Teliospore Germination of Soybean Rust Fungus (Phakopsora pachyrhizi Syd.)

Teliospores of the soybean rust fungus (Phakopsora pachyrhizi Syd.) germinated after treatment with 10—12 wetting and drying cycles at room temperature. Spores swelled, vacuolated and germinated with a slightly curved basidium bearing 1—4 sterigmata with basidiospores. High germination rates were observed when telia were stored at 5 °C for 5—6 months before breaking dormancy.     

Secretion of Extracellular Enzymes by Verticillium psalliotae Treschow and Verticillium lecanii (Zimm.) Viégas During Growth on Uredospores of the Soybean Rust Fungus (Phakopsora pachyrhizi Syd.) in Liquid Cultures

Two isolates of the mycoparasite Verticillium psalliotae grew rapidly in liquid cultures on autoclaved uredospores of the soybean rust fungus (Phakopsora pachyrhizi Syd.) as sole carbon source and secreted β-l,3-glucanase, chitinase, and protease activities into the medium. One isolate of Verticillium lecanii grew slowly, failed to produce measurable chitinase activity and secreted lower specific activities of β-l,3-glucanase and protease, compared with V. psalliotae. The tested isolates of V. psalliotae and V. lecanii produced comparable levels of lipolytic activity. Amylolytic activity was secreted by V. lecanii but not by V. psalliotae. The isolates of V. psalliotae and V. lecanii used in our experiments differed clearly in protein and protease pattern, determined by electrophoresis on polyacrylamide gels. The results indicate that the rapid growth of V. psalliotae on autoclaved uredospores in liquid culture and on uredosori is probably based primarily on nutrients made available to the mycoparasite by activities of β-1,3-glucanases, chitinases and proteases.     

Development of Infection Structures by the Direct-Penetrating Soybean Rust Fungus (Phakopsora pachyrhizi Syd.) on Artificial Membranes

The development of infection structures by the directly infecting soybean rust fungus of different artificial membranes was followed by light and scanning electron microscopy. On water agar uredospores developed germ tubes without appressoria. On dialysis membranes more than 80% of the uredospores formed appressoria. With low frequencies (1–7%) also primary hyphae and/or penetration hyphae were present. When cellulose nitrate membrane filters with pore diameters ≤ 0.2 μm were used, uredospores germinated but showed a strongly reduced appressoria formation. Membranes with pores ≥ 0.1 μm allowed a development of infection structures similar to that on dialysis membranes. In experiments with paraffin oil incorporated into collodion membranes more than 90% of the uredospores formed appressoria, about 50% of the appressoria developed hyphae. Ungerminated spores and germ tubes always contained 2 nuclei. In fully developed appressoria 4 nuclei were present. Compared with stomata entering rust fungi appressoria formation by Phakopsora pachyrhizi occurred more frequently and seemed to be less dependent on specific stimuli. Moreover, in most cases only few of the appressoria formed penetration or primary hyphae. The induction of these structures seemed to be dependent on further unknown stimuli.     

Defense Reactions in Host and Nonhost Plants Against the Soybean Rust Fungus (Phakopsora pachyrhizi Syd.)

Growth and development of two races of the soybean rust fungus (Phakopsora pachyrhizi Syd.) were compared on host and nonhost plants. Both groups had several lines of defense, each of which could stop a part of attacking uredospores. Germ tubes and appressoria were produced equally well on hosts and nonhosts. A reduced formation of penetration hyphae contributed to the resistance of nonhosts and resistant host genotypes. In the epidermal cells of wheat and barley leaves, lower frequencies of penetration hyphae coincided with the production of papillae-like structures which were not penetrated. The last line of defense of all nonhosts was localized in the epidermal cell where the growth of the penetration hyphae was arrested definitively. The colony development in these species was suppressed completely. In highly resistant host genotypes the last defense reaction occurred later as a hypersensitive cell collapse which interrupted the growth of the rust colony.     

Lectin Binding Studies on the Cell Walls of Soybean Rust (Phakopsora pachyrhizi Syd.)

Walls of uredospores, infection structures, intercellular hyphae and haustoria of the soybean rust fungus (Phakopsora pachyrhizi) were studied by electron microscopy using gold-labeled wheat germ lectin (WGL) and Concanavalin A (ConA) as cytochemical probes. Receptors for WGL (probably chitin) were detected in all fungal walls included in this study. WGL-binding occurred throughout the entire walls (uredospores, appressorial cone, penetration hyphae, haustorial mother cells) or only to the inner wall layers (germ tubes, appressoria, intercellular hyphae).     

