Studies of the life cycle of <i>Diplocarpon rosae</i> Wolf on roses and the effectiveness of fungicides on pathogenesis

The blackspot disease of roses caused by Diplocarpon rosae Wolf teleomorph (anamorph Marssonina rosae) is a widespread and important disease on outdoor grown roses. The development of D. rosae in rose leaves is not clearly understood and no detailed well-documented photographs of the fungus development in the host are available. The control of this pathogen heavily relies on fungicides. The objective of this study was to provide a detailed growth pattern the D. rosae inside the rose leaves and to test the effectiveness of fungicides, the strobilurins and azoles, on the various fungal structures formed by this fungus. The aggressiveness of various Kenyan and Germany isolates was investigated. A German isolate was used to study the life cycle of D. rosae. The life cycle of the fungus was studied using different staining techniques for the light microscopy and the conventional preparation for electron microscopy: A conidium germinated to form a germ tube, from which an appressorium was sometimes formed. A brown ring that was presumably melanized was formed at the point of penetration of the host cuticle. Upon penetration an infection vesicle was formed in the subcuticular region. Primary hyphae spread from the infection vesicle into the subcuticular and intercellular regions in the host. The subcuticular, intercellular and intramural hyphae formed haustoria of varying shapes in the epidermal cells. The intercellular hyphae also formed haustoria in palisade mesophyll cells. Intracellular hyphae were formed just before the formation of the reproduction structures. Brown heavily ornamented structures formed in the overwintering leaves, opened in spring of the following year to release small thin-walled structures. No ascospores were formed in the subepidermal apothecia-like structures. The strobilurins completely inhibited germination of the conidia of D. rosae but the azoles did not. The azoles were able to destroy the fungal structures in the host even when they were applied after the fungus was fully established in the host but the effectiveness of the strobilurins was limited. Protective and curative tests of the effectiveness of the fungicides were carried out with 100 ppm of the active ingredients. The study showed that D. rosae is a hemibiotroph: The biotrophic phase is marked by the formation haustoria and the necrotrophic phase by the formation of the intracellular hyphae. The weather conditions in Germany may not be suitable for the development of the ascospores in the subepidermal apothecia. The strobilurins were only effective when applied protectively. The azoles were effective when applied pre and post inoculation.Untersuchungen zum Lebenszyklus von Diplocarpon rosae Wolf an Rosen und zum Einfluss von Fungiziden auf die Pathogenese Der Sternrußtau (Schwarzfleckigkeit), Hauptfruchtform Diplocarpon rosae Wolf, (Neben-fruchtform Marssonina rosae), ist eine häufige und weit verbreitete Krankheit an Freilandrosen. Bisher war weder die Entwicklung des Pilzes im Blattgewebe geklärt, noch waren in der Literatur fotografische Aufnahmen zur endophytischen Pilzentwicklung zu finden. Die Bekämpfung des Pilzes wird überwiegend mit Fungiziden durchgeführt. Die Untersuchungen vorliegender Arbeit umfassen sowohl mikroskopische Studien zur exakten Ausbreitung des Erregers im Rosenblatt als auch die Wirkung unterschiedlicher Fungizide, insbesondere Strobilurine und Azole, auf pilzliche Strukturen. Zunächst wurde die Aggressivität verschiedener Isolate aus Kenia und Deutschland getestet. An einem deutschen Isolat konnte der Entwicklungszyklus von D. rosae mit verschiedenen Färbetechniken lichtmikroskopisch sowie nach konventioneller Präparation elektronenmikro-skopisch studiert werden: Nach der Konidienkeimung entsteht eine Keimhyphe, von der aus meist ein Appressorium gebildet wird. Auf der Wirtskutikula zeigt sich dann an der Stelle der späteren Penetration ein brauner Ring, der vermutlich melanisiert ist. Nach der Penetration bildet sich in der subkutikulären Region ein Infektionsvesikel. Primäre Hyphen durchdringen von diesem Infektionsvesikel ausgehend subkutikuläre und interzelluläre Wirtsgewebezonen. Subkutikuläre, interzelluläre und intramurale Hyphen bilden in den epidermalen Zellen Haustorien unterschiedlicher Formen. Interzelluläre Hyphen produzieren ebenfalls Haustorien im Palisadenparenchym. Intrazelluläre Hyphen entstehen erst kurz vor der Bildung der Reproduktionsorgane. Braune, stark ornamentierte Strukturen werden vom Pilz auf den überwinternden Blättern gebildet. Diese öffnen sich im Frühjahr des Folgejahres und entlassen dünnwandige Organe. In diesen subepidermalen Apothezien-ähnlichen Strukturen entstehen aber keine Ascosporen. Strobilurine hemmten die Konidienkeimung vollständig, während Azole einen weniger starken Einfluss auf die Keimung hatten. Dagegen zerstörten die Azole alle Pilzstrukturen im Inneren des Gewebes, auch wenn sie sich bereits gut etabliert hatten. Diese Reaktion konnte bei Einsatz der Strobilurine nicht so deutlich beobachtet werden. Alle Fungizidtests zur protektiven und kurativen Wirkung wurden mit jeweils 100 ppm reinem Wirkstoff durchgeführt. Die Untersuchungen zeigten, dass D. rosae eine hemibiotrophe Lebensweise besitzt: die biotrophe Phase besteht aus der Haustorienbildung und die nekrotrophe Phase aus der Bildung interzellulärer Hyphen. Die deutschen Witterungsbedingungen eignen sich offenbar nicht für eine subepidermale Apothezienbildung. Strobilurine zeigten nur nach protektiver Applikation eine Wirkung. Azole wirkten sowohl bei prä- als auch bei post-inokulativer Behandlung

