Isolation by ConA binding of haustoria from different rust fungi and comparison of their surface qualities

Summary Rust haustoria isolated from infected leaf tissue strongly bind to ConA. This property was exploited to purify them by affinity chromatography on a ConA-Sepharose macrobead column. Haustoria were obtained with more than 90% purity and yields of up to 50%. Binding of haustoria to the column was partially inhibited by a ConA-specific sugar, methyl -D-mannopyranoside. Compared to ConA,Lens culinaris agglutinin and wheat germ agglutinin were less efficient affinity ligands. Using ConA-Sepharose, rust haustoria from a variety of sources could be isolated with equal efficiency, indicating that they have similar carbohydrate surface properties. The haustoria maintained their typical shape after the isolation procedure, which suggests a rather rigid wall structure. The morphology of haustoria was characteristic both for a given species and the nuclear condition of the rust mycelium. Electron microscopy of isolated haustoria revealed an intact haustorial wall surrounded by a fibrillar layer presumably derived from the extrahaustorial matrix. The matrix thus appears to represent a layer with gel-like properties which is rich in ConA-binding carbohydrates and connected to the haustorial wall but not to the host-derived extrahaustorial membrane.Abbreviations ConA Concanavalin A - LCA Lens culinaris agglutinin - WGA wheat germ agglutinin - FITC fluorescein isothiocyanate - DAPI 4,6-diamidinophenylindol×2 HCl     

植物病原真菌的自噬

作为真核生物中普遍存在的现象,自噬不但实现了对细胞内物质的降解和回收利用,而且与植物病原真菌早期侵染阶段的附着胞发育、膨压升高、菌丝体形成、完成侵染等一系列过程密切相关,并且发挥了重要的作用。本文归纳了植物病原真菌自噬的相关基因和自噬过程;总结了自噬对病原真菌生长发育、致病力的调控和影响;概括了病原真菌自噬所涉及的信号通路;阐明了自噬影响植物病原真菌侵染过程的主要分子机制。为今后以自噬相关基因或蛋白作为靶点来筛选抑制病原真菌侵染的新型药物提供新的策略和思路。     

New geographical distribution and host records of rust fungi from northern Thailand

Ten species of rust fungi (Crossopsora 2, Maravalia 1, Pileolaria 1, Puccinia 1, Ravenelia 1, Sphaerophragmium 1, Uredo 2, and Uromyces 1) are newly recorded together with six new host plants in Thailand.Contribution no. 194, Laboratory of Plant Parasitic Mycology, Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan     

拮抗植物病原真菌的纤维素/木质素降解菌株的筛选及鉴定

【背景】秸秆还田在改善土壤肥力和丰富营养等方面有重要作用,但是也存在秸秆难以快速降解利用和病原真菌病害威胁的问题。【目的】为解决还田秸秆的降解和病原真菌病害问题,从长期秸秆还田地区采集样品,从中筛选出具有降解秸秆和抑菌功能的菌株。【方法】采用稀释分离法、苯胺蓝染色法和刚果红染色法等对秸秆高效降解菌株进行筛选,通过16S rRNA基因测序及构建系统发育树进行菌株鉴定。采用对峙培养法测定筛选到的秸秆降解菌株对玉米大斑病菌(Setostphaeria turcica)、梨黑斑病菌(Alternaria kikuchiana)、马铃薯早疫病菌(Alternaria solani)、链格孢属真菌(Alternaria alternata) ACCC38230和ACCC38231等5种供试植物病原真菌的抑制作用。以玉米大斑病菌(Setostphaeria turcica)为后期供试植物病原真菌,测定拮抗菌株代谢产物的抑菌能力;通过观察拮抗菌株粗提液对玉米大斑病菌分生孢子萌发和菌丝生长的影响,从而测定菌株对病原真菌的抑制作用。【结果】从秸秆还田土壤中分离筛选获得3株高效降解纤维素及木质素菌株并命名为JY122、ZY133和JY215。通过形态学观察和16S rRNA基因测序鉴定上述菌株全部为芽孢杆菌属(Bacillus)。通过系统发育树分析结果表明,JY122与蜡样芽孢杆菌(Bacillus cereus)相似性为99.4%,ZY133与枯草芽孢杆菌(Bacillus subtilis)相似性为100%,JY215与贝莱斯芽孢杆菌(Bacillus velezensis)相似性为99.1%。平板对峙实验结果显示,JY122、ZY133和JY215菌株对不同种属的植物病原真菌均有较强的抑制作用,抑制率高达43.74%-67.54%。此外,上述芽孢杆菌代谢产物具有抑菌活性及很强的热稳定性,经95℃处理后依然具有良好的抑菌效果。【结论】筛选获得的JY122、JY133和JY215菌株具有高效降解纤维素/木质素能力,抑制多种植物病原真菌生长,代谢产物抑菌能力强且热稳定性高。这为玉米秸秆还田提供菌株资源,也为进一步解决秸秆还田难点提供了新方法和思路。     

