Isolation and Characterization of fungal causal agents of black bark disease of apple using the “apple test”

In den letzten, häufig von Trockenheit geprägten Jahren hat sich der Schwarze Rindenbrand an Apfel- und Birnenbäumen in verschiedenen Teilen Deutschlands zunehmend zu einem Problem entwickelt. Ziel der vorliegenden Arbeit war es daher, methodische Grundlagen zur Isolierung der Erreger aus Apfelbäumen und ihrer Charakterisierung im Labor zu erarbeiten. Die Experimente beinhalteten die klassische Isolierung durch Auslegen von erkranktem Gewebe auf Agarmedien sowie ein in der Literatur als „Apfeltest“ beschriebenes Verfahren, bei dem befallene Rinde in Apfelfrüchte gesteckt wird. In der Folge reichern sich Pathogene im Gewebe an und können aus den entstehenden Faulstellen isoliert werden.Aus dem Stammholz eines im Wipfelbereich welkenden Baumes wurden nach Auslegen auf Agarmedium Trametes versicolor, Diaporthe eres sowie Diplodia seriata (bekannt als einer von mehreren Erregern des Schwarzen Rindenbrandes) isoliert. Diese Art wurde auch nach Auslegen befallener Rinde auf Kartoffel-Dextrose-Agar erhalten, ebenso wie Diplodia malorum. Durch Einstecken befallener Rindenstücke in Apfelfrüchte wurden weitere Isolate von D. malorum, drei Isolate von Diplodia bulgarica sowie Lambertella corni-maris, Penicillium sp. und Sclerotinia sclerotiorum gewonnen. Im Biotest mit künstlicher Inokulation mit Reinkulturen riefen die drei letztgenannten Isolate, alle Diplodia-Isolate sowie Diaporthe eres Fruchtfäulen hervor. Die Diplodia-Arten wurden darüber hinaus hinsichtlich der Geschwindigkeit des Hyphenwachstums und der Sporenmorphologie charakterisiert.During the previous years characterized by summer-drought, black canker disease of apple and pear has become a problem in different parts of Germany. The aim of the current work was therefore to develop basic techniques for isolation of the inciting pathogens from apple trees and their characterization in the laboratory. The experiments included the standard isolation of putative pathogens by placing symptomatic tissue on agar media as well as a method described in the literature as „apple test”. For the latter, symptomatic bark pieces are inserted into apple fruits and colonization of the apple tissue is visible as rotting spots. Candidate pathogens can be easily isolated from the colonized apple tissue.After placement of tissue from the trunk of the top of an apple tree expressing wilt symptoms, Trametes versicolor, Diaporthe eres and Diplodia seriata (one of several agents known to cause black canker disease) were isolated. Together with Diplodia malorum, the latter species was also obtained after placement of symptomatic bark pieces on potato dextrose agar. Insertion of diseased bark into apple fruits led to the isolation of further isolates of D. malorum, three isolates of Diplodia bulgarica, Lambertella corni-maris, Penicillium sp. and Sclerotinia sclerotiorum. After inoculation with pure cultures into apple fruits, the latter three, all Diplodia-isolates and Diaporthe eres were pathogenic and caused fruit rots. The Diplodia isolates were further characterised regarding speed of hyphal growth and spore morphology

Regulation of seed germination in the close Arabidopsis relative Lepidium sativum : a global tissue-specific transcript analysis

The completion of germination in Lepidium sativum and other endospermic seeds (e.g. Arabidopsis [Arabidopsis thaliana]) is regulated by two opposing forces, the growth potential of the radicle (RAD) and the resistance to this growth from the micropylar endosperm cap (CAP) surrounding it. We show by puncture force measurement that the CAP progressively weakens during germination, and we have conducted a time-course transcript analysis of RAD and CAP tissues throughout this process. We have also used specific inhibitors to investigate the importance of transcription, translation, and posttranslation levels of regulation of endosperm weakening in isolated CAPs. Although the impact of inhibiting translation is greater, both transcription and translation are required for the completion of endosperm weakening in the whole seed population. The majority of genes expressed during this process occur in both tissues, but where they are uniquely expressed, or significantly differentially expressed between tissues, this relates to the functions of the RAD as growing tissue and the CAP as a regulator of germination through weakening. More detailed analysis showed that putative orthologs of cell wall-remodeling genes are expressed in a complex manner during CAP weakening, suggesting distinct roles in the RAD and CAP. Expression patterns are also consistent with the CAP being a receptor for environmental signals influencing germination. Inhibitors of the aspartic, serine, and cysteine proteases reduced the number of isolated CAPs in which weakening developed, and inhibition of the 26S proteasome resulted in its complete cessation. This indicates that targeted protein degradation is a major control point for endosperm weakening

<i>DELAY OF GERMINATION 1</i> mediates a conserved coat-dormancy mechanism for the temperature- and gibberellin-dependent control of seed germination

Seed germination is an important life-cycle transition because it determines subsequent plant survival and reproductive success. To detect optimal spatiotemporal conditions for germination, seeds act as sophisticated environmental sensors integrating information such as ambient temperature. Here we show that the DELAY OF GERMINATION 1 (DOG1) gene, known for providing dormancy adaptation to distinct environments, determines the optimal temperature for seed germination. By reciprocal gene-swapping experiments between Brassicaceae species we show that the DOG1-mediated dormancy mechanism is conserved. Biomechanical analyses showthat thismechanism regulates the material properties of the endosperm, a seed tissue layer acting as germination barrier to control coat dormancy. We found that DOG1 inhibits the expression of gibberellin (GA)-regulated genes encoding cell-wall remodeling proteins in a temperature-dependent manner. Furthermore we demonstrate that DOG1 causes temperature-dependent alterations in the seed GA metabolism. These alterations in hormone metabolism are brought about by the temperature-dependent differential expression of genes encoding key enzymes of the GA biosynthetic pathway. These effects of DOG1 lead to a temperature-dependent control of endosperm weakening and determine the optimal temperature for germination. The conserved DOG1-mediated coat-dormancymechanismprovides a highly adaptable temperature-sensing mechanism to control the timing of germination.</p

