorium scitamineum is the fungus that causes sugarcane smut. This fungus has the potential to infect all sugarcane species unless a species is resistant to biotrophic fungal pathogens. However, it remains unclear how the fungus breaks through the cell walls of sugarcane and causes the formation of black or gray whip-like structures on the sugarcane plants.

Results

Here, we report the first high-quality genome sequence of S. scitamineum assembled de novo with a contig N₅₀ of 41 kb, a scaffold N₅₀ of 884 kb and genome size 19.8 Mb, containing an estimated 6,636 genes. This phytopathogen can utilize a wide range of carbon and nitrogen sources. A reduced set of genes encoding plant cell wall hydrolytic enzymes leads to its biotrophic lifestyle, in which damage to the host should be minimized. As a bipolar mating fungus, a and b loci are linked and the mating-type locus segregates as a single locus. The S. scitamineum genome has only 6 G protein-coupled receptors (GPCRs) grouped into five classes, which are responsible for transducing extracellular signals into intracellular responses, however, the genome is without any PTH11-like GPCR. There are 192 virulence associated genes in the genome of S. scitamineum, among which 31 expressed in all the stages, which mainly encode for energy metabolism and redox of short-chain compound related enzymes. Sixty-eight candidates for secreted effector proteins (CSEPs) were found in the genome of S. scitamineum, and 32 of them expressed in the different stages of sugarcane infection, which are probably involved in infection and/or triggering defense responses. There are two non-ribosomal peptide synthetase (NRPS) gene clusters that are involved in the generation of ferrichrome and ferrichrome A, while the terpenes gene cluster is composed of three unknown function genes and seven biosynthesis related genes.

Conclusions

As a destructive pathogen to sugar industry, the S. scitamineum genome will facilitate future research on the genomic basis and the pathogenic mechanisms of sugarcane smut.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-996) contains supplementary material, which is available to authorized users.     

Free Amino Acids in Roots of Infected Cotton Seedlings Resistant and Susceptible to Meloidogyne incognita

Quantities of free amino acids in segments of cotton roots resistant and susceptible to Meloidogyne incognita were compared. Following infection, the root-knot susceptible cultivar, M8, had greater percentage increases of certain individual free amino acids than the resistant cultivar, Clevewilt, but the sum total of free amino acids was greatest in the resistant cultivar. More free amino acids were present in infected than in noninfected plants of both cultivars. The overall concn of glycine declined over the I 0-day period following inoculation. The concns of the aromatic amino acids, tyrosine and phenylalanine, varied as functions of infection, cultivar, and time of harvest. Proline in susceptible M8 increased nearly 2000-fold 10 days after infection, when considerable thickening of syncytial walls is occurring.     

Differential accumulation of γ‐aminobutyric acid in elicited cells of two rice cultivars showing contrasting sensitivity to the blast pathogen

Intracellular free amino acid pools were quantified in suspension cultured cells of a blast‐sensitive and a blast‐resistant rice genotype at increasing times after treatment with Magnaporthe oryzae cell wall hydrolysates. Besides some expected variations in free phenylalanine, a remarkable early increase of γ‐aminobutyric acid (GABA) levels was evident in both cultivars. Glutamate decarboxylase activity and protein levels were unaffected. GABA homeostasis was recovered in the sensitive cultivar 48 h after the treatment. In contrast, a further GABA accumulation and a general increase of most amino acids was found at this later stage in the resistant genotype, which showed a larger decrease in cell viability as a consequence of elicitor addition. Data support a recently hypothesised role of GABA metabolism in the plant response to fungal pathogens.     

Sugar cane arginase competes with the same fungal enzyme as a false quorum signal against smut teliospores

Sugar cane cultivars resistant to smut produce a pool of glycoproteins after experimental infection, one of them develops arginine activity. This arginase induces cytoagglutination of smut teliospores but impedes germination. Teliospores also secrete a fungal arginase that accelerates their own germination. This fungal arginase binds to teliospore cell walls. The affinity of this arginase for the ligand shows to be higher than that found for plant arginase. In fact, fungal arginase removes sugar cane arginase previously bound to their ligands in the cell wall whereas the inverse process is practically negligible. The enzymatic activity is required to the binding of the protein to teliospore cell wall but it is not related to the germination process. Thus, it can be concluded that the binding of fungal arginase to smut teliospores activates a signal transduction cascade that enhances germination.     

