Carmen Büttner

Impact of Piriformospora indica on tomato growth and on interaction with fungal and viral pathogens

Piriformospora indica is a root endophytic fungus with plant-promoting properties in numerous plant species and induces resistance against root and shoot pathogens in barley, wheat, and Arabidopsis. A study over several years showed that the endophyte P. indica colonised the roots of the most consumed vegetable crop tomato. P. indica improved the growth of tomato resulting in increased biomass of leaves by up to 20%. Limitation of disease severity caused by Verticillium dahliae by more than 30% was observed on tomato plants colonised by the endophyte. Further experiments were carried out in hydroponic cultures which are commonly used for the indoor production of tomatoes in central Europe. After adaptation of inoculation techniques (inoculum density, plant stage), it was shown that P. indica influences the concentration of Pepino mosaic virus in tomato shoots. The outcome of the interaction seems to be affected by light intensity. Most importantly, the endophyte increases tomato fruit biomass in hydroponic culture concerning fresh weight (up to 100%) and dry matter content (up to 20%). Hence, P. indica represents a suitable growth promoting endophyte for tomato which can be applied in production systems of this important vegetable plant not only in soil, but also in hydroponic cultures.

Male Phyllotreta striolata (F.) produce an aggregation pheromone: Identification of male-specific compounds and interaction with host plant volatiles

The chrysomelid beetle Phyllotreta striolata is an important pest of Brassicaceae in Southeast Asia and North America. Here, we identified the aggregation pheromone of a population of P. striolata from Taiwan, and host plant volatiles that interact with the pheromone. Volatiles emitted by feeding male P. striolata attracted males and females in the field. Headspace volatile analyses revealed that six sesquiterpenes were emitted specifically by feeding males. Only one of these, however, elicited an electrophysiological response from antennae of both sexes. A number of host plant volatiles, e.g., 1-hexanol, (Z)-3-hexen-1-ol, and the glucosinolate hydrolysis products allyl isothiocyanate (AITC), 3-butenyl isothiocyanate, and 4-pentenyl isothiocyanate also elicited clear responses from the antenna. The active male-specific compound was identified as (+)-(6R,7S)-himachala-9,11-diene by chiral stationary phase gas-chromatography with coupled mass spectrometry, and by comparison with reference samples from Abies nordmanniana, which is known to produce the corresponding enantiomer. The pheromone compound was synthesized starting from (–)-α-himachalene isolated from Cedrus atlantica. Under field conditions, the activity of the synthetic pheromone required concomitant presence of the host plant volatile allyl isothiocyanate. However, both synthetic (+)-(6R,7S)-himachala-9,11-diene alone and in combination with AITC were attractive in a two-choice laboratory assay devoid of other natural olfactory stimuli. We hypothesize that P. striolata adults respond to the pheromone only if specific host volatiles are present. In the same laboratory set up, more beetles were attracted by feeding males than by the synthetic stimuli. Thus, further research will be necessary to reveal the components of a more complex blend of host or male-produced semiochemicals that might enhance trap attractiveness in the field.

Analysis of expressed genes of the bacterium 'Candidatus phytoplasma Mali' highlights key features of virulence and metabolism.

‘Candidatus Phytoplasma mali’ is a phytopathogenic bacterium of the family Acholeplasmataceae assigned to the class Mollicutes. This causative agent of the apple proliferation colonizes in Malus domestica the sieve tubes of the plant phloem resulting in a range of symptoms such as witches’- broom formation, reduced vigor and affecting size and quality of the crop. The disease is responsible for strong economical losses in Europe. Although the genome sequence of the pathogen is available, there is only limited information on expression of selected genes and metabolic key features that have not been examined on the transcriptomic or proteomic level so far. This situation is similar to many other phytoplasmas. In the work presented here, RNA-Seq and mass spectrometry shotgun techniques were applied on tissue samples from Nicotiana occidentalis infected by ‘Ca. P. mali’ strain AT providing insights into transcriptome and proteome of the pathogen. Data analysis highlights expression of 208 genes including 14 proteins located in the terminal inverted repeats of the linear chromosome. Beside a high portion of house keeping genes, the recently discussed chaperone GroES/GroEL is expressed. Furthermore, gene expression involved in formation of a type IVB and of the Sec-dependent secretion system was identified as well as the highly expressed putative pathogenicity–related SAP11-like effector protein. Metabolism of phytoplasmas depends on the uptake of spermidine/putescine, amino acids, co-factors, carbohydrates and in particular malate/citrate. The expression of these transporters was confirmed and the analysis of the carbohydrate cycle supports the suggested alternative energy-providing pathway for phytoplasmas releasing acetate and providing ATP. The phylogenetic analyses of malate dehydrogenase and acetate kinase in phytoplasmas show a closer relatedness to the Firmicutes in comparison to Mycoplasma species indicating an early divergence of the Acholeplasmataceae from the Mollicutes.

