Katarzyna Sikora

Evaluating the persistence of Phlebiopsis gigantea (Fr.: Fr.) Jülich mycelium and fruiting bodies in pine stumps after root-rot protection treatments

Ocena występowania grzybni i owocników Phlebiopsis gigantea (Fr.: Fr.) Jülich w pniakach sosnowych po wykonaniu zabiegu ochronnego przed hubą korzeni Evaluating the persistence of Phlebiopsis gigantea (Fr.: Fr.) Jülich mycelium and fruiting bodies in pine stumps after root-rot protection treatments Commercial preparations Rotstop and PgSuspension, used in Scandinavia and the United Kingdom, are EU-licensed biocontrol agents against root rot that contain isolates of the fungus Phlebiopsis gigantea. The composition of these two products differs from that of PgIBL, previously use in Poland, hence the need to verify their effectiveness under Polish site and stand conditions. Stumps were treated with the commercial products and subsequently checked after one year for the persistence of mycelia and fruiting bodies. Molecular methods were used on the mycelia present in the stumps to confirm that their genetic identity was consistent with fungal isolates from the preparations. Treatments with EU-licensed preparations gave satisfactory results - in about 70-80% of the stumps mycelia were present under the bark. Inoculation with Rotstop preparations was most effective during autumn 2008 (effectiveness of 82%), while PgSuspension application was most effective during spring 2008 (effectiveness of 77%). The development of the mycelia in pine stumps from the two preparations investigated (containing preserved dormant spores) was similar to that of preparation PgIBL (formulated from living mycelium within a growing medium), in terms of their relative effectiveness measured as the percentage of stumps supporting under-bark mycelia (which ranged between 70-90%, depending on the treatment term).

The plasticity of fungal interactions

Their hyphal structure, the common events of hybridization and horizontal gene transfer, as well as intimate associations with prokaryotes (including endobiotic bacteria) and cooperation with eukaryotes have made fungi very flexible at the genetic, physiological, and ecological levels. It is manifested with the fungal ability to perfectly exploit existing nutrient sources and plastically fit into a changing environment. Although the links between fungi and other ecosystem components are rarely clearly visible and unambiguous, fungi can be ecosystem buffers playing a homeostatic role throughout global ecosystems, reacting to changes in various ways, not only by modifications of gene expression but also by nuclear status and “extended phenotype”. The goal of this review is to underline some ecological interactions involving fungi and other organisms and to indicate high fungal plasticity in terms of ontogenetic perspective.

Using laser micro-dissection and qRT-PCR to analyze cell type-specific gene expression in Norway spruce phloem

The tangentially oriented polyphenolic parenchyma (PP) and radially organized ray parenchyma in the phloem are central in the defense of conifer stems against insects and pathogens. Laser micro-dissection enables examination of cell-specific defense responses. To examine induced defense responses in Norway spruce stems inoculated with the necrotrophic blue-stain fungus Ceratocystis polonica, RNA extracted from laser micro-dissected phloem parenchyma and vascular cambium was analyzed using real-time RT-PCR (qRT-PCR) to profile transcript levels of selected resistance marker genes. The monitored transcripts included three pathogenesis-related proteins (class IV chitinase (CHI4), defensin (SPI1), peroxidase (PX3), two terpene synthesis related proteins (DXPS and LAS), one ethylene biosynthesis related protein (ACS), and a phenylalanine ammonia-lyase (PAL). Three days following inoculation, four genes (CHI4, PAL, PX3, SPI1) were differentially induced in individual cell and tissue types, both close to the inoculation site (5 mm above) and, to a lesser degree, further away (10 mm above). These resistance marker genes were all highly induced in ray parenchyma, supporting the important role of the rays in spruce defense propagation. CHI4 and PAL were also induced in PP cells and in conducting secondary phloem tissues. Our data suggests that different cell types in the secondary phloem of Norway spruce have overlapping but not fully redundant roles in active host defense. Furthermore, the study demonstrates the usefulness of laser micro-dissection coupled with qRT-PCR to characterize gene expression in different cell types of conifer bark.

