Conversion of Helminthosporium sacchari Toxin to Toxoids by beta-Galactofuranosidase from Helminthosporium

Helminthosporium sacchari produces a host-selective toxin and structurally related nontoxic compounds, here referred to as `toxoids.' Toxin and the three toxoids were each isolated to a high level of purity and were hydrolyzed under acidic conditions. The released galactose was measured by a galactose oxidase/peroxidase assay. Toxin was found to contain four units of galactose per molecule, as previously reported. Toxoids I, II, and III contained one, two, and three units of galactose, respectively. In cultures of the fungus, toxin concentration peaked at 3 weeks, followed by a rapid decline; as toxin levels fell, the total amount of toxoids increased. An enzyme with β-galactofuranosidase activity was found in small amounts in the cultures of H. sacchari; the enzyme converted toxin to the toxoids in vitro. β-Galactofuranosidase was previously known from very few micro-organisms; therefore, several pathogenic Helminthosporia and other fungi were tested for production. β-Galactofuranosidase activity in culture filtrates and mycelia of H. victoriae, H. maydis, H. carbonum, and H. turcicum was much greater than in filtrates and mycelium of H. sacchari. More work is needed to determine the significance of enzyme production by these fungi. No β-galactofuranosidase was evident from Fusarium oxysporum and Cladosporium cucumerinum.     

Interconversions of aglycone and host-selective toxin from Helminthosporium sacchari

Helminthosporium sacchari, a fungus that causes disease in sugarcane, produces oligosaccharide-sesquiterpene toxins (HS toxins A, B, and C) that are required for infection and disease development. Two free sesquiterpenes were isolated from mycelium but not from culture fluids of the fungus. One sesquiterpene was identified by HPLC and mass spectrometry as an aglycone of HS toxin C and could be obtained by enzymatic hydrolysis of this toxin. The other sesquiterpene appeared to be the 2-keto form of the first compound. The aglycone from toxin C hydrolysis was labelled with tritium by successive treatments with active manganese dioxide, sodium boro[³H]hydride, and lithium aluminium hydride. The labelled compound was fed to cultures of H. sacchari, radioactivity was incorporated into HS toxin C and into lower molecular weight homologues. The results suggest a metabolic route (aglycone → metabolite Y, → HS toxin → metabolite X) for the biosynthesis of HS toxin; metabolites X and Y are lower molecular weight homologues of the toxin.     

Biological Activity of the Isomeric Forms of Helminthosporium sacchari Toxin and of Homologs Produced in Culture

The effect of Helminthosporium sacchari (HS) toxin isomers and related, pathogen-produced compounds on dark CO₂ fixation in HS-susceptible sugar cane leaf slices was investigated. HS toxin consists of a mixture of three isomeric bis-5-O-(β-galactofuranosyl)-β-galactofuranosides (A, B, and C) differing in the position of one double bond in the sesquiterpene aglycone. Maximum inhibition of dark CO₂ fixation in susceptible sugar cane (CP52-68) occurred within 30 to 40 minutes, and amounts necessary to reach 50% inhibition values typically were approximately 1.7 micromolar for natural toxin mixture ( 2:3:5 mixture of isomers A:B:C) and 4, 6, and 0.7 micromolar for isomers A, B, and C, respectively. Other fractions from cultures of the pathogen consist of comparable mixtures of sesquiterpene isomers but have only 1, 2, or 3 galactofuranose units (HS₁, HS₂, HS₃) or two α-glucopyranose units as well as four β-galactofuranose units (HS₆). The lower toxin homologs were not toxic to clone CP52-68, but protected sugar cane from the effects of toxin. Minimum ratios of protectant: toxin giving 95% protection were approximately 50:1, 6:1, and 12:1 for HS₁, HS₂, and HS₃, respectively. HS₂ and HS₃ protected when added up to 12 minutes after toxin as well as when added with or before toxin. Some common plant galactopyranosides were not toxic and did not protect at 500:1 molar excess. The sample of HS₆ was toxic at 500 micromolar, and did not protect against HS toxin. With the availability of purified, homogeneous preparations of HS toxin, homologs, and chemically modified or synthetic analogs, the dark CO₂ fixation assay should prove to be a useful tool for understanding the mode of action of HS toxin.     