A model system for plant-virus interaction—infectivity and seed transmission of Cherry leaf roll virus (CLRV) in Arabidopsis thaliana

The wide natural incidence of Cherry leaf roll virus (CLRV) in deciduous forest trees and nurseries in northern Europe is believed to have occurred, apart from occasional mechanical spread and transmission through grafting, mainly by seed transmission. The mode of the vertical transmission and its role in the epidemiology of the virus has not been investigated, basically due to the inconvenient host-pathogen combinations studied to date. With the aim of obtaining an appropriate system for identification of viral genes and products participating in infection processes and seed transmission of CLRV, we performed infection and seed transmissibility tests with CLRV in Arabidopsis thaliana plants. Two phylogenetically and serologically different CLRV isolates were tested. Both of them were found able to infect A. thaliana plants, exhibited clear symptoms of the infection and spread systemically in the plants. Infection of the seeds and of a remarkable number of seedlings generated from infected seeds was possible for two consecutive generations. These results, for first time, report seed transmission of CLRV in the model plant A. thaliana and allow the assumption to be made of embryo invasion during seed transmission. Furthermore, first indications are given that genetically diverse CLRV isolates exhibit different abilities for vertical transmission in A. thaliana. The CLRV-A. thaliana model system is suitable for investigating viral invasion of developing plant organs and meristematic tissue, a prerequisite for successful virus dissemination via vertical transmission through seed.

Zucchini yellow mosaic virus - incidence and sources of virus infection in field-grown cucumbers and pumpkins in the Spreewald, Germany.

In a survey in field-grown cucumber (Cucumis sativus L.) of the Spreewald cultivation area, Germany, from 2001 to 2004, infection with zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), cucumber mosaic virus (CMV) and cucumber green mottle mosaic virus (CGMMV) were recorded. ZYMV was of most relevance, owing to its damaging effects and frequency. Pumpkins (Cucurbita pepo L. and Cucurbita maxima Duchesne) were also found to be infected.In glasshouse experiments with 27 fresh and pickle cucumber cultivars frequently cultivated in the area, all cultivars proved (after mechanical inoculation) to be susceptible to ZYMV. ZYMV-infected cucumbers showed leaf chlorosis and mosaic, as well as deformation of leaves and fruits. Mechanical inoculation with ZYMV, in the early developmental stages of cucumber plants, reduced yields by up to 70%. Mixed infection with ZYMV and CMV resulted in synergistic effects, increasing the severity of damage. Fruit and leaf deformation also occurred in cucumber cultivars exhibiting a documented tolerance to CMV.The widespread weeds fat hen (Chenopodium album L.) and hoary alison (Berteroa incana (L.) DC.) were identified as possible sources of natural ZYMV infection in the Spreewald region. ZYMV transmission through seed seems to be of no importance, and possible only if fresh seeds from diseased cucumbers or pumpkins are used (commercially available seed is not contaminated). Uptake of ZYMV through contaminated soil is not a major mode of natural virus transmission, even though it proved possible in baiting experiments. Epidemiologic and economic implications of ZYMV infections of cucumber and pumpkin plants are discussed.ZusammenfassungIn Untersuchungen zum Auftreten von Viren im Freilandanbau von Gurken im Spreewald wurden in den Jahren 2001 bis 2004 Zucchinigelbmosaikvirus (ZYMV), Wassermelonenmosaikvirus (WMV), Gurkenmosaikvirus (CMV) und das Grünscheckungsmosaikvirus der Gurke (CGMMV) nachgewiesen. Von besonderer Bedeutung erwies sich ZYMV im Untersuchungszeitraum durch sein häufiges Auftreten und die großen Schäden. Das Virus war sowohl in Gurken (Cucumis sativus L.) als auch in Kürbissen (Cucurbita pepo L. und Cucurbita maxima Duchesne) nachzuweisen.Versuche mittels mechanischer Inokulation ergaben für alle 27 geprüften Gurkensorten eine Anfälligkeit gegenüber ZYMV. Die Blätter ZYMV-infizierter Pflanzen zeigten Chlorosen und Mosaik, die Früchte waren deformiert. Pflanzen, die in frühen Entwicklungsstadien durch mechanische Inokulation infiziert wurden, reagierten mit Ertragsdepressionen von bis zu 70 Prozent. Bemerkenswert ist, dass eine Mischinfektion von ZYMV und CMV sichtbare Schäden an Sorten verursacht, welche als tolerant gegenüber CMV beschrieben werden.Auf der Suche nach möglichen Infektionsquellen von ZYMV in der Region Spreewald wurden 23 verschiedene Unkrautarten getestet. Natürliche Infektionen mit dem Virus wurden in Weißem Gänsefuß (Chenopodium album L.) und Graukresse (Berteroa incana (L.) Dc) gefunden. Eine übertragung von ZYMV durch kontaminiertes Saatgut wurde bisher nur dann nachgewiesen, wenn frisches Saatgut aus erkrankten Gurken bzw. Kürbissen verwendet wird. An kommerziell erhältlichem Saatgut wurde dagegen keine Kontamination festgestellt. Die übertragbarkeit von ZYMV durch kontaminierten Boden wurde durch Fangpflanzen belegt, da in virusfreien Testpflanzen, die in ZYMV kontaminiertem Boden kultiviert wurden, später ZYMV nachgewiesen werden konnte. Die epidemiologische und wirtschaftliche Bedeutung einer ZYMV-Infektion von Gurken und Kürbissen wird diskutiert.

