Production of branches from unsuccessful primary germ tubes of conidia of Erysiphe graminis f. sp. tritici on wheat leaves

Confusion exists regarding nomenclature and possible significance of outgrowths produced by conidial primary germ tubes (PGT) of Erysiphe graminis f. sp. tritici Em. Marchai on wheat leaves. Production of these outgrowths has been taken as a criterion of successful host-parasite relationship. However, such outgrowths have been observed to have developed from 12.3 % of the unsuccessful PGT on a resistant wheat and 9.2 % of the unsuccessful PGT on a susceptible wheat.It is proposed that these outgrowths may be called PGT branches which may develop whether the penetration by PGT is successful or not. In case the PGT penetration is successful, then such a PGT branch may be called a secondary hypha. These branches from unsuccessful PGT may also produce host responses in the form of halos in the host cell-walls or may also penetrate the host cells successfully and produce apparently healthy (normal) haustoria. Both PGT branches and the secondary hyphae may be called functional when they produce either host responses in the form of halos or penetrate successfully and produce haustoria, or produce both halos and haustoria.     

Effect of Host Plant Resistance on Haustorium Formation in Cereal Rust Fungi

Haustoria of Puccinia triticina (wheat leaf rust fungus) and P. hordei (barley leaf rust fungus) were isolated from susceptible and partially resistant wheat lines, and susceptible, hypersensitive and partially resistant barley lines. Haustoria were counted and measured. The size of haustoria was similar in the partially resistant and susceptible genotypes but haustoria were smaller in the hypersensitive barley line L94+Pa7. The number of haustoria was reduced in both partially and hypersensitive lines when compared with susceptible ones. Therefore it seems that the reduction in the number of haustoria is a consequence of the resistance that can be attributable either to early abortion of infection units or reduced colony growth. The reduction of the number of haustoria was more pronounced in the adult plant stage.     

Release of the extracellular matrix from conidia ofBlumeria graminis in relation to germination

The release of extracellular matrix (ECM) and the emergence of germ tubes from conidia ofBlumeria graminis were studied by light microscopy and micromanipulation. More prompt and frequent ECM release was confirmed on an artificial hydrophobic substratum than on an artificial hydrophilic substratum. Conidia initially incubated on the hydrophilic substratum were transferred by micromanipulation to either the hydrophobic or the hydrophilic substrata. Immediately after transfer onto the hydrophobic substratum, 75% of conidia released ECM, whereas only 16% did so upon transfer to the hydrophilic substratum. Conidia transferred onto the hydrophobic substratum produced a primary germ tube (PGT) more promptly and frequently than those transferred to the hydrophilic substratum. Thus, conidia recognize and respond to substratum hydrophobicity perhaps immediately after contact. When inoculated onto either isolated barley cuticle or the hydrophobic artificial substratum, 2/3 of the conidia produced a PGT from their polar regions. By contrast, on the hydrophilic substratum 2/3 of conidia did so from the side region. These results show that substratum hydrophobicity affects the location of PGT emergence from conidia. Furthermore, the study indicates that very rapid recognition of surface hydrophobicity by conidia promotes ECM release and this in turn may influence the location of PGT emergence.     

Analysis of wheat resistance to the cereal aphidSitobion avenae using electrical penetration graphs and flow charts combined with correspondence analysis