Resistance to Melanaphis sacchari in the sugarcane cultivar R 365

This study focuses on the resistance of sugarcane, Saccharum spec. (Poaceae), to the sugarcane aphid, Melanaphis sacchari (Zehntner) (Hemiptera: Aphididae), which vectors Sugarcane yellow leaf virus (SCYLV). Resistance was characterized in cultivar R 365, using a 3‐year field trial and laboratory experiments on potted plantlets and excised leaves. R 365 reduced aphid populations in the field by antixenosis and antibiosis. Using the electrical penetration graph technique, we detected delayed aphid salivation in phloem and inhibition of passive phloem sap uptake in R 365. The resistance factors also proved to be effective against the corn leaf aphid, Rhopalosiphum maidis (Fitch) (Hemiptera: Aphididae), another vector of SCYLV.     

Selective Toxins and Analogs Produced by Helminthosporium sacchari: Production, Characterization, and Biological Activity

Helminthosporium sacchari toxin and several lower molecular weight, nontoxic analogs were isolated from culture filtrates. Three isomers of the toxin (A, B, and C), each with four galactose units, were separated by high performance liquid chromatography. Isomer C had the highest and isomer A had the lowest toxicity to H. sacchari-susceptible sugarcane; resistant clones were not affected. Each toxin isomer was partially digested with a β-galactofuranosidase and the resulting analogs (seven from each toxin isomer) were separated by reverse phase high performance liquid chromatography and identified. Each isomer of the analogs with 3 galactose units per mole also was partially digested and the arrangement of galactose units was determined. The compound with one galactose attached to position 2 of the bicyclic sesquiterpene and with 2 galactose units attached to position 13 (analog A_1,2) was highly toxic to some but not to all clones of H. sacchari-susceptible sugarcane. Toxin analogs protected sensitive tissue against active toxin; protective effects of the analogs differed, but at least a 10-fold excess of analog was required. Analog C_2,1 was more effective at preventing toxin C-induced electrolyte losses than was any other analog. Each of the 3-galactose analog isomers protected better than did any of the 2-galactose compounds. The 1,1 analogs did not protect as well as did the 2,0 or 0,2 analogs. Thus, the sesquiterpene isomer, the number of galactose units, and the galactose arrangement pattern determine the effectiveness of the compound in induction of electrolyte loss and in prevention of toxininduced loss from sugarcane tissues.     

alpha-Galactoside Binding Proteins from Plant Membranes: Isolation, Characterization, and Relation to Helminthosporoside Binding Proteins of Sugarcane

α-Galactoside binding proteins were isolated from cellular membranes of mint and tobacco as well as two clones of sugarcane which differ in their sensitivity to helminthosporoside, a toxic galactoside. Sodium trichloroacetate was used to disrupt membranes after which the proteins were purified using a melibiose-Sepharose-6B affinity column. Proteins from mint, tobacco, and susceptible sugarcane had equal electrophoretic mobilities, whereas resistant sugarcane protein migrated more slowly. Pretreatment of the proteins with fluorescamine caused them to migrate with the tracking dye. Each of the proteins had molecular weights of about 100,000 and each was shown to be oligomeric. Gel filtration revealed that aqueous solutions of these membrane proteins contained a mixture of size species which included a high molecular weight multimer and lower molecular weight oligomers. The relative abundance of the oligomers was dependent upon protein concentration: the lower concentrations yielded higher relative amounts of oligomers (Kenfield and Strobel 1980 Biochim Biophys Acta 600: 705-712). Also, the binding activity of these receptors was inversely proportional to protein concentration. At low protein concentration (4 micrograms per milliliter), the K_d's of each of the proteins for galactinol, raffinose, and helminthosporoside was about 10 micromolar. At high protein concentrations (100 micrograms per milliliter), mint and resistant sugarcane proteins failed to bind α-galactosyl ligands, whereas proteins from tobacco and susceptible sugarcane exhibited a markedly decreased binding activity compared to that at lower protein concentrations. Binding proteins from susceptible sugarcane were mixed with receptors from either resistant sugarcane or mint at low protein concentrations, then assayed for binding activity. Such mixtures showed a concentration-dependent decrease in binding activity analogous to the activity of homogeneous protein solutions. Bovine serum albumin, a nonsubunit protein, had no effect on the binding activity of the protein from susceptible sugarcane. Thus, receptors from diverse plants can associate in vitro and form functional oligomers. The amino acid composition of each of the binding proteins was similar but not identical. The significance of these results is discussed in regard to regulation of carbohydrate transport and sensitivity to phytotoxins.     

