Anna Maria Pirttilä

The Hidden World within Plants: Ecological and Evolutionary Considerations for Defining Functioning of Microbial Endophytes

SUMMARY All plants are inhabited internally by diverse microbial communities comprising bacterial, archaeal, fungal, and protistic taxa. These microorganisms showing endophytic lifestyles play crucial roles in plant development, growth, fitness, and diversification. The increasing awareness of and information on endophytes provide insight into the complexity of the plant microbiome. The nature of plant-endophyte interactions ranges from mutualism to pathogenicity. This depends on a set of abiotic and biotic factors, including the genotypes of plants and microbes, environmental conditions, and the dynamic network of interactions within the plant biome. In this review, we address the concept of endophytism, considering the latest insights into evolution, plant ecosystem functioning, and multipartite interactions.

Expression of Genes Involved in Anthocyanin Biosynthesis in Relation to Anthocyanin, Proanthocyanidin, and Flavonol Levels during Bilberry Fruit Development

The production of anthocyanins in fruit tissues is highly controlled at the developmental level. We have studied the expression of flavonoid biosynthesis genes during the development of bilberry (Vaccinium myrtillus) fruit in relation to the accumulation of anthocyanins, proanthocyanidins, and flavonols in wild berries and in color mutants of bilberry. The cDNA fragments of five genes from the flavonoid pathway, phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase, were isolated from bilberry using the polymerase chain reaction technique, sequenced, and labeled with a digoxigenin-dUTP label. These homologous probes were used for determining the expression of the flavonoid pathway genes in bilberries. The contents of anthocyanins, proanthocyanidins, and flavonols in ripening bilberries were analyzed with high-performance liquid chromatography-diode array detector and were identified using a mass spectrometry interface. Our results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. At the early stages of berry development, procyanidins and quercetin were the major flavonoids, but the levels decreased dramatically during the progress of ripening. During the later stages of ripening, the content of anthocyanins increased strongly and they were the major flavonoids in the ripe berry. The expression of flavonoid pathway genes in the color mutants of bilberry was reduced. A connection between flavonol and anthocyanin synthesis in bilberry was detected in this study and also in previous data collected from flavonol and anthocyanin analyses from other fruits. In accordance with this, models for the connection between flavonol and anthocyanin syntheses in fruit tissues are presented.

Isolation of high quality RNA from bilberry (Vaccinium myrtillus L.) fruit

A simple and efficient method is described for isolating high quality RNA from bilberry fruit. The procedure is based on the use of hexadecyltrimethyl ammonium bromide (CTAB), polyvinylpyrrolidone (PVP), and β-mercaptoethanol in an extraction buffer in order to eliminate the polysaccharides and prevent the oxidation of phenolic compounds. This method is a modification of the one described for pine trees, and yields high-quality RNA suitable for cDNA based methodologies. This method is applicable for a variety of plant tissues.

Detection of Intracellular Bacteria in the Buds of Scotch Pine (Pinus sylvestris L.) by In Situ Hybridization

ABSTRACT Bacterial isolates were obtained from pine (Pinus sylvestris L.) tissue cultures and identified asMethylobacterium extorquens and Pseudomonas synxantha. The existence of bacteria in pine buds was investigated by 16S rRNA in situ hybridization. Bacteria inhabited the buds of every tree examined, primarily colonizing the cells of scale primordia and resin ducts.

DNA isolation methods for medicinal and aromatic plants

Several protocols described for plant DNA isolation fail to produce good quality DNA from medicinal herbs and aromatic plants. These plants contain exceptionally high amounts of secondary metabolites that interfere with DNA isolation. To address this problem, we developed 2 DNA isolation methods for sundew and tarragon that produce DNA suitable for molecular biological applications. One of the methods also is applicable for milfoil and Siberian ginseng.

Identification of phenolic compounds from lingonberry (Vaccinium vitis-idaea L.), bilberry (Vaccinium myrtillus L.) and hybrid bilberry (Vaccinium x intermedium Ruthe L.) leaves.

Phenolic compounds from leaves of lingonberry (Vaccinium vitis-idaea L.), bilberry (Vaccinium myrtillus L.), and the natural hybrid of bilberry and lingonberry (Vaccinium x intermedium Ruthe L., hybrid bilberry) were identified using LC/TOF-MS and LC/MS/MS after extraction from the plant material in methanol in an ultrasonicator. The phenolic profiles in the plants were compared using the LC/TOF-MS responses. This is the first thorough report of phenolic compounds in hybrid bilberry. In total, 51 different phenolic compounds were identified, including flavan-3-ols, proanthocyanidins, flavonols and their glycosides, and various phenolic acid conjugates. Of the identified compounds, 35 were detected in bilberry, 36 in lingonberry, and 46 in the hybrid. To our knowledge, seven compounds were previously unreported in Vaccinium genus and many of the compounds are reported for the first time from bilberry and lingonberry.