The rust fungi (Uredinales) on ferns in South Africa

The rust fungi (Uredinales, basidiomycota) occuring on ferns (Pteridophyta) in South Africa are described, illustrated and keyed out. All species belong to the pucciniastraceous genera Milesina (M. blechni), Uredinopsis (U. pteridis) or to the related uredinial anamorph genus Milesia (M. nervisequa, M. cf. magellanica, M. silvae-knysnae). Milesia silvae-knysnae on Polystichum pungens is new to science; it probably belongs to the teleomorph genus Milesina. Milesina blechni is reported from South Africa for the first time on the new hosts Blechnum punctulatum and Rumohra adiantoides; it has hitherto been known only from the Northern Hemisphere on Blechnum spicant. Rust specimens collected on Asplenium aethiopicum and A. rutifolium were tentatively assigned to Milesia magellanica which has been known so far only from southern Chile. Hyalopsora neocheilanthis, Milesina neoexigua and M. neovogesiaca are proposed as new names for Hyalopsora cheilanthis, Milesia exigua and M. vogesiaca. It is discussed that the pucciniastraceous fern rusts could have reached South Africa either by migration (M. blechni) or by long-distance air dispersal. In the absence of their gametophyte hosts, species of Abies (Pinaceae), the rusts have to propagate in South Africa by urediniospores infecting fern to fern. Taxonomical novelties Milesia silvae-knysnae R. Berndt Milesina neoexigua R. Berndt Milesina neovogesiaca R. Berndt Hyalopsora neocheilanthis R. Berndt     

Components in the haustorial wall of the flax rust fungus,Melampsora lini, are labelled by three anti-calmodulin monoclonal antibodies

Summary Immunolocalisation studies, using flax leaf material infected with the flax rust fungus,Melampsora lini, and isolated haustorial complexes, have shown that three anti-calmodulin monoclonal antibodies bind to the haustorial wall of the fungus. The epitopes recognised by these antibodies are inserted into the wall during the early stages of haustorium development and remain in the wall throughout the life of the haustorium. The epitopes are present in both compatible and incompatible reactions and are oligosaccharide in nature. The results provide evidence for molecular differentiation within the haustorial complex ofM. lini.Abbreviations BMM butyl-methylmethacrylate - CaM calmodulin - FITC fluorescein isothiocyanate - MAb monoclonal antibody     

Bacterial effects on arbuscular mycorrhizal fungi and mycorrhiza development as influenced by the bacteria, fungi, and host plant