Phytophthora quercina infections in elevated CO2 concentrations

Abstract In the last decades, a new wave of oak decline has been observed in Poland. The most important pathogenic organisms involved in this phenomenon are probably soil-borne pathogens Phytophthoragenus, especially P. quercina. In this work, we sought to test the influence of elevated CO2 concentration on the susceptibility of oaks (Quercus robur L.) to infection by P. quercina. In order to test the susceptibility of oak fine roots to infection, we applied phosphite-based fertiliser Actifos in 0.6% concentration. One-year-old oak seedlings were grown for one year in greenhouse with either an ambient atmosphere (400 ppm CO2) or an elevated (800 ppm) concentration of CO2. Oaks grown at the elevated CO2 concentration developed longer shoots as proved by statistically significant differences. However, there was no difference in the development of root systems. The application of Actifos had a positive significant effect on the development of shoots and the surface area of fine roots under the elevated CO2 concentration.

Effect of CO2 enhancement on beech (Fagus sylvatica L.) seedling root rot due to Phytophthora plurivora and Phytophthora cactorum

Abstract Global climate change is associated with higher concentrations of atmospheric carbon dioxide (CO2). The ongoing changes are likely to have significant, direct or indirect effects on plant diseases caused by many biotic agents such as phytopathogenic fungi. This study results showed that increased CO2 concentration did not stimulate the growth of 1-year-old beech Fagus sylvatica L seedlings but it activated pathogenic Phytophthora species (P. plurivora and P. cactorum) which caused significant reduction in the total number of fine roots as well as their length and area. The results of the greenhouse experiment indicated that pathogens once introduced into soil survived in pot soil, became periodically active (in sufficient water conditions) and were able to damage beech fine roots. However, the trees mortality was not observed during the first year of experiment. DNA analyses performed on soil and beech tissue proved persistence of introduced Phytophthora isolates.

Molecular analysis of Phytophthora species found in Poland

Abstract Pathogens of Phytophthora genus are common not only in forest nurseries and stands, but also in water courses. Species of Phytophthora spread with plants for plantings (and soil attached to them) and with water courses as well, attacking the plants growing in riparian ecosystems. Several specialized organisms damaging only one tree species were identified like P. alni on alders or P. quercina on oaks. Some Phytophthora species can develop on several hosts like P. plurivora and P. cactorum on oaks, beeches, alders, ashes and horse chestnuts. Other oomycetes like P. gallica species was found for the first time in Poland in water used for plant watering in forest nursery. Species P. lacustris and P. gonapodyides were found in superficial water. Phytophthora species P. polonica was identified in the declining alder stands for the first time in the world, and P. taxon hungarica and P. megasperma were found in the rhizosphere of seriously damaged ash stands for the first time in Poland. The most often isolated species were P. plurivora (clade 2) with frequency 37% and P. lacustris with frequency 33% (clade 6). The best represented clade 6 revealed the occurrence of 6 species: P. gonapodyides, P. lacustris, P. megasperma, P. sp. raspberry, P. taxon hungarica and P. taxon oak soil.

Four different Phytophthora species that are ableto infect Scots pine seedlings in laboratory conditions

Abstract To investigate susceptibility of young Scots pine seedlings to four Phytophthora species: Phytophthora cactorum, Phytophthora cambivora, Phytophthora plurivora and Phytophthora pini; seven-day-old seedlings of Scots pine (15 seedlings per experiment) were infected using agar plugs of the respective species. Control group also consisted of 15 seedlings and was inoculated with sterile agar plugs. Results unambiguously show that after 4.5 days, all seedlings show clear signs of infection and display severe symptoms of tissue damage and necrosis. Moreover, three and two seedlings in the P. cactorum and P. cambivora infected seedlings groups, respectively, collapsed. The length of largest necrosis measured 13.4±3.90 mm and was caused by P. cactorum. To rule out any putative contamination or infection by secondary pathogens, re-isolations of pathogens from infection sites were performed and were positive in 100% of plated pieces of infected seedlings. All re-isolations were, however, negative in the case of the control group. Detailed microscopic analyses of infected tissues of young seedlings confirmed the presence of numerous Phytophthora species inside and on the surface of infected seedlings. Therefore, our results suggest Phytophthora spp. and mainly P. cactorum and P. cambivora as aggressive pathogens of Scots pine seedlings and highlight a putative involvement of these species in the damping off of young Scots pine seedlings frequently observed in forest nurseries.