The behaviour ofSitobion avenae (F.), was compared on resistant wheat lines ofTriticum monococcum (L.) and a susceptible variety ofTriticum aestivum (L.). Firstly, stylet penetration activities were monitored with the Electrical Penetration Graph (EPG) technique and subsequently analysed using flow charts combined with correspondence analysis. Plant resistance was shown to be associated with repeated penetrations without access to either the xylem or the phloem, and with numerous failures in starting a sustained sap ingestion (as represented by pattern E2). Access to sieve elements of the phloem did not seem to be much affected on resistant plants but it took the aphid three times as long to produce a sap ingestion pattern when maintained on the resistant lineT. monococcum n^o 44 (Tm44) as compared with aphids maintained on susceptible plants. As a result the total time spent in ingesting from sieve elements was reduced by 72% on Tm44. Secondly, direct observations of freely-moving apterous adults were performed. Aphids did not discriminate between resistant and susceptible wheat during the first 30 min of access to test leaves, but only 4 out of 25 aphids were still probing after eight hours on resistant Tm44. The relevance of these results to possible location of the resistance factor(s) are discussed. Although detection of plant resistance before sieve elements are reached can not be rigorously excluded, the factors involved inT. monococcum resistance toS. avenae undoubtedly occur within the phloem vessels.     

Polymorphic change of appressoria by the tomato powdery mildew Oidium neolycopersici on host tomato leaves reflects multiple unsuccessful penetration attempts

The appressorial shapes of the powdery mildews are an important clue to the taxonomy of the powdery mildew fungi, but the conidia of the tomato powdery mildew Oidium neolycopersici KTP-01 develop non-lobed, nipple-shaped, and moderately lobed or multilobed appressoria on the same leaves. To remove this ambiguity, we performed consecutive observations of sequential appressorial development of KTP-01 conidia with a high-fidelity digital microscope. Highly germinative conidia of KTP-01, collected from conidial pseudochains formed on the tomato leaves, were inoculated into host tomato and nonhost barley leaves or an artificial hydrophobic membrane (Parafilm). Events from germination initiation to appressorium formation were synchronous in all conidia on all materials used for inoculation, but post-appressorial behaviors varied among the materials. Appressoria on the membrane-stuck glass slide formed several projections at different portions of the appressoria to repeat unsuccessful penetration attempts. Similar unsuccessful penetration behavior by KTP-01 conidia was observed in the inoculations into leaves of barley plants, wild tomato species Lycopersicon peruvianum LA2172 (carrying the Ol-4 gene for powdery mildew resistance), and a susceptible host tomato (Lycopersicon esculentum) that had been inoculated with the barley powdery mildew (Blumeria graminis f. sp. hordei, race 1) conidia. On the barley leaves, all penetrations of KTP-01 were impeded by the papillae formed beneath the sites of the appressorial projections. On both the wild tomato and the race 1-inoculated cultivated tomato plants, KTP-01 conidia were prevented from forming functional haustoria by hypersensitive epidermal cell death; this hypersensitive reaction involved the Ol-4 gene in the wild tomato plants or the 'induced resistance' acquired by the nonpathogenic conidia previously inoculated into the cultivated tomato plants. All these KTP-01 conidia produced several projections on the appressoria during the repeated unsuccessful penetration attempts and eventually exhibited multilobed appressoria. On the host tomato leaves inoculated singly with KTP-01 conidia, fewer than 20% of the conidia located appressoria on the central part of target epidermal cells and succeeded in forming functional haustoria at the first penetration attempt without forming an appressorial projection. These conidia exhibited non-lobed appressoria. The remaining conidia, however, whose appressoria were located on/near the border of the target epidermal cells, were more likely to fail to penetrate at the first penetration, and then to develop additional projections for subsequent penetrations. Most conidia succeeded in forming functional haustoria at the second to fourth penetration attempts, but a few conidia failed to produce haustoria at all attempted penetrations. Eventually, the conidia that succeeded at the second penetration possessed a single appressorial projection (exhibiting the nipple-shaped appressoria), whereas the remaining conidia exhibited moderately lobed appressoria with two to four appressorial projections and multilobed appressoria, with more projections. Thus, the present study revealed that the basic shape of appressoria of KTP-01 was the non-lobed type, and that polymorphic changes of the appressoria occurred as a result of successive production of projections during repeated unsuccessful penetration attempts.     