Bacterial strains from mycorrhizal roots (three belonging to Comamonadaceae and one to Oxalobacteraceae) and from non-mycorrhizal roots (two belonging to Comamonadaceae) of Medicago truncatula and two reference strains (Collimonas fungivorans Ter331 and Pseudomonas fluorescens C7R12) were tested for their effect on the in vitro saprophytic growth of Glomus mosseae BEG12 and on its colonization of M. truncatula roots. Only the Oxalobacteraceae strain, isolated from barrel medic mycorrhizal roots, and the reference strain P. fluorescens C7R12 promoted both the saprophytic growth and root colonization of G. mosseae BEG12, indicating that they acted as mycorrhiza helper bacteria. Greatest effects were achieved by P. fluorescens C7R12 and its influence on the saprophytic growth of G. mosseae was compared to that on Gigaspora rosea BEG9 to determine if the bacterial stimulation was fungal specific. This fungal specificity, together with plant specificity, was finally evaluated by comparing bacterial effects on arbuscular mycorrhizal symbiosis when each of the fungal species was inoculated to two different plant species (M. truncatula and Lycopersicon esculentum). The results obtained showed that promotion of saprophytic growth by P. fluorescens C7R12 was expressed in vitro towards G. mosseae but not towards G. rosea. Bacterial promotion of mycorhization was also expressed towards G. mosseae, but not G. rosea, in roots of M. truncatula and L. esculentum. Taken together, results indicated that enhancement of arbuscular mycorrhiza development was only induced by a limited number of bacteria, promotion by the most efficient bacterial strain being fungal and not plant specific.     

Rust fungi (Uredinales) found in Marshall Islands and Pohnpei

Uromyces wedeliae, Coleosporium plumeriae, andUredo guettardae are reported for the first time from Kwajalein Atoll, Republic of Marshall Islands, and Pohnpei, Federal States of Micronesia.     

Effect of brassicaceous plants on the survival and infectivity of insect pathogenic fungi

Studies were conducted to investigate potentialinteractions between brassicaceous plants, theisothiocyanates they produce and insectpathogenic fungi. Studies in vitro showedthat 100 ppm of 2-phenylethyl isothiocyanatecompletely inhibited growth of Metarhiziumanisopliae and Tolypocladiumcylindrosporum. T. cylindrosporum wassignificantly inhibited by 10 ppm 2-phenylethylisothiocyanate, but not by 1 ppm. M.anisopliae was not inhibited by 10 or 1 ppm2-phenylethyl isothiocyanate. The effect offreshly grated rutabaga on virulence of M.anisopliae to Galleria mellonella larvaewas also tested, resulting in reduced infectionby M. anisopliae. To study the effect ofplants in the Brassicaceae under morefield-like conditions fungi were added to soilwithout plants or at the bases of two differentplant species, Barbarea vulgaris and Eruca vesicaria sativa. Soil was sampled at 5,10 and 15 days to determine the concentrationof colony forming units (CFUs). Treatments withplants did not have fewer CFUs than thecontrol. Instead significantly fewer CFUsresulted when M. anisopliae was added topots with soil only than to pots with plants.There were no significant differences betweenthe two plant species in the number of CFUs atday 5. For days 10 and 15, however,significantly more M. anisopliae CFUswere found in pots containing E. vesicariasativa than B. vulgaris. T.cylindrosporum was less affected by thedifferent treatments than M. anisopliae.Our studies demonstrated that whileisothiocyanates can inhibit insect pathogenicfungi in Petri dishes, when using a morerealistic fungus/plant/soil microcosm no fungalinhibition was found.     

Three species of rust fungi (Uredinales) from Okinawa,Japan

Three species of rust fungi were reported based on the specimens collected in the islands of the Yaeyama group and Okinawa, Japan.Puccinia tarennicola onTarenna gracilipes andUredo daphniphylli onDaphniphyllum teijsmannii were described as new species.Villebrunea frutescens was added to the host plants ofUredo pipturi. Contribution No. 115, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Ecological genetic interactions between a clonal host plant (Spartina pectinata) and associated rust fungi Puccinia seymouriana and Puccinia sparganioides

The spatial scale of genetic diversity among patches of a host plant could affect the likelihood of pathogen adaptation to the host. If host patches are genetically distinct, pathogen adaptation to local host genotypes may occur. To study this issue, we focused on the ecological and genetic interactions between two rust fungi, Puccinia seymouriana and P. sparganioides, and the clonal prairie grass, Spartina pectinata. In a field transplant experiment, disease levels differed among plants from different patches, suggesting variation in resistance. Over a 4.5-km scale, disease levels were not higher on plants transplanted back into their source patch as opposed to other locations, providing no evidence for local adaptation in the pathogen. However, on the spatial scales examined (ranging from 0.2 km to 120 km), there was no relationship between the physical distance separating host patches and their similarity in isozyme banding patterns, implying that plants from more distant patches are not necessarily more genetically distinct than plants from nearby patches. Plants derived from the most distant location had, on average, the lowest mean number of pustules at the end of the summer, suggesting the need for reciprocal transplant studies to be performed on a larger spatial scale.     