Pathogenicity of Phytophthora isolates originatingfrom several woody hosts in Bulgaria and Poland

Abstract Our aim was to examine the virulence of eight Phytophthora isolates belonging to three species (Phytophthora cryptogea, Phytophthora plurivora and Phytophthora quercina) obtained from diverse European ecosystems (in Bulgaria, Poland and Germany) towards three forest tree hosts – English oak (Quercus robur L.), Turkey oak (Quercus cerris L.) and European beech (Fagus sylvatica L.). All plants grown from seeds in a greenhouse conditions were artificially inoculated under the stem bark with Phytophthora cultures. The tested isolates turned to be more aggressive to Turkey oaks than to English oak trees. In case of European beech, the isolates of P. cryptogea and P. plurivora exposed various virulence. The potential hazard of the introduced foreign isolates for the oak and beech forests in Poland and Bulgaria is discussed. Amongst the tested isolates, P. quercina P290 from German highly infected Bulgarian Turkey oaks; therefore, its negative potential impact on Bulgarian oak forests could be considered as high (if unintentionally introduced). Also, two Bulgarian isolates belonging to P. cryptogea and P. plurivora are risky for Polish beech forests, if exposed to the pathogen. The observed pathogenicity of the tested Phytophthora species proved their potential as important contributors to decline of valuable forest ecosystems dominated by oaks (Q. robur and Q. cerris) or beech (F. sylvatica), in both Poland and Bulgaria. We found that investigated Phytophthora pathogens could develop in the living plant stem tissues without causing any disease symptoms, which is another demonstration that phytosanitary control by simple observation of plant material is not effective.

Molecular detection of oomycetes species in water courses

Abstract In Poland, about 20% of forest nurseries use irrigation water coming from natural superficial reservoirs, presumed to be the first source of infection caused by harmful pathogens belonging to the Oomycota class, especially Phytophthora genus and Pythium genus. The forest nursery is the only place where forest managers can react before pathogens leave it with asymptomatic plants or soil attached to their roots. The aim of this research was detection and identification phytopathogens in water samples. In order to recognise genus Phytophthora or Pythium in water collected from 33 places in five different forest districts in Poland, two DNA-based approaches of identification were applied: (i) the TaqMan probes, and (ii) sequencing of the ITS6/4 region. The genomic DNA was obtained from 17 of 33 investigated water samples. TaqMan probes helped to identify 8 oomycetes present in 17 water samples. Based on ITS rDNA sequencing data, pathogens were identified in 17 cases, and this to the genus level (6 cases) and to the species level (11 cases). In total five Oomycetes species were identified, i.e. 3 Pythium species (Py. citrinum, Py. angustatum, Py. helicoides) and two Phytophthora species (P. lacustris sp. nov. - former taxon Salixsoil, P. gallica sp. nov.).

Antrodia gossypium, Phlebiopsis gigantea and Heterobasidion parviporum: in vitro growth and Norway spruce wood block decay

ABSTRACT Polymerase chain reaction-amplified and sequenced isolates of Antrodia gossypium, Phlebiopsis gigantea and Heterobasidion parviporum from decaying Norway spruce wood blocks after three and six months, which exhibited linear growth, were investigated. P. gigantea strains showed the fastest growth, whereas A. gossypium growth was five times slower. The differences between the mean daily increment of A. gossypium and the other examined isolates (except Hp2) were statistically significant. There were also significant differences in wood decay between densities over time. These results were confirmed by the decay acceleration index (DAI) and decay activity index, which were positively correlated with wood density regardless of the fungus species. The registered P. gigantea strains (Rotstop and PG Suspension) exhibited a strong decomposition ability (28% after six months); the weight loss caused by A. gossypium after six months of decay (15.2%) was similar to the results of P. gigantea (GB) after just three months (13.2%). All tested H. parviporum isolates showed rather rapid growth and equally strong wood decay (20–25%) compared to those of P. gigantea. DAI showed that A. gossypium may significantly contribute to wood decomposition over time, particularly in less dense wood samples. The use of both saprotrophs as biological agents against root pathogens is discussed.