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     

Ultrastructural comparison of incompatible and compatible interactions in the barley powdery mildew disease

Summary In the powdery mildew disease of barley,Erysiphe graminis f. sp.hordei forms an intimate relationship with compatible hosts, in which haustoria form in epidermal cells with no obvious detrimental effects on the host until late in the infection sequence. In incompatible interactions, by contrast, the deposition of papillae and localized host cell death have been correlated with the cessation of growth byE. g. hordei. With the advent of improved, low temperature methods of sample preparation, we felt that it was useful to reevaluate the structural details of interactions between barley andE. g. hordei by transmission electron microscopy. The haustoria that develop in susceptible barley lines appear highly metabolically active based on the occurrrence of abundant endoplasmic reticulum, Golgi-like cisternae, and vesicles. In comparison, haustoria found in the resistant barley line exhibited varying signs of degradation. A striking clearing of the matrix and loss of cristae were typical early changes in the haustorial mitochondria in incompatible interactions. The absence of distinct endoplasmic reticulum and Golgi-like cisternae, the formation of vacuoles, and the occurrence of a distended sheath were characteristic of intermediate stages of haustorial degeneration. At more advanced stages of degeneration, haustoria were dominated by large vacuoles containing membrane fragments. This process of degeneration was not observed in haustoria ofE. g. hordei developing in the susceptible barley line.Abbreviations b endoplasmic reticulum extension, blebbing - er endoplasmic reticulum - f fibrillar material - g Golgi-like structure - h haustorium - hb haustorial body - hcw haustorial cell wall - hcy haustorial cytoplasm - hf haustorial finger - hocw host cell wall - hocy host cytoplasm - 1 lipid-like droplet - m mitochondrion - mt microtubule - mve multivesicular body - n nucleus - p papilla - ph penetration site of an infection peg - pl plasma membrane - s sheath - sm extrahaustorial membrane - v vacuole - ve vesicle     

Evaluation of the Entomopathogenic Fungi Beauveria bassiana and Paecilomyces fumosoroseus for Microbial Control of the Silverleaf Whitefly, Bemisia argentifolii

Collaborative research was conducted at the USDA-ARS Subtropical Agricultural Research Center in southern Texas to assess the microbial control potential of Beauveria bassiana and Paecilomyces fumosoroseus against Bemisia whiteflies. Laboratory assays demonstrated the capacity of both pathogens to infect Bemisia argentifolii nymphs on excised hibiscus leaves incubated at relative humidities as low as 25% at 23 ± 2°C (ca. 35% infection by B. bassiana and P. fumosoroseus resulted from applications of 0.6–1.4 × 10³ conidia/mm² of leaf surface). In small-scale field trials using portable air-assist sprayers, applications at a high rate of 5 × 10¹³ conidia in 180 liters water/ha produced conidial densities of ca. 1–2.5 × 10³ conidia/mm² on the lower surfaces of cucurbit leaves. Multiple applications of one isolate of P. fumosoroseus and four isolates of B. bassiana made at this rate at 4- to 5-day intervals provided >90% control of large (third- and fourth-instar) nymphs on cucumbers and cantaloupe melons. The same rate applied at 7-day intervals also provided >90% control in zucchini squash, and a one-fourth rate (1.25 × 10¹³ conidia/ha) applied at 4- to 5-day intervals reduced numbers of large nymphs by >85% in cantaloupe melons. In contrast to the high efficacy of the fungal applications against nymphs, effects against adult whiteflies were minimal. The results indicated that both B. bassiana and P. fumosoroseus have strong potential for microbial control of nymphal whiteflies infesting cucurbit crops.     

Carbohydrate involvement during infection of maize by Helminthosporium maydis

Germination and germ tube length of Helminthosporium maydis conidia did not exhibit much difference on fixed decolourized and living green leaves. However, appressoria, penetrations and colonizations were much less on decolourized host leaves and were enhanced significantly when sugars were added in the infection court. Few leached conidia germinated on the decolourized host leaves and appressoria, penetrations and colonizations effected on them by leached conidia were almost negligible. The presence of exogenous sugars and leaf leachates enabled the leached conidia to accomplish some penetrations and colonizations. Carbohydrate content of decolourized leaves and leached conidia was much less than the green leaves and non-leached conidia, respectively. Carbohydrates accumulated at the infection sites/green islands which also exhibited higher chlorophyll content.     

