Local and systemic changes in glucosinolates in Chinese and European cultivars of oilseed rape (Brassica nap us L.) after inoculation with Sclerotinia sclerotiorum (stem rot)

Four Chinese Brassica napus lines, generated through a breeding programme to identify Sclerotinia sclerotiorum tolerant and susceptible lines, and three European varieties were analysed for changes in glucosinolates (qualitative and quantitative), and general host reactions, after localised inoculation with a UK S. sclerotiorum isolate. Plants at the fifth leaf stage were either singly inoculated (third leaf) or were inoculated once (third leaf) and then challenged a second time (seventh leaf) 7 days after the first inoculation. The results showed very distinct reactions in the different lines and cultivars to the fungus, both locally and systemically. Of the European lines B. napus cv. Bienvenu showed good resistance (small lesions and less host damage) both 3 and 7 days post-inoculation. Capricorn was the most susceptible followed by Cobra; the third leaves of these cultivars were showing strong chlorotic and necrotic reactions by day 3 and lesions were well developed. By day 7 the third leaves of Capricorn were completely rotten whilst Cobra still had a little healthy tissue. Inoculation of the four Chinese lines showed that two had moderate resistance (014 and 020 — slightly less resistant than Bienvenu) and two were very susceptible (016 and 024 — similar reactions to Capricorn and Cobra), based on lesion size and host tissue damage. Glucosinolate induction in line 014 was good both locally and systemically, with clear local and systemic induction of indolylglucosinolates and 2-phenylethylglucosinolate both 3 and 7 days post-inoculation. Line 020 did not show no particular increases in glucosinolates after inoculation either locally or systemically. In line 016 there was a small local increase and a large systemic reduction in total glucosinolates. Inoculation of line 024 caused no major local changes in glucosinolates and again a big reduction in glucosinolates systemically. The dual inoculation system, with lines 014 and 016, produced comparable results, with line 014 showing good local and systemic induction of glucosinolates (after the first inoculation) and a further local and systemic induction after the second inoculation. This induction in pre-inoculated line 014 plants was associated with a reduction in lesion size of the second inoculum. Line 016 responded poorly both locally and systemically, and there were no real decrease in the lesion size of the second inoculum. It appears that in line 014 glucosinolate induction may be an important part of resistance, whereas in line 020 there are clearly other non-glucosinolate factors involved. The poor local and systemic induction of glucosinolates in lines 016 and 024, and subsequent susceptibility, implies that glucosinolate induction may be an important marker of resistance to S. sclerotiorum in oilseed rape.     

aspS encoding an unusual aspartyl protease from Sclerotinia sclerotiorum is expressed during phytopathogenesis

The gene aspS encoding an aspartyl protease has been cloned from Sclerotinia sclerotiorum by screening a genomic library with a PCR-amplified fragment of the gene. The open reading frame of 1368 bp interrupted by one intron would encode a preproprotein of 435 amino acids. The catalytic aspartyl residues characteristic of aspartyl proteases are conserved; however, the active-site motif (DSG) in the N-terminal lobe is unusual in that Ser replaced Thr used in the active-site motif (DTG) of the C-terminal lobe and in all other fungal aspartyl proteases. RT-PCR revealed that aspS expression in axenic culture is not subjected to catabolite repression and demonstrated that aspS is expressed from the beginning of infection of sunflower cotyledons.     