In vitro strategies for selection of eye-spot resistant sugarcane lines using toxins of Helminthosporium sacchari

In vitro strategies were applied for the selection of eye-spot resistant variants from susceptible sugarcane cultivar Co 419 Different selective units (callus and leaf) of the susceptible cultivar were subjected to sub-lethal to lethal doses of toxins (culture filtrate and partially purified toxin) of H. sacchari, with the objective of improving the efficacy of in vitro selection protocols. All the selective units gave more or less similar response with culture filtrate, but a distinct response was observed when leaf was subjected to partially purified toxin treatment. The response was characterised by the degree of resistance exhibited by the regenerated seedlings.     

玉米种质资源抗矮花叶病鉴定

采用苗期人工接毒和全生育期田间蚜虫传毒自然感染相结合的方法,于2002-2005年对893份玉米种质资源进行了由甘蔗花叶病毒(SCMV)引起的矮花叶病田间抗性鉴定。结果表明,表现抗病类型(高抗~中抗)材料有66份,占鉴定材料总数的7.4%。其中,表现高抗矮花叶病的有10份材料,表现抗病的有10份材料,表现中抗的有46份材料。鉴定材料表现为4种反应类型:①兼抗病毒汁液摩擦接种和蚜虫传毒类型,其中抗性突出的有赤L031、哲4678、宁74、赤L022、哲357和2019等6份自交系和农家种白苞谷;②抗病毒汁液摩擦接种但不抗蚜虫传毒类型;③抗蚜虫传毒但不抗病毒汁液摩擦接种类型;④不抗病毒汁液摩擦接种和蚜虫传毒类型。     

Description of Gibberella sacchari and neotypification of its anamorph Fusarium sacchari

We described the teleomorph of Fusarium sacchari as Gibberella sacchari, sp. nov. This species can be separated from other species of Gibberella on the basis of the longer, narrower ascospores found in G. sacchari and by sexual cross fertility. Female-fertile mating type tester strains were developed that can be used for making sexual crosses with this heterothallic fungus under laboratory conditions. The anamorph, Fusarium sacchari, was neotypified.     

甘蔗非生物胁迫抗性研究进展

甘蔗是世界上重要的糖料和能源作物,对于我国食糖产业发展具有举足轻重的作用。但是,我国甘蔗种植面积正不断减少,呈现出向高海拔、土壤贫瘠等生产条件差的地方转移的趋势,因此遭受的逆境胁迫程度日益加深,严重影响了甘蔗的生长发育及产量形成。如何解决逆境胁迫下甘蔗的产量问题是目前生产上面临的重要课题。目前最好的解决办法还是培育高抗逆品种,为了给甘蔗抗性品种选育提供参考,本文对甘蔗的各种逆境,如低温、干旱、高盐、重金属等伤害与抗逆性的生理生化机制及甘蔗抗逆相关功能基因的挖掘研究进行了综述,以期系统地了解甘蔗逆境研究现状,并提出了甘蔗抗逆育种需要开展的关键工作,以期为甘蔗抗逆相关研究方向的设定、抗性机理和抗性品种的选育提供借鉴。     