Genetic transfer of resistance to powdery mildew and of an associated biochemical marker from Aegilops ventricosa to hexaploid wheat

Summary Resistance to powdery mildew, caused by the fungus Erysiphe graminis f.sp. tritici, has been transferred from Aegilops ventricosa (genomes D^vM^v) to hexaploid wheat (Triticum aestivum, ABD). In two transfer lines, H-93-8 and H-93-35, the resistance gene was linked to a gene encoding protein U-1, whereas one line, H-93-33, was resistant but lacked the molecular marker, and another line, H-93-1, was susceptible but carried the gene for U-1, indicating that the original M^v chromosome from Ae. ventricosa, carrying the two genes, had undergone recombination with a wheat chromosome in the last two lines.     

马铃薯对晚疫病水平抗性的组织细胞学观察

以马铃薯晚疫病水平抗性品种LBr-12和感病品种费乌瑞它为材料,采用普通光学和电子显微镜技术,系统研究了马铃薯与晚疫病菌(致病疫霉)互作的组织细胞学反应特征。观察结果显示:(1)接种后,水平抗性材料LBr-12出现过敏反应,病菌被限制在侵染点的几个细胞中,菌丝产生较少的分支和吸器。(2)感病品种费乌瑞它被侵染细胞呈蔓延趋势,菌丝产生较多的分支和吸器。(3)电镜观察发现,抗病品种上病菌的胞间菌丝、吸器母细胞、吸器在细胞和亚细胞水平均发生了一系列异常变化,包括原生质的电子致密度加深、液泡增多变大、菌丝细胞壁不规则增厚、细胞器排列紊乱及解体、吸器母细胞及吸器形态异常、病菌最终畸形坏死,同时发现抗病品种受病菌侵染时可迅速产生结构防卫反应,形成的细胞壁沉积物使胞壁极度增厚或细胞膜上产生乳突状结构。     

Fusarium nisikadoi, a new species from Japan

A new species ofFusarium, F. nisikadoi, isolated fromPhyllostachys nigra var.henonis (bamboo) andTriticum aestivum (wheat) in Japan, is described, illustrated and discussed. This species is differentiated from other known species of the genus by the following characteristics: whitish colony color, long zigzag-like chains of 0–3(-5)-septate clavate conidia, intermixed with pyriform conidia, produced mostly from monophialides and rarely from polyphialides in the aerial mycelium, very long and slender sporodochial conidia, and no chlamydospores. The long chains of septate conidia are known only in this species of the genusFusarium. The conidiophores on the aerial mycelium sometimes proliferate sympodially. The species is tentatively placed in the form-sectionLiseola.     

Comparison of categories of resistance in wheat and barley genotypes against biotype 2 of the Russian wheat aphid, Diuraphis noxia (Kurdjumov)

The Russian wheat aphid Diuraphis noxia (Kurdjumov) (Homoptera: Aphididae) is a global pest of wheat and barley. This arthropod is difficult to manage with pesticides or biological control agents due to the aphid’s ability to seek shelter in rolled leaves and also to develop virulent biotypes. During the past 20 years, the use of aphid-resistant cereal cultivars has proven to be an economically and ecologically beneficial method of protecting crops from D. noxia damage. Our research reports the results of experiments to determine the categories of D. noxia biotype 2 resistance present in Cereal Introduction Triticeae (CItr) 2401, and a barley genotype (IBRWAGP4-7), compared to control resistant and susceptible wheat and barley genotypes. CItr2401 and IBRWAGP4-7 exhibit no antixenosis, but both genotypes demonstrated antibiosis to D. noxia in the form of reduced aphid populations. Reduced leaf dry weight change, a measure of plant tolerance of D. noxia feeding, was significantly less in CItr2401 and IBRWAGP4-7 plants than in plants of susceptible control varieties. However, tolerance was negated when a tolerance index was calculated to correct for differences in aphid populations. Barley IBRWAGP4-7 is a new source of D. noxia biotype 2 resistance. D. noxia foliar leaf damage and population growth were significantly less on IBRWAGP4-7 plants than on plants of the susceptible barley variety Morex. IBRWAGP4-7 plants were equal in resistance to plants of the resistant barley STARS 9301 and wheat genotype CItr2401. Handling editor: Heikki Hokkanen     