Colonization of Soybean Cyst Nematode Females,Cysts, and Gelatinous Matrices by the Fungus Verticillium lecanii

Heterodera glycines was grown in monoxenic culture on soybean roots and then inoculated with the antagonistic fungus Verticillium lecanii. Use of root explant cultures allowed evaluation of the fungus-nematode interaction with the nematode attached to roots or removed from the host, and avoided contamination with other fungi. From 16 hours to 14 days following inoculation, female and cyst samples were examined with the light microscope, or prepared for either conventional or low-temperature scanning electron microscopy. Within 16 hours, hyphae had begun colonizing the gelatinous matrices (GM). The fungus proliferated in the GM of some specimens within a week, but was rarely seen in unhatched eggs. Fungus penetration holes in female and cyst walls were observed 3 days after inoculation; penetration through nematode orifices was not seen at that time. More cysts than females were colonized at the earliest sampling dates. Specimens associated with external hyphae exhibited variable internal colonization, ranging from no fungal penetration to extensive mycelial growth.     

Kinetic Studies on the Control of the Bean Rust Fungus (Uromyces phaseoli L.) by an Inhibitor of Polyamine Biosynthesis

α-Difluoromethylornithine (DFMO), a specific and irreversible inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase, effectively inhibits mycelial growth of several phytopathogenic fungi on defined media in vitro and provides systemic protection of bean plants against infection by Uromyces phaseoli L. race 0 (MV Rajam, AW Galston 1985 Plant Cell Physiol 26: 683-692; MV Rajam et al. 1985 Proc Natl Acad Sci USA 82: 6874-6878). We now find that application of 0.5 millimolar DFMO to unifoliolate leaves of Pinto beans up to 3 days after inoculation with uredospores of U. phaseoli completely inhibits the growth of the pathogen, while application 4 or 5 days after inoculation results in partial protection against the pathogen. Spores do not germinate on the surface of unifoliolate leaves treated with DFMO 1 day before infection, but addition of spermidine to the DFMO treatments partially reverses the inhibitory effect. The titer of polyamines in bean plants did not decline after DFMO treatment; rather, putrescine and spermidine contents actually rose, probably due to the known but paradoxical stimulation of arginine decarboxylase activity by DFMO.     

Degradation of Chlorbromuron and Related Compounds by the Fungus Rhizoctonia solani

The ability of the soil fungus Rhizoctonia solani to degrade phenyl-substituted urea herbicides was investigated. The fungus was able to transform chlorbromuron [3-(3-chloro-4-bromophenyl)-1-methyl-1-methoxyurea] to the demethylated product [3-(3-chloro-4-bromophenyl)-1-methoxyurea], which was isolated and identified. Evidence was obtained that further degradation of chlorbromuron occurred. Several other phenylurea compounds (chloroxuron, diuron, fenuron, fluometuron, linuron, metobromuron, neburon, and siduron) were also metabolized by the fungus, indicating that R. solani may possess a generalized ability to attack this group of herbicides.     

Glucosinolate Biosynthesis: Sulfation of Desulfobenzylglucosinolate by Cell-Free Extracts of Cress (Lepidium sativum L.) Seedlings

After removal of myrosinase activity by concanavalin A-Sepharose 4B chromatography, cell-free extracts of light-grown cress (Lepidium sativum L.) seedlings, catalyzed the sulfation of desulfobenzylglucosinolate (K_m, 0.23 millimolar) to benzylglucosinolate using PAPS (K_m, 1 millimolar) as sulfur donor. Sulfotransferase activity, which was optimal at pH 9.0, was stimulated by MgCl₂, MnCl₂, β-mercaptoethanol, and dithiothreitol and was inhibited by ZnSO₄ and SH-reagents. The enzyme also sulfated desulfoallyglucosinolate to allylglucosinolate (sinigrin) but was inactive towards all phenylpropanoids and flavonoids tested.     

Detoxification of Formaldehyde by the Spider Plant (Chlorophytum comosum L.) and by Soybean (Glycine max L.) Cell-Suspension Cultures

The phytotoxicity of formaldehyde for spider plants (Chlorophytum comosum L.), tobacco plants (Nicotiana tabacum L. cv Bel B and Bel W3), and soybean (Glycine max L.) cell-suspension cultures was found to be low enough to allow metabolic studies. Spider plant shoots were exposed to 7.1 [mu]L L-1 (8.5 mg m-3) gaseous [14C]-formaldehyde over 24 h. Approximately 88% of the recovered radioactivity was plant associated and was found to be incorporated into organic acids, amino acids, free sugars, and lipids as well as cell-wall components. Similar results were obtained upon feeding [14C]formaldehyde from aqueous solution to aseptic soybean cell-suspension cultures. Serine and phosphatidylcholine were identified as major metabolic products. Spider plant enzyme extracts contained two NAS+-dependent formaldehyde dehydrogenase activities with molecular mass values of about 129 and 79 kD. Only the latter enzyme activity required glutathione as an obligatory second cofactor. It had an apparent Km value of 30 [mu]M for formaldehyde and an isoelectric point at pH 5.4. Total cell-free dehydrogenase activity corresponded to 13 [mu]g formaldehyde oxidized h-1 g-1 leaf fresh weight. Glutathione-dependent formaldehyde dehydrogenases were also isolated from shoots and leaves of Equisetum telmateia and from cell-suspension cultures of wheat (Triticum aestivum L.) and maize (Zea mays L.). The results obtained are consistent with the concept of indoor air decontamination with common room plants such as the spider plant. Formaldehyde appears to be efficiently detoxified by oxidation and subsequent C1 metabolism.     