植物病原真菌抑制杂草的活性筛选及菌株CY-H的活性代谢产物

【目的】分离并鉴定具有较好除草活性的植物病原真菌菌株,进一步分离活性化合物,为开发新型生物源除草剂奠定一定的基础。【方法】采用固体培养基接种法分离纯化植物病原真菌,通过形态学特征观察和5.8S rDNA测序鉴定目标菌株;在活性筛选追踪下,对活性组分进行追踪分离及纯化,经波谱分析确定活性单体化合物结构;采用培养皿生物分析法测定活性单体化合物的除草活性及对常见作物的安全性。【结果】茶叶致病菌CY-H具有较好的除草活性,其发酵液对稗草和反枝苋根的抑制率分别为94.6%和77.3%。CY-H被鉴定为间座壳属菌(Diaporthe sp.)。从CY-H菌中分离得到单体化合物CY1被鉴定为cytosporaphenones C。在供试浓度为100μg/mL时,CY1具有较好的抑制反枝苋根的活性,抑制率为57.1%,且其对小麦和油菜的安全性较好,抑制率均在20%左右。【结论】茶叶致病菌CY-H具有开发成微生物源除草剂的潜力。     

Quantitative estimation of the surface carbohydrates on the infection structures of rust fungi with enzymes and lectins

Lectins of Triticum vulgaris (WGA), Concanavalia ensiformis (ConA), Phaseolus vulgaris (PHA), Lotus tetragonolobus (LTA), Arachis hypogaea (PNA), Ricinus communis (RCA I), Griffonia simplicifolia (GSA II) and the enzymes endo-(13)--D-glucanase, exo-(13)--D-glucanase and laminarinase were tested for binding to the infection structures of Puccinia coronata and Uromyces appendiculatus. The enzymes and lectins were labeled with fluorescein and the fluorescence was measured with a microscope photometer. GSA II and ConA bound to all parts of the two rust fungi to a certain extent. The germ tubes of P. coronata bound at least two times more WGA than did the germ tubes of U. appendiculatus. The appressoria of both rust fungi additionally bound exo-(13)--glucanase, endo-(13)--glucanase and laminarinase. The substomatal vesicle and the infection hypha of both rust fungi mainly bound the glucanases. Furthermore, the substomatal vesicle of U. appendiculatus bound PHA. No obvious binding with LTA, RCA I and PNA was observed. Binding generally could be inhibited by appropriate haptens. Binding to uredospores generally appeared unspecific. The results indicate that the germ tubes have chitin on their outer surfaces, the appressoria chitin and glucans and the substomatal vesicles and infection hyphae mainly glucans. Compared to P. coronata, U. appendiculatus has more terminal linked glucose residues or the glucan has more (13)--linkages. Also, U. appendiculatus has N-acetylgalactosamine or a similar sugar on the surface of the substomatal vesicle.Abbreviations ConA Concanavalia ensiformis agglutinin - FITC fluorescein isothiocyanate - GSA II Griffonia simplicifolic agglutimin II - LTA Lotus tetragonolobus agglutinin - PBS phosphate buffered saline - PNA Peanut agglutinin - RCA I Ricinus communis agglutinin I - PHA Phaseolus vulgaris agglutinin - WGA Wheat germ agglutinin     