The molecular initiation and subsequent acquisition of disease resistance in plants

Interactions between disease resistance (R) genes in plants and their corresponding pathogen avirulence (Avr) genes are the key determinants of whether a plant is susceptible or resistance to a pathogen attack. Evidence has emerged that these gene-for-gene interactions in the perception of pathogenic invasions and development of acquired resistance in plants involve different molecular and hormonal transduction pathways, which are still poorly understood. It has become apparent that plants actively produce several phytohormones such as ethylene, jasmonate, salicylic acid, and reactive oxygen intermediates prior to upregulation of R genes. The physiological role of these molecules in plant resistance to pathogens is beginning to attract attention. The use of transgenic plants in recent attempts, including development of mutants with altered R genes, has provided new insights into the mechanisms involved in pathogen perception, signal transduction and subsequent resistance to disease in plants. This review tries to summarize current knowledge of pathogen-related genes in plants, and how they can be use to improve disease resistance in agronomically valuable plants. It also describes the molecular basis of defense mechanisms in plants under pathogen attack. (African Journal of Biotechnology: 2003 2(2): 26-32

A home made kit for plasmid DNA mini-preparation

Many methods have been used to isolate plasmid DNA, but some of them are time consuming especially when extracting a large number of samples. Here, we developed a rapid protocol for plasmid DNA extraction based on the alkaline lysis method of plasmid preparation (extraction at pH 8.0). Using this new method, a good plasmid preparation can be made in approximately one hour. The plasmids are suitable for any subsequent molecular applications in the laboratory. By applying the recommendations to avoid contaminations and to maximize the plasmid yield and quality during extraction, this protocol could be a valuable reference especially when analyzing a large number of samples. (African Journal of Biotechnology: 2003 2(4): 87

The maize ALDH protein superfamily: linking structural features to functional specificities

<p>Abstract</p> <p>Background</p> <p>The completion of maize genome sequencing has resulted in the identification of a large number of uncharacterized genes. Gene annotation and functional characterization of gene products are important to uncover novel protein functionality.</p> <p>Results</p> <p>In this paper, we identify, and annotate members of all the maize aldehyde dehydrogenase (ALDH) gene superfamily according to the revised nomenclature criteria developed by ALDH Gene Nomenclature Committee (AGNC). The maize genome contains 24 unique <it>ALDH </it>sequences encoding members of ten ALDH protein families including the previously identified male fertility restoration <it>RF2A </it>gene, which encodes a member of mitochondrial class 2 ALDHs. Using computational modeling analysis we report here the identification, the physico-chemical properties, and the amino acid residue analysis of a novel tunnel like cavity exclusively found in the maize sterility restorer protein, RF2A/ALDH2B2 by which this protein is suggested to bind variably long chain molecular ligands and/or potentially harmful molecules.</p> <p>Conclusions</p> <p>Our finding indicates that maize ALDH superfamily is the most expanded of plant <it>ALDHs </it>ever characterized, and the mitochondrial maize RF2A/ALDH2B2 is the only plant ALDH that harbors a newly defined pocket/cavity with suggested functional specificity.</p