Modification of competence for in vitro response to Fusarium oxysporum in tomato cells. II. Effect of the integration of Agrobacterium tumefaciens genes for auxin and cytokinin synthesis

We have studied the effect of a change in the endogenous hormone equilibria on the competence of tomato (Lycopersicon esculentum) cells to defend themselves against the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Calluses from cvs Davis and Red River, respectively resistant and susceptible to Fusarium and transgenic for an auxin- or cytokinin-synthesizing gene from Agrobacterium tumefaciens, were used. The integration of Agrobacterium hormone-related genes into susceptible cv Red River can bring the activation of defense processes to a stable competence as assessed by the inhibition of mycelial growth in dual culture and gem-tube elongation of Fusarium conidia, the determination of callose contents, peroxidase induction and ion leakage in the presence of fusaric acid. This is particularly true when the transformation results in a change of phytohormone equilibria towards an higher cytokin in concentration. On the contrary, in resistant cv Davis the inhibition of both fungal growth in dual culture and conidia germination is higher when the hormone balance is modified in favour of the auxins. No significant effect was observed for ion leakage and peroxidase induction, probably because of a constitutive overproduction of cytokinins in Davis cells.     

Molecular differentiation in pea powdery-mildew haustoria

Monoclonal antibodies have been raised against haustorial complexes isolated from pea (Pisum sativum L.) leaves infected by the biotrophic powdery mildew fungus Erysiphe pisi D.C. Immuno-localisation studies, using isolated haustorial complexes and infected pea leaf material, have shown that one of the antibodies, designated UB7, binds to fungal wall and plasma membranes present in both haustoria and mycelia. However, a second antibody, UB8, binds specifically to the haustorial plasma membrane, and does not label fungal plasma membranes in mycelia. Western blotting and antigen-modification techniques have shown that UB8 recognises a protein epitope of a 62-kDa antigen. A reduction in molecular weight of this component after endo-F treatment indicates that the antigen is an N-linked glycoprotein. UB7 also recognises a 62-kDa glycoprotein, which is susceptible to endo-F treatment, and the antibody binds to a carbohydrate epitope. Differences in molecular weights of the products after endo-F treatment of antigens show that the 62-kDa glycoproteins recognised by the antibodies are distinct molecules, in accordance with the localisation results. Overall, the results provide evidence for molecular differentiation associated with the development of haustoria in a biotrophic infection.Abbreviations ehm extrahaustorial membrane - ELISA enzyme-linked immunosorbent assay - HC haustorial complex - hpm haustorial plasma membrane - IIF indirect immunofluorescence - MAb monoclonal antibody - M_r apparent molecular weight - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We thank Mr. D. Mills and Mr. P. Stanley for help with the EM immunogold techniques. This work was supported by an Agricultural and Food Research Council grant and a studentship from the Science and Engineering Research Council.     