独蒜兰提取物对植物病原真菌的抑菌活性

【目的】研究独蒜兰假鳞茎乙醇提取物对植物病原真菌的抑菌作用,为植物源杀菌剂的开发提供依据。【方法】采用菌丝生长速率法,研究独蒜兰假鳞茎乙醇提取物对15种植物病原菌的抑制活性;以西瓜尖孢镰刀菌作为供试菌,进一步研究该提取物对病原真菌的菌丝干重、细胞膜、过氧化氢酶（CAT）、过氧化物酶（POD）和超氧化物歧化酶（SOD）等的影响。【结果】独蒜兰假鳞茎提取物对辣椒疫霉病菌、西瓜尖孢镰刀菌、番茄灰霉病菌和非洲隐地疫霉菌的抑菌效果明显,其EC₅₀值分别为0.849、0.782、0.813和1.161 mg·mL^-1;经独蒜兰提取物处理后的西瓜尖孢镰刀菌菌丝干重随着药剂浓度的增加而减少;细胞膜丙二醛含量和相对电导率增加;菌丝体细胞内CAT、POD和SOD 3种保护酶活性增加。【结论】独蒜兰假鳞茎提取物对植物病原真菌具有较好的抑菌活性,其抑菌作用可能与其干扰菌丝生长、使菌丝细胞膜正常功能受损等有关。     

山芝麻提取物对10种植物病原真菌的抑菌活性初探

该文采用菌丝生长速率法,研究了山芝麻根、茎和叶不同溶剂萃取物在1.5 mg·mL^-1浓度下对10种植物病原真菌菌丝生长的抑制活性,用孢子萌发法测试了山芝麻根石油醚相和乙酸乙酯相萃取物对香蕉炭疽病菌分生孢子萌发的抑制作用,用离体法测试了山芝麻根石油醚相和乙酸乙酯相萃取物对香蕉炭疽病的防治效果,并通过气相与质谱联用技术(Gas Chromatography-Mass Spectrometer,GC-MS)分析了山芝麻根石油醚相和乙酸乙酯相萃取物的主要成分,测试了其中8种主要化合物对香蕉炭疽病菌菌丝生长的抑制活性。结果表明:山芝麻各部分萃取相对10种植物病原真菌菌丝生长均具有不同程度的抑制作用。其中:在1.5 mg·mL^-1浓度下,山芝麻根石油醚相和乙酸乙酯相萃取物对香蕉炭疽病菌菌丝生长的抑制率分别为87.00%和86.14%,其EC50分别为0.062 mg·mL^-1和0.052 mg·mL^-1;浓度在2、4、8 mg·mL^-1时,山芝麻根石油醚相和乙酸乙酯相萃取物对香蕉炭疽病菌分...     

Mycorrhizal fungi and the nutrient ecology of three oldfield annual plant species

Summary Three oldfield annual species (Abutilon theophrasti Medic., Ambrosia artemisiifolia L. and Setaria lutescens (Weigel) Hubb.) were investigated. All three developed substantial mycorrhizal infections when inoculated with Glomus etunicatum Becker & Gerd. Mycorrhizal infection dramatically increased phosphorus content and dry weight of both Abutilon and Ambrosia, but did not significantly affect dry weight and only modestly increased phosphorus content of Setaria. These results were consistent with a lower level of infection and much greater root density in Setaria than in the other species. When Abutilon was grown in the presence of Setaria, mycorrhizal infection had no effect on Abutilon phosphorus content or dry weight. The depressive effect of Setaria on the response to inoculation in Abutilon was probably not caused by water soluble allelopathic chemicals from Setaria roots, but soil leachate from Abutilon plants did inhibit infection in other Abutilon plants. The data were consistent with the hypothesis that the very high root density and effective soil exploitation of Setaria reduced the benefit from mycorrhizal infection in Abutilon via phosphorus depletion in a large proportion of the available soil volume. Furthermore, even if mycorrhizal infection were capable of increasing phosphorus content of Abutilon in the presence of Setaria, the very high competitive ability of Setaria for nitrogen in the soil could have reduced the benefit of an enhanced phosphorus content. Carbon isotope ratios were reduced in Abutilon by mycorrhizal infection, indicating a possible reduction in water use efficiency.     