The evolution of seeds

The evolution of the seed represents a remarkable life-history transition for photosynthetic organisms. Here, we review the recent literature and historical understanding of how and why seeds evolved. Answering the \u27how\u27 question involves a detailed understanding of the developmental morphology and anatomy of seeds, as well as the genetic programs that determine seed size. We complement this with a special emphasis on the evolution of dormancy, the characteristic of seeds that allows for long \u27distance\u27 time travel. Answering the \u27why\u27 question involves proposed hypotheses of how natural selection has operated to favor the seed life-history phenomenon. The recent flurry of research describing the comparative biology of seeds is discussed. The review will be divided into sections dealing with: (1) the development and anatomy of seeds; (2) the endosperm; (3) dormancy; (4) early seed-like structures and the transition to seeds; and (5) the evolution of seed size (mass). In many cases, a special distinction is made between angiosperm and gymnosperm seeds. Finally, we make some recommendations for future research in seed biology

Peroxidases identified in a subtractive cDNA library approach show tissue-specific transcript abundance and enzyme activity during seed germination of Lepidium sativum

The micropylar endosperm is a major regulator of seed germination in endospermic species, to which the close Brassicaceae relatives Arabidopsis thaliana and Lepidium sativum (cress) belong. Cress seeds are about 20 times larger than the seeds of Arabidopsis. This advantage was used to construct a tissue-specific subtractive cDNA library of transcripts that are up-regulated late in the germination process specifically in the micropylar endosperm of cress seeds. The library showed that a number of transcripts known to be up-regulated late during germination are up-regulated in the micropylar endosperm cap. Detailed germination kinetics of SALK lines carrying insertions in genes present in our library showed that the identified transcripts do indeed play roles during germination. Three peroxidases were present in the library. These peroxidases were identified as orthologues of Arabidopsis AtAPX01, AtPrx16, and AtPrxIIE. The corresponding SALK lines displayed significant germination phenotypes. Their transcripts were quantified in specific cress seed tissues during germination in the presence and absence of ABA and they were found to be regulated in a tissue-specific manner. Peroxidase activity, and particularly its regulation by ABA, also differed between radicles and micropylar endosperm caps. Possible implications of this tissue-specificity are discussed

Location of Pathogen Inoculum in the Potting Substrate Influences Damage by Globisporangium ultimum, Fusarium culmorum and Rhizoctonia solani and Effectiveness of Control Agents in Maize Seedlings

The aim of this study was to determine the impact of the location of the pathogen inoculum on damage caused by Globisporangium (syn. Pythium) ultimum, Fusarium culmorum and Rhizoctonia solani in pot tests with maize. For this purpose, pathogen inoculum was added to potting substrate, and the resulting mix was used to fill the whole pot volume, the upper half, or the lower half of pots. The remaining volume was filled with non-inoculated substrate. In a second experimental approach, maize seeds were germinated in non-inoculated potting substrate and the seedlings were transferred to inoculated substrate. The seeds were untreated, treated with the chemical thiram, or treated with a bacterial or a fungal biocontrol agent. With each of the pathogens, the damage to the developing maize seedlings was the strongest when the seeds germinated in the inoculated potting substrate. When only the roots were in contact with the inoculum, there was limited damage by R. solani and F. culmorum, and no damage by G. ultimum. This implies that in experiments with artificial inoculation, the seeds should always be in immediate contact with the inoculum if a strong pathogenic effect is desired. Conversely, seed treatments must, in the first place, be able to protect the spermosphere, while the requirement to protect the roots at a distance from the seed seems to depend on the pathogen

In vivo cell wall loosening by hydroxyl radicals during cress (Lepidium sativum L.) seed germination and elongation growth

Loosening of cell walls is an important developmental process in key stages of plant life cycles, including seed germination, elongation growth and fruit ripening. Here we report direct in vivo evidence for hydroxyl radical (•OH)-mediated cell wall loosening during plant seed germination and seedling growth. We used electron paramagnetic resonance (EPR)-spectroscopy to show that •OH is generated in the cell wall during radicle elongation and weakening of the endosperm of cress (Lepidium sativum L., Brassicaceae) seeds. Endosperm weakening precedes for radicle emergence, as demonstrated by direct biomechanical measurements. By 3H-fingerprinting we showed that wall polysaccharides are oxidised in vivo by the developmentally regulated action of apoplastic •OH in radicles and endosperm caps: the production and action of •OH increased during endosperm weakening and radicle elongation and were inhibited by the germination-inhibiting hormone abscisic acid. Both effects were reversed by gibberellin. Distinct and tissue-specific target sites of •OH attack on polysaccharides were evident. In vivo •OH attack on cell wall polysaccharides were not only evident in germinating seeds, but also in elongating maize (Zea mays L., Poaceae) seedling coleoptiles. We conclude that plant cell wall loosening by •OH is a controlled action of this type of reactive oxygen species (ROS)