椪柑精油及其主要抑菌组分对菌核青霉的抑制作用

采用水蒸气蒸馏法提取椪柑(Citrus reticulate Blanco)果皮精油,并用GC-MS对其成分进行分析,共鉴定出46种成分,主要包括柠檬烯(57.67%)、β-芳樟醇(5.36%)、2-蒈烯(4.47%)、β-蒎稀(4.31%)、γ-松油烯(4.08%)、α-蒎烯(3.27%)、1,2-二异丙烯基环丁烷(2.87%)、β-月桂烯(2.77%)、α-侧柏烯(2.40%)、β-水芹烯(2.24%)、癸醛(1.80%)和香茅醇(1.49%)等。采用污染食物技术(poisoned food technique)和液体培养法(liquid culture)测定了不同浓度椪柑精油以及主要抑菌组分对菌核青霉的抑制作用。结果表明:不同浓度(0.5~20μl/ml)的椪柑精油对菌核青霉生长均有一定的抑制,且抑菌效果与浓度呈正相关。无论是采用污染食物技术还是液体培养法,20μl/ml椪柑精油均能完全抑制菌核青霉生长;随着培养 时间延长到7d, 20μl/ml椪柑精油抑菌效果有所下降,但仍能显著抑制菌核青霉生长。采用污染食物技术考察了椪柑精油中7种常见抑菌成分对菌核青霉的影响。结果表明,0.04μl/ml柠檬醛和1.07μl/ml β-芳樟醇能显著抑制菌核青霉生长,而其他组分无明显作用。实验结果表明,椪柑精油的抑菌作用可能归功于其所含的柠檬醛和β-芳樟醇。     

Effect of Carbon Source on Production of Lytic Enzymes by the Sclerotial Parasites Trichoderma atroviride and Coniothyrium minitans

Three isolates of Trichoderma atroviride and two isolates of Coniothyrium minitans known to efficiently degrade sclerotia of Sclerotinia sclerotiorum were cultured on minimal medium with sucrose, carboxymethyl cellulose (CMC), xylan, laminarin, colloidal chitin or powdered sclerotia as carbon source. The activity of endochitinase, endo‐β‐1,3‐glucanase, endoxylanase and endocellulase in culture filtrates was determined after 7 and 15 days of culture using dye‐labelled substrates. The strongest inducers of chitinase were colloidal chitin and sclerotia powder. Chitinase activity appeared to be faster induced in the isolates of T. atroviride than in the isolates of C. minitans, but the maximum level of activity present in culture filtrates of the two species was similar. With CMC and xylan as carbon source, concurrent production of the corresponding enzymes was observed for the Trichoderma isolates. The isolates of C. minitans produced cellulase on xylan but not on CMC, whereas xylanase was produced on both carbon sources. Laminarin induced the formation of glucanases in the three isolates of T. atroviride but not the isolates of C. minitans. However, in the sclerotia‐containing cultures of C. minitans glucanase activity was even higher than in the respective cultures of the Trichoderma isolates. During the 31‐day study period, the pattern of enzyme production in shake cultures containing sclerotia powder was very similar for the isolates of T. atroviride and C. minitans. Glucanase activity generally reached a peak 24 days after inoculation of the flasks, whereas the activity of chitinase, cellulase and xylanase remained fairly constant throughout the experiment.     

Genotypic characteristics in populations of Sclerotinia sclerotiorum from New York State,USA

White mould, caused by the fungus Sclerotinia sclerotiorum, is one of the most destructive diseases of beans globally. In New York State, USA, white mould causes substantial losses in soybean, snap, dry and succulent baby lima beans, which are grown successively in intensive crop rotations. Management strategies for white mould in these crops are reliant upon the prophylactic use of fungicides. No complementary information on the genetic structure of the populations of S. sclerotiorum in New York State, USA is available. Twenty isolates of S. sclerotiorum were collected from symptomatic bean plants within each of 10 fields across New York State, USA in 2014. Eight microsatellite (SSR) markers were used to characterise the genotypic diversity of the hyphal‐tipped isolates. Twenty‐four multilocus genotypes (MLGs) were detected within the population but one MLG was most prevalent. Although STRUCTURE analysis identified two subpopulations, these subpopulations were not associated with geographic location, suggesting no spatial structure to the population. In addition, the pathogen populations were predominantly clonal, with some evidence of infrequent outcrossing. These findings may assist in understanding the durability of management strategies for white mould and support the selection of representative isolates for host resistance screening for pathogen populations in the sampling area.     