Volatiles of two chemotypes of Majorana syriaca L. (Labiatae) as olfactory cues for the honeybee

Summary A study of olfactory discrimination by honeybee has been set up in order to understand the role of volatiles of the aromatic plant Majorana syriaca in attracting pollinating insects. The honeybee's response to volatiles from leaves and inflorescences of two M. syriaca chemotypes, which differ in the thymol carvacrol ratio of their volatiles, was tested using a bioessay method based on associative conditioning and recruitement techniques. Behavioural data show that a honeybee identifies and reacts selectively to olfactory signals from leaves and from inflorescences of the two chemotypes. Such data suggest that the volatiles from all parts of the aromatic plant M. syriaca may have a role in attracting pollinators. The vegetative parts produce a volatile emission which attracts the pollinators from a distance towards the whole plant. At close range these are directed by the flower signal which is amplified by the volatiles of bracts and leaves in the inflorescence.     

Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin,fusicoccin

The high-affinity fusicoccin-binding protein (FCBP) was solubilized from plasma-membrane vesicles prepared from leaves of Vicia faba L. by aqueous two-phase partitioning. Conditions for the solubilization of intact FCBP-radioligand complexes were worked out. About 60–70% of the complexes can be solubilized with 50–60 mM nonanoyl-N-methylglucamide in the presence of 1 mg· ml^-1 soybean phosphatidylcholine, type IV S, and 20% (v/v) glycerol at pH 5.5. The slow dissociation of the radioligand, 9-nor-fusicoccin-8-alcohol-[³H] from the FCBP at low temperatures permits the purification of FCBP-radioligand complexes at 4–10° C by fast protein liquid chromatography on anion-exchange and gel permeation columns. The FCBP, extracted from plasma membranes with cholate and chromatographed in the presence of this detergent, gave an apparent molecular mass (M_r) of 80±20 kDa on gel permeation columns under the conditions used. By comparison of the elution profiles of the fraction most enriched in FCBP-radioligand complexes with polypeptide patterns obtained on sodium dodecyl sulfate-polyacrylamide gels, a polypeptide with an M_r of approx. 34kDa co-separated with the radioactivity profile. A second, faint band of approx. 31 kDa was sometimes also observed co-electrophoresing. Photoaffinity labeling of plasma-membrane vesicles with the new compound 9-nor-8[(3,5-[³H]-4-azidobenzoy)ethylenediamine]-fusicoccin ([³H]ABE-FC) and subsequent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis labeled a single band with an M_r of 35±1 kDa. Labeling in this band was strongly reduced when the membranes were incubated with [³H]ABE-FC in the presence of 0.1–1 M fusicoccin. From our data, we conclude (i) that the 34-35-kDa polypeptide represents the FCBP and (ii) that in detergent extracts of plasma membranes this polypeptide is probably present as a di- or trimeric structure.Abbreviations ABE-FC [(4-azidobenzoyl)-ethylenediamine]-fusicoccin - ABE-NHS (4-azidobenzoyl)-N-hydroxysuccinimide ester - FC fusicoccin - FCBP fusicoccin-binding protein - FCol 9-norfusicoccin-8-alcohol - MAB monoclonal antibody - Mega-9(10) nonanoyl(decanoyl)-N-methylglucamide - M_r apparent molecular mass - PMSF phenylmethyl-sulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Temperature and seedling age affect susceptibility of perennial ryegrass seedlings to pathogenic fungi

Summary Effects of temperature and seedling age on survival of perennial ryegrass (Lolium perenne L.) seedlings grown on sand-wheat wholemeal cultures of different isolates ofFusarium spp. (9 isolates),Pythium spp. (9 isolates), andChaetomium spp. (1 isolate) are reported. Some isolates were virulent over the whole range of temperatures tested (7.5–27.5°C). The virulence of others depended on temperature. Most isolates were less virulent at intermediate temperatures (12.5–22.5°C) than at higher or lower temperatures. At 25°C ryegrass seedlings were susceptible to fungal attack for only a limited period after germination commenced. This period differed for different fungi, but for most isolates tested, seedlings were resistant after 2–3 days.