Molecular identification of Trichogramma species from Pakistan, using ITS-2 region of rDNA

Molecular techniques were used to distinguish six Trichogramma species (Hymenoptera, Trichogrammatidae) collected from different ecological zones of Pakistan: T. chilonis (Ishii), T. chilotraeae (Nagaraja and Nagarkatti), T. evanescens (Westwood), T. pintoi (Voegele), T. euproctidis (Girault, 1911), T. siddiqi (Nasir and Schöller). Electrophoresis of PCR-amplified ribosomal DNA internal transcribed spacer 2 (ITS-2) followed by restriction endonuclease digestions was applied. The restriction enzyme analysis was carried out in order to obtain species specific banding patterns. To optimize the amplification and sequence reaction, two new primers were designed. The sequence of each species distinctly differed in length and in nucleotide composition. Thus, the results showed that this technique is a good tool to identify cryptic Trichogramma species, which are otherwise difficult to distinguish on the basis of morphological characters. Here we provide a dichotomous molecular key for Trichogramma species of Pakistan for easy and quick species identification.

High genetic diversity at the inter-/intra-host level of Cherry leaf roll virus population associated with the birch leaf-roll disease in Fennoscandia

ABSTRACT A viral epidemic associated with the Cherry leaf roll virus (CLRV) has emerged in Betula species in Fennoscandia, exhibiting quick and effective spread during the last 15 years. A population genetics approach is chosen in order to characterise the virus diversity and the sources of genetic variation aiming to investigate the epidemiology of the pathogen. In a CLRV population from Rovaniemi urban parks and a population that occurred after infecting young Betula seedlings with scions from the original Finnish trees, the genetic diversity is found to be remarkably high, mixed infections by CLRV variants from different phylogenetic groups are detected in single trees, while recombination is evidenced to occur. The estimated genetic variability is high and the CLRV haplotypes detected exhibit clear clustering and belong to different phylogenetic groups. The structure of the viral population reveals a pathogen with high evolutionary potential assumed to carry on its effective spread.

Binding of RDX to Cell Wall Components of Pinus sylvestris and Picea glauca and Three-Year Mineralisation Study of Tissue-Associated RDX Residues

Contamination of soils with the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX, Research Department Explosive) as a result of military applications is a large-area problem globally. Since coniferous trees dominate the vegetation of large areas of military land in Central Europe, particularly in Germany, the long-term fate of 14C-RDX in the conifers Scots pine and Dwarf Alberta spruce was studied. Acetic acid was the most effective solvent for the removal of extractable RDX residues from homogenates of RDX-laden tree material (85%, 80–90% and 64–80% for roots, wood and needles, respectively). On average, only a fifth of RDX-derived 14C was bound in non-extractable residues (NER). Within the main cell wall compartments, lignin was the dominant binding site for NER (needles: 32–62%; roots: 38–42%). Hemicellulose (needles: 11–18%; roots: 6–11%) and cellulose (needles: 12–24%; roots: 1–2%) were less involved in binding and a considerable proportion of NER (needles: 15–24%; roots: 59–51%) was indigestible. After three-year incubation in rot chambers, mineralisation of tree-associated 14C-RDX to 14CO2 clearly dominated the mass balance in both tree species with 48–83%. 13–33% of 14C-RDX-derived radioactivity remained in an unleachable form and the remobilisation by water leaching was negligible (< 2%).