Changes in Cell Wall-bound Phenolic Compounds and Lignin in Roots of Date Palm Cultivars Differing in Susceptibility to Fusarium oxysporum f. sp. albedinis

The roots of date palm contain four cell wall‐bound phenolic acids identified as p‐hydroxybenzoic, p‐coumaric, ferulic and sinapic acids. The ferulic acid represents the major phenolic compound since it constitutes 48.2–55.8% of cell wall‐bound phenolic acids. All these phenolic acids were present in the resistant cultivar (BSTN) and the susceptible cultivar (JHL). However, the pre‐infection contents of p‐coumaric, ferulic and sinapic acids were greater in the resistant cultivar than in the susceptible one. For the contents of p‐hydroxybenzoic acid, there was no significant difference between the resistant cultivar and the susceptible cultivar. Similarly, the pre‐infection contents of lignin were approximately equal for both cultivars. Inoculation of the date palm roots by Fusarium oxysporum f. sp. albedinis induced important modifications to the contents of the cell wall‐bound phenolic compounds and lignin, which made it possible to distinguish between resistant and susceptible cultivars. The post‐infection contents of cell wall‐bound phenolic compounds underwent a rapid and intense increase with a maximum accumulation on the tenth day for p‐hydroxybenzoic acid (1.54 μmol/g), p‐coumaric acid (2.77 μmol/g) and ferulic acid (2.64 μmol/g) and on the fifteenth day for sinapic acid (1.85 μmol/g). The maximum contents accumulated in the resistant cultivar were greater than those in the susceptible cultivar, namely, 11 times for p‐hydroxybenzoic acid, 2.6 times for p‐coumaric acid, 1.8 times for ferulic acid and 12.3 times for sinapic acid. In the susceptible cultivar, p‐coumaric acid and ferulic acid contents also increased after inoculation although they did not reach the pre‐infection contents of the resistant cultivar. The contents of p‐hydroxybenzoic acid in the susceptible cultivar roots did not present post‐infection modification and those of sinapic acid decreased instead. The lignin contents increased in both cultivars with a maximum accumulation on the fifteenth day. However, the maximum contents accumulated in the resistant cultivar roots were 1.5 times greater than those of the susceptible cultivar. These results showed clear differences between the resistant BSTN and the susceptible JHL cultivars. The implication of cell wall‐bound phenolic compounds and lignin in the resistance of date palm to F. oxysporum f. sp. albedinis appears to be dependent on the speed and intensity of their accumulation with greater contents in the first stage of infection.     

高粱对丝黑穗病的抗性及遗传研究

1981—1985年在人工接种的条件下,对1239份高粱品种资源,进行了抗丝黑穗病性鉴定。与此同时,用17个抗性不同的品种系,按照不完全双列杂交设计,进行了高粱对丝黑穗病的抗性遗传研究。结果表明,高粱对丝黑穗病的抗性遗传方式因品种而异。有的品种系具数量性状遗传特点,有的则具有质量性状遗传特点。抗病性属数量性状遗传的品种系,其抗性主要是受加性基因控制。     

Sporisorium scitamineum colonisation of sugarcane genotypes susceptible and resistant to smut revealed by GFP‐tagged strains

Sporisorium scitamineum is the causal agent of sugarcane smut disease. The fungus establishes a biotrophic interaction with sugarcane tissues, and unlike smut fungi of other monocot hosts, the primary meristem of sugarcane plants develops a whip‐like structure instead of a tumour‐like galls emerging from floral structures (tassels and ears). We examined (GFP)‐tagged S. scitamineum infecting tissues of three sugarcane genotypes with distinct responses to smut (susceptible, intermediate resistant and resistant). Mating compatible haploid cells gfp‐expressing were obtained by Agrobacterium tumefaciens‐mediated transformation (ATMT) using the integrative vector pFAT‐gfp. Regardless of the inoculation method (drop inoculation and hypodermal syringe inoculation), all genotypes were colonised by the fungus. GFP‐tagged strains of opposite mating reaction were able to: (a) grow in vitro as fluorescent yeast‐like cells; (b) generate infectious dikaryon; (c) penetrate sugarcane tissues; (d) colonise tissues by growing a filamentous network; and (e) form the characteristic highly branched hyphae within host cells. Fungal colonisation 160 DAI revealed an association of the fungus with vascular vessels disrupting their organisation in all three genotypes analysed. However, the resistant plants did not develop whips spanning the experiment time. The first whips emerged 76 DAI from plants of the susceptible genotype whereas for intermediate resistant plants whips were detected at 137 DAI. These whips were dissected and fluorescent sporogenesis and teliospore maturation were analysed. In vitro germination of recovered teliospores revealed after meiosis the formation of a three‐celled hyphal filament, where the fourth cell was likely maintained in the teliospore coat. These cells showed independent segregation of the gfp marker, as a result of gfp insertions in different chromosomes of each compatible haploid strain. This work presents the complete fungal life cycle of GFP‐marked S. scitamineum to study developmental stages in planta.