Comparison of responses of ascospores and mycelium by ELISA with anti-mycelium and anti-ascospore antisera for the development of a method to detect Sclerotinia sclerotiorum on petals of oilseed rape

The goal of this project was the development of a serological test for the detection of Sclerotinia sclerotiorum on oilseed rape petals. Since the fungus exists in two forms on petals, as ascospores and mycelium, the responses of anti-mycelium and anti-ascospore antisera against these two kinds of antigens were compared. Two anti-mycelium sera, Smy and Smy', were produced against mycelial soluble extracts at different concentrations (0.3 mg and 0.1 mg of protein ml^-1). Smy gave the greatest response level with eight S. sclerotiorum mycelial extracts tested. It had a very superior level of recognition to that of anti-ascospore serum, Ssp, when mycelium was tested as antigen. In contrast, Ssp and Smy were equally reactive when exposed to ascospores but the sensitivity of the assay was low. For each antiserum, the solution from which ascospores had been removed reacted similarly to the original suspension containing ascospores. A collection of fungi likely to be found on oilseed rape petals was examined. Cross-reactions were produced with both antisera, especially with Botrytis cinerea for which greater cross-reactivities were produced with Smy. The cross-absorption of antiserum Smy with a mycelial extract of B. cinerea considerably reduced this cross-reaction. The choice of antiserum for the development of a reliable detection system is discussed.     

Enzymatic oxalate decarboxylation in isolates of Sclerotinia sclerotiorum

Abstract Sclerotinia sclerotiorum isolates B24 (virulent) and SS41 (hypovirulent) possess oxalate decarboxylase. Production was regulated by composition and pH of culture medium and required the presence of oxalate or its precursor, succinic acid, as inducers. Mycelia of both isolates contain equivalent amounts of enzyme.     

菌核病防治研究进展

菌核病是一种寄主种类广泛的重大植物病害,可侵染450多种重要作物和草类,在我国每年给油菜、大豆以及多种蔬菜带来10～30亿元的损失.介绍了菌核病的症状、危害以及致病机理等,概述了主要的防治措施,并报道了国内外在关于菌核病生物防治、转基因育种、分子机理等方面的研究进展.     

两株毒力不同的核盘菌产草酸、果胶酶的比较

对强弱毒力不同的核盘菌Ep-1PNA5和Ep-1PN的主要致病因子草酸和果胶酶的产生进行了比较研究。结果发现强毒力的Ep-1PNA5和弱毒力Ep-1PN都可产生草酸,并且在发病油菜活体组织上,弱毒力Ep-1PN的病组织中的草酸含量高于Ep-1PNA5病组织;两个菌株在发病油菜活体组织上的果胶酶产量没有差异,但在诱导培养基中Ep-1PN菌株比Ep-1PNA5的果胶酶产量高。这一结果表明弱毒力Ep-1PN菌株毒力的衰退并不是因为其所携带的dsRNA因子抑制了草酸和果胶酶所产生的。     

海洋真菌菌核青霉FS50固体发酵代谢产物

采用硅胶柱、反相硅胶柱、凝胶柱、薄层制备和HPLC等色谱技术对1株分离自南海沉积物的海洋真菌菌核青霉Penicillium sclerotiorum FS50的大米发酵提取物进行分离纯化,从中分离得到7个化合物。通过波谱数据分析,化合物1-7分别被鉴定为:6,8‐dihydroxyisocoumarin‐3‐butyl formate(1),6,8‐dihydroxyisocoumarin‐3‐carboxylic acid(2),6‐methoxy‐8‐hydroxyisocoumarin‐3‐carboxylic acid(3),(+)‐sclerotiorin(4),4‐methyl‐5,6‐dihydropyren‐2‐one(5),5‐羟甲基糠醛(6),胡萝卜甙(7)。化合物1为新化合物,化合物2,3,6为首次从青霉属中分离得到。