Ability of the larval ectoparasitoid Habrobracon hebetor (Say, 1836) (Hymenoptera: Braconidae) to locate the rice moth Corcyra cephalonica (Stainton, 1865) (Lepidoptera: Pyralidae) in bagged and bulk stored rice

The potential of using Habrobracon hebetor for the inundative biological control of Corcyra cephalonica in rice stored in jute bags and in bulk was assessed. Host-finding in the situation of bag storage was tested by preparing small jute bags measuring 18 · 16 cm filled with 5 kg of un-infested rice grains. In the first experiment, 60 larvae of C. cephalonica were released into the bag. The bag was placed in an empty climatized room with an area of 12.3 m2. Sixty H. hebetor adults (sex ratio of 1 male: 2 females) were released at a distance of 1.6 m away from the jute bag. The number of H. hebetor adults that entered the jute bag was counted daily for 8 days by opening the bag and counting the parasitoids. Only 3% of the parasi- toids entered the jute bag. In a second experiment, 60 C. cephalonica larvae were placed inside a wire gauze cage within the jute bag. Ten H. hebetor adults were released into the jute bag. The bag was sealed. After 1 week the cage was removed, and the number of C. cephalonica as well as F1 generation of H. hebetor that emerged was recorded. H. hebetor was able to significantly reduce the number of C. cephalonica emerging from the bagged rice; the corrected mortality due to the parasitoid release was 92.13%. The emergence of H. hebetor progeny started on day 9 after the introduction of the parasitoids and continued until day 21.Host-finding in the situation of bulk rice was tested in glass cylinders (50 cm height, Ø = 25 cm) containing caged larvae at horizontal depths of 7 and 14 cm. H. hebetor was able to find its host C. cephalonica in both depths; significantly more F1 H. hebetor adults emerged from hosts placed 7 cm deep. The implications of these results with respect to the suitability of H. hebetor for the biological control of C. cephalonica in bagged and bulk rice are discussed.Zusammenfassungp]Das Potential der Mehlmottenschlupfwespe Habrobracon hebetor für die inundative biologische Bekämpfung der Reismotte Corcyra cephalonica in Reis gelagert in Jutesäcken oder geschuttet wurde untersucht. Die Wirtsfindung bei Sacklagerung wurde mit Hilfe kleiner Jutesacke mit den Maßen 18 · 16 cm getestet, die mit 5 kg unbefallenem Reis gefüllt wurden. In einem ersten Experiment wurden sechzig Reismottenlarven in den Sack entlassen. Der Sack wurde in einen leeren Klimaraum mit einer Grundfläche von 12,3 m2 gelagert. Sechzig Imagines von H. hebetor (Geschlechterverhältnis ein Männchen zu zwei Weibchen) wurden in 1.6 m Entfernung des Sacks freigelassen. Die Anzahl H. hebetor in den Säcken wurde über 8 Tage täglich gezählt, indem die Säcke geöffnet wurden. Nur 3% der Parasitoide drangen in den Jutesack ein. In einem zweiten Experiment wurden 60 Larven von C. cephalonica gekäfigt und im Innern des Jutesacks deponiert. Zehn Imagines von H. hebetor wurden in den Sack entlassen, welcher daraufhin verschlossen wurde. Nach 1 Woche wurde der Käfig entnommen und die Anzahl geschlüpfter C. cephalonica und F1 H. hebetor gezählt. H. hebetor reduzierte signifikant die Anzahl von C. cephalonica; die korrigierte Mortalität betrug 92.13%. Der Schlupf der Nachkommen von H. hebetor begann am 9. Tag nach Freilassung und hielt bis zum 21. Tag an. Die Wirtsfindung in geschüttetem Reis wurde in Glaszylindern (Höhe 50 cm, Ø = 25 cm) getestet, in denen in 7 and 14 cm Tiefe gekäfigte Reismottenlarven eingebracht wurden. H. hebetor konnte die Wirte in beiden Tiefen erreichen; signifikant mehr F1 H. hebetor schlüpften von Wirten in 7 cm Tiefe. Die Bedeutung der Ergebnisse für die Eignung von H. hebetor für die biologische Bekämpfung der Reismotte in geschüttetem und gesacktem Reis wird diskutiert.

Control of root zone pH is not effective in preventing Pythium aphanidermatum disease in cucumber

Pythium aphanidermatum is the most wide-spread root pathogen in greenhouse cucumber. Although environmental condi-tions are known to affect the disease severity and many in vitro studies have documented these effects, only few in vivo experiments have been conducted, mainly focusing on the effect of temperature. In the present study, the impact of the pH in the root environment was investigated. Mature cucumber plants were grown in aerated nutrient solution at pH values of 4.0, 5.5 and 7.0, and inoculated with P. aphanidermatum. Experiments were carried out at temperatures of 20, 25 and 30°C. No clear effect of root zone pH on mycelium density in the roots measured by means of an indirect ELISA, and growth reduction of inoculated cucumber plants could be observed, probably due to different responses of the mycelium, oospores and zoospores of P. aphanidermatum to pH.