The epidemiology of leaf smut disease of dahlia caused by Entyloma calendulae f. sp. dahliae (Syd.) Viegas

Entyloma calendulae f. sp.dahliae overwinters in the soil as teliospores or in dead leaf tissue. It was not seed — transmitted and did not survive in tubers although it occurred in soil attached to tubers. Teliospore germination decreased from the end of November to a minimum between January and March. Spores exposed to wintry conditions remained dormant longer than those in the laboratory. In spring and early summer some overwintered spores germinated in the soil to give rise to conidia resembling those normally produced on leaves. Fresh outbreaks of the disease occurred in July from spores which had overwintered in the soil. The host tissues nearest to the ground are infected first and the disease remains more severe on the lower part of the plant than on the upper leaves. New tissues were infected throughout the summer, giving a maximum in early autumn. Many more conidia were present in the air on wet windy days than on dry calm ones.

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Zusammenfassung Entyloma calendulae f. sp.dahliae überwintert in Erdboden als Teliosporen oder im Gewebe toter Blätter. Es war nicht durch Samen übertragen und überlebt nicht im Knollen, obwohl es in der an den Knollen haftenden Erde vorkommt. Teliosporenkeimung nahm vom Ende November bis zu einem Minimum zwischen Januar und März ab. Sporen, die winterlichen Bedingungen ausgesetzt waren, blieben längerer Zeit dormant, denn diejenigen im Laboratorium. Im Frühjahr und Frühsommer keimten manche überwinterte Sporen in der Erde, um Konidien zu entwickeln, die denen normalerweise an den Blättern vorkommenden ähnlich waren. Ein neuer Ausbruch der Krankheit kam im Juli vor, die durch die im Boden über-winterten Sporen verursacht wurden. Das Wirtsgewebe, nähst dem Boden, ist erst infiziert und die Krankheit verbleibt schwerer im unteren Teil der Pflanze, denn in den obersten Blättern. Das neue Gewebe wurde durch den Sommer infiziert und die Höhe wurde im Frühherbst erreicht. Vielmehr Sporen waren in der Luft an nassen, windigen Tagen vorhanden, denn in ruhigem, trockenem Wetter.

     

Initial Steps in the Degradation of Methoxychlor by the White Rot Fungus Phanerochaete chrysosporium

The white rot fungus Phanerochaete chrysosporium mineralized [ring-(sup14)C]methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] and metabolized it to a variety of products. The three most prominent of these were identified as the 1-dechloro derivative 1,1-dichloro-2,2-bis(4-methoxyphenyl)ethane, the 2-hydroxy derivative 2,2,2-trichloro-1,1-bis(4-methoxyphenyl)ethanol, and the 1-dechloro-2-hydroxy derivative 2,2-dichloro-1,1-bis(4-methoxyphenyl)ethanol by comparison of the derivatives with authentic standards in chromatographic and mass spectrometric experiments. In addition, the 1-dechloro-2-hydroxy derivative was identified from its (sup1)H nuclear magnetic resonance spectrum. The 1-dechloro and 2-hydroxy derivatives were both converted to the 1-dechloro-2-hydroxy derivative by the fungus; i.e., there was no requirement that dechlorination precede hydroxylation or vice versa. All three metabolites were mineralized and are therefore likely intermediates in the degradation of methoxychlor by P. chrysosporium.     

Identification of a Degradation Intermediate of the Momilactone A Rice Phytoalexin by the Rice Blast Fungus

We have already shown that major rice diterpene phytoalexin, momilactone A, was detoxified by Magnaporthe oryzae. We report here the identification by NMR, MS, and chemical synthesis of 3,6-dioxo-19-nor-9β-pimara-7,15-diene (1) as the degradation intermediate. Compound 1 exhibited similar antifungal activity to that of momilactone A, indicating 1 to be a precursor of possible detoxified compounds.     

Control of White Grubs (Melolontha melolontha L.) by Treating Adults with the Fungus Beauveria brongniartii

Having developed a method to contaminate the breeding sites of the cockchafer with the aid of infected females, two large field trials were carried out, one in 1985 and the other in 1988. Blastospores were applied by helicopter at 26 sites of woodland borders, where the swarming beetles aggregate. The mean dose amounted to 2.0-3.7 × 10¹⁴ spores ha^-1. The infection rates of the treated adults ranged from 30 to 99%. The treatment reduced the average reproduction rate from 5.09 to 2.15 second instar larvae/adult. The development of the treated populations was followed at 15 sites in perennial grassland by examining samples taken in the winter season. Increased infection rates were observed in the generation following the treatment. The population density 6 and 9 years after the treatment, corresponding to two and three generations of the beetle respectively, decreased at 13 sites by more than 50% and at four sites by more than 80%. The population densities remained high at only two sites, although the fungus was present. During this period, the area of heavy feeding damage by the adults at woodland borders decreased from 17.8 to 3.9 km. At the same time, damage increased outside the treated area from 27 to 31 km. The advantages and disadvantages of this method of breeding site contamination are discussed.