不同基因型高粱的理化特性与抗蚜性的相关性研究

通过对抗蚜的河农16和感蚜品系Tx623B、Tx622B、Tx3197B、千三、晋五及杂交种的物理特性、组织结构、生理生化特性的对比分析,研究了不同基因型高粱理化特性与抗蚜性的相关性.结果表明,高粱抗蚜品系叶片表面较感蚜品系的细胞排列整齐、致密、表皮细胞直径较感蚜品系小、叶片薄、叶片表面光滑、叶片颜色浅.感虫后各品系过氧化物酶(POD)、多酚氧化酶(PPO)活性均有升高,与蚜虫的诱导存在相关性,但不同品系感蚜前后均不存在趋势性变化;苯丙氨酸解氨酶(PAL)酶活性感虫前在抗虫的河农16及杂交种中具有高水平表达,并与其他感蚜品系存在显著差异;蚜虫侵害诱导后PAL酶活性均有上升,但抗蚜品系中表现稳定,酶活性变化幅度与叶片损伤程度存在相关性,PAL酶活性与高粱的抗蚜性存在明显的相关性.感虫前后氨基酸含量及其变化与高粱品系抗蚜性间无相关性;在感蚜虫前,抗蚜品系及其杂交种可溶性糖含量显著高于感蚜品系,接虫后感蚜品系可溶性糖含量升高显著.     

植物抗病育种中的标记辅助选择与甘蔗

抗病基因的分子标记具有稳定、准确、高效的特点,通过对基因型而不是表型的直接选择,抗病基因分子标记应用于辅助选择可大大加快常规育种进程,提高育种效率。本文评述了植物抗病育种中标记辅助选择的发展概况,介绍了各种DNA分子标记技术及植物抗病基因连锁标记的筛选方法,重点介绍了甘蔗抗病基因分子标记研究的现状,展望了分子标记辅助选择在甘蔗抗病育种中的前景。     

Force Required to Detach Conidia of Helminthosporium maydis

The force required to break the conidium-conidiophore attachment in Helminthosporium maydis was measured by centrifugation and by a small jet of air. The force which removed half the spores from their stalks was found by both methods to be about 1 × 10⁻² dynes. The corresponding speed of the air jet was about 10 m/sec. Although spores are removed over a range of applied force, most of this spread is accounted for by the variation in spore size. Therefore, the apparent strength of the attachment of conidia to conidiophores, though relatively large, is surprisingly uniform.     

Heat-induced antigen retrieval: mechanisms and application to histochemistry

Since the introduction of the fluorescence-labeled antibody method by Coons et al. [Immunological properties of antibody containing a fluorescent group. Proc Soc Exp Biol Med 47, 200-2002], many immunohistochemical methods have been refined to obtain high sensitivity with low background staining at both light and electron microscopic levels. Heat-induced antigen retrieval (HIAR) reported by Shi et al. in the early 1990s has greatly contributed to immunohistochemical analysis for formalin-fixed and paraffin-embedded (FFPE) materials, particularly in the field of pathology. Although antigen retrieval techniques including enzyme digestion, treatment with protein denaturants and heating have been considered tricky and mysterious techniques, the mechanisms of HIAR have been rapidly elucidated. Heating cleaves crosslinks (methylene bridges) and add methylol groups in formaldehyde-fixed proteins and nucleic acids and extends polypeptides to unmask epitopes hidden in the inner portion of antigens or covered by adjacent macromolecules. In buffers having an appropriate pH and ion concentration, epitopes are exposed without entangling the extended polypeptides during cooling process, since polypeptides may strike a balance between hydrophobic attraction force and electrostatic repulsion force. Recent studies have demonstrated that HIAR is applicable for immunohistochemistry with various kinds of specimens, i.e., FFPE materials, frozen sections, plastic-embedded specimens, and physically fixed tissues at both the light- and electron-microscopic levels, and have suggested that the mechanism of HIAR is common to aldehyde-fixed and aldehyde-unfixed materials. Furthermore, heating has been shown to be effective for flow cytometry, nucleic acid histochemistry (fluorescein in situ hybridization (FISH), in situ hybridization (ISH), and terminal deoxynucleotidyl transferase-mediated nick labeling (TUNEL)), and extraction and analysis of macromolecules in both FFPE archive materials and specimens processed by other procedures. In this article, we review mechanism of HIAR and application of heating in both immunohistochemistry and other histochemical reactions.