Modeling-Dependent Protein Characterization of the Rice Aldehyde Dehydrogenase (ALDH) Superfamily Reveals Distinct Functional and Structural Features

The completion of the rice genome sequence has made it possible to identify and characterize new genes and to perform comparative genomics studies across taxa. The aldehyde dehydrogenase (ALDH) gene superfamily encoding for NAD(P)+-dependent enzymes is found in all major plant and animal taxa. However, the characterization of plant ALDHs has lagged behind their animal- and prokaryotic-ALDH homologs. In plants, ALDHs are involved in abiotic stress tolerance, male sterility restoration, embryo development and seed viability and maturation. However, there is still no structural property-dependent functional characterization of ALDH protein superfamily in plants. In this paper, we identify members of the rice ALDH gene superfamily and use the evolutionary nesting events of retrotransposons and protein-modeling–based structural reconstitution to report the genetic and molecular and structural features of each member of the rice ALDH superfamily in abiotic/biotic stress responses and developmental processes. Our results indicate that rice-ALDHs are the most expanded plant ALDHs ever characterized. This work represents the first report of specific structural features mediating functionality of the whole families of ALDHs in an organism ever characterized

The role of plant growth promoting rhizobacteria in plant drought stress responses.

Climate change has exacerbated the effects of abiotic stresses on plant growth and productivity. Drought is one of the most important abiotic stress factors that interfere with plant growth and development. Plant selection and breeding as well as genetic engineering methods used to improve crop drought tolerance are expensive and time consuming. Plants use a myriad of adaptative mechanisms to cope with the adverse effects of drought stress including the association with beneficial microorganisms such as plant growth promoting rhizobacteria (PGPR). Inoculation of plant roots with different PGPR species has been shown to promote drought tolerance through a variety of interconnected physiological, biochemical, molecular, nutritional, metabolic, and cellular processes, which include enhanced plant growth, root elongation, phytohormone production or inhibition, and production of volatile organic compounds. Therefore, plant colonization by PGPR is an eco-friendly agricultural method to improve plant growth and productivity. Notably, the processes regulated and enhanced by PGPR can promote plant growth as well as enhance drought tolerance. This review addresses the current knowledge on how drought stress affects plant growth and development and describes how PGPR can trigger plant drought stress responses at the physiological, morphological, and molecular levels

Evaluating the effects of mefenoxam on taxonomic and functional dynamics of nontarget fungal communities during carrot cultivation.

Ridomil Gold SL (45.3% a.i. mefenoxam) is a widely used chemical fungicide for the control of oomycetes. However, its impact on fungal communities remains unexplored. Therefore, the goal of this study was to examine the effects of mefenoxam on the temporal dynamics of fungal taxonomic and functional diversities during carrot cultivation under four treatment groups: mefenoxam application with and without Pythium inoculation, and untreated control groups with and without Pythium inoculation. Our in vitro sensitivity assay showed that the maximum recommended concentration of mefenoxam, 0.24&nbsp;ppm, did not suppress the mycelial growth of P. irregulare. At 100&nbsp;ppm, mycelial growth was only reduced by 11.4%, indicating that the isolate was resistant to mefenoxam. MiSeq sequencing data revealed transient taxonomic variations among treatments 2 weeks post-treatment. Mortierella dominated the fungal community in the mefenoxam-Pythium combination treatment, as confirmed through PCR using our newly designed Mortierella-specific primers. Conversely, mefenoxam-Pythium combination had adverse effects on Penicillium, Trichoderma, and Fusarium, and decrease the overall alpha diversity. However, these compositional changes gradually reverted to those observed in the control by the 12th week. The predicted ecological functions of fungal communities in all Pythium and mefenoxam treatments shifted, leading to a decrease in symbiotrophs and plant pathogen functional groups. Moreover, the community-level physiological profiling approach, utilizing 96-well Biolog FF microplates, showed discernible variations in the utilization of 95 diverse carbon sources among the treatments. Notably, arbutin, L-arabinose, Tween 80, and succinamic acid demonstrated a strong positive association with Mortierella. Our findings demonstrate that a single application of mefenoxam at its recommended rate triggers substantial taxonomic and functional shifts in the soil fungal community. Considering this impact, the conventional agricultural practice of repeated mefenoxam application is likely to exert considerable shifts on the soil ecosystem that may affect agricultural sustainability