Survival of Neozygites cf. floridana (Zygomycetes: Entomophthorales) in mummified cassava green mites and the viability of its primary conidia

The survival of Neozygites cf. floridana (Weiser and Muma) as dry hyphal bodies in mummified cassava green mites, Mononychellus tanajoa (Bondar), at 5.0% RH in the dark was affected by storage temperature. Survival of the fungus in mummies kept at 24±1.0°C could be demonstrated for 6–7 months. When stored at 4°C, the fungus sporulated from 90% of the mummies liberating an average of 186.9 primary conidia per mummy even after a storage period of 16 months, when the experiment was terminated. The temperature, humidity and light condition significantly affected the viability of primary conidia. The percent viability across all factors dropped from 98.4% after 0 h (beginning of the experiment) to 23.4% after a 1 h exposure to the conditions tested. Lower temperatures maintained higher viabilities with 86.3% of the conidia surviving after 18 h at 18°C, whereas almost all conidia died after 12 h at 33°C. Conidia survived less than 1 h when exposed to SDs (saturation deficit) of 2.0 mm Hg or higher at any tested temperature.     

Isolation of haustoria ofPuccinia graminis f.sp.tritici from wheat leaves

Haustoria of the wheat stem rust fungus,Puccinia graminis f.sp.tritici, race 32, were isolated from two different infected wheat cultivars. Yield of haustoria from the highly susceptible wheat cv. “Little Club” was 10 times higher than that of the resistant wheat line “Prelude × Eagle” carrying the Sr26 gene for resistance toP. graminis f.sp.tritici. Tests for the integrity of haustoria using the stain methylene blue showed that up to 91% of the haustoria were undamaged.     

Resistance to the cereal cyst nematode (Heterodera avenae Woll.) transferred from the wild grassAegilops ventricosa to hexaploid wheat by a “stepping-stone” procedure

Transfer of resistance toHeterodera avenae, the cereal cyst nematode (CCN), by a stepping-stone procedure from the wild grassAegilops ventricosa to hexaploid wheat has been demonstrated. The number of nematodes per plant was lower, and reached a plateau much earlier, in the resistant introgression line H93-8 (1–2 nematodes per plant) than in the recipient H10-15 wheat (14–16 nematodes per plant). Necrosis (hypersensitive reaction) near the nematode, little cell fusion, and few, often degraded syncytia were observed in infested H93-8 roots, while abundant, well-formed syncytia were present in the susceptible H10-15 wheat. Line H93-8 was highly resistant to the two Spanish populations tested, as well as the four French races (Fr1-Fr4), and the British pathotype Hall, but was susceptible to the Swedish pathotypes HgI and HgIII. Resistance was inherited as though determined by a single quasi-dominant factor in the F₂ generations resulting from crosses of H93-8 with H10-15 and with Loros, a resistant wheat carrying the geneCre1 (syn.Ccn1). The resistance gene in H93-8 (Cre2 orCcn2) is not allelic with respect to that in Loros. RFLPs and other markers, together with the cytogenetical evidence, indicate that theCre2 gene has been integrated into a wheat chromosome without affecting its meiotic pairing ability. Introduction ofCre2 by backcrossing into a commercial wheat backgroud increases grain yield when under challenge by the nematode and is not detrimental in the absence of infestation.     

不同抗性短枝木麻黄种源苗木接种青枯病菌后酚类物质含量的变化

为了解短枝木麻黄(Casuarina equisetifolia)抗青枯病的机理,对接种青枯病菌(Ralstonia solanacearum)后短枝木麻黄的单宁和黄酮含量变化进行了分析。结果表明,不同抗性短枝木麻黄种源小枝的总酚和单宁含量呈现不同的变化趋势,高抗、中抗种源均呈现先升高后降低的变化趋势,峰值均约为126 mg g–1,但中抗种源的峰值出现时间较晚,而易感种源则呈逐渐升高趋势。抗、感种源木麻黄接种青枯菌后,小枝中缩合单宁含量均呈现逐渐升高的趋势,但高抗种源的缩合单宁含量均显著高于易感种源,增加70.33%。抗性种源黄酮含量呈S型上升趋势,易感种源则持续缓慢升高。这表明接种青枯病菌后,抗、感短枝木麻黄种源表现出不同的防御特征,次生物质含量增幅越大,抑菌抗氧化能力越强,短枝木麻黄表现出的抗性越强。