Methane formation in aerobic environments

Methane (CH_4), the second principal anthropogenic greenhouse gas after CO_2, is the most abundant reduced organic compound in the atmosphere and plays a central role in atmospheric chemistry. Therefore a comprehensive understanding of its sources and sinks and the parameters that control emissions is prerequisite to simulate past, present and future atmospheric conditions. Until recently biological CH_4 formation has been associated exclusively with anoxic environments and methanogenic activity. However, there is growing and convincing evidence of alternative pathways in the aerobic biosphere including terrestrial plants, soils, marine algae and animals. Identifying and describing these sources is essential to complete our understanding of the biogeochemical cycles that control CH_4 in the atmospheric environment and its influence as a greenhouse gas.

Root endophytes along a primary succession gradient in northern Finland

Primary successional vegetation gradients are characterized by changes in the soil microbial communities. However, information on possible shifts of the root endophytes along these gradients is scarce. The objective of the current study was to identify root endophytic fungi from a primary successional gradient on land uplift seashore of a geographically isolated island area. We applied a sequencing approach by amplifying the ITS region with fungal specific primers. We used mainly an isolate-based method, and to compare the abundance of culturable and unculturable endophytes, direct sequencing of one representative plant specimen Deschampsia flexuosa was also carried out. A total of 38 cultured endophytic strains were sequenced from Empetrum nigrum (Empetraceae), Vaccinium vitis-idaea (Ericaceae) and Deschampsia flexuosa (Poaceae). Out of these, 27 were identified as Phialocephala fortinii, three as Mollisia minutella, four as Phialophora sp., one as Ascomycetes sp. and three remained unidentified. The strains clustered into five clades in the phylogram, mostly irrespective of the successional stages and hosts from which they had been isolated. The early successional seashore dune ridge plants however, seemed to host a distinct fungal taxon, Phialophora sp. Culture-independent methods were applied on a root sample of a mid-successional Deschampsia flexuosa specimen and a total of 16 clones were randomly selected and sequenced. Out of 16 sequences, 13 were identified as unculturable strains and three showed closest similarity to a basidiomycete Cortinarius callisteus. The unculturable sequences were grouped into two main clades and were different from any culturable isolate in this study. Our results suggest that (i) P. fortinii dominates the isolate data at mid to late successional stages, (ii) roots of the ericaceous plants and the grass Deschampsia flexuosa are colonized by the same endophytic fungi in this ecosystem, and (iii) unculturable endophytes are common and potentially more abundant than the culturables. To our knowledge, this is the first report of the molecular phylogenies of the DSE in the mid-boreal zone and also the first report of the unculturable root endophytes of D. flexuosa.

Bioactivity and genetic diversity of endophytic fungi in Rhododendron tomentosum Harmaja

Eighty-seven endophytic fungi were isolated from asymptomatic leaf tissues of Rhododendron tomentosum. Most of the isolates were non-sporulating and therefore difficult to identify exclusively based on morphological characters. Eighteen isolates that were morphologically distinct were selected for identification by sequencing the ITS region. For culture-independent analysis, the DNA was isolated from the surface-sterilized leaves of R. tomentosum and the ITS region was amplified using fungal specific primers ITS1F and ITS4, cloned, and 11 clones were randomly selected and sequenced. The phylogenetic analysis was performed with MEGA4 on a total of 49 sequences, including 18 endophytic isolates and 11 unculturables obtained in this study, and 20 sequences from Genbank, which were distributed in four clusters. The culturable and unculturable endophytes formed separate clades and were clearly distinguishable with no overlap within the groups. Endophytic fungi are a well-recognized source of bioactive compounds and therefore antibacterial and antioxidant activities of the isolates of R. tomentosum were studied. All isolates were grown in two different media, enriched (MEB) and depleted (DM), and screened for antibacterial and antioxidant activities. As a result, 10% produced antibacterials and 14% antioxidants, in total 24% of the isolates had biological activity. Antioxidant broth of TRT59 was partially purified using HPLC. The majority of antibacterial compounds were produced in DM media and antioxidants in MEB media. Therefore, it is advisable to test various media for production of antibacterials and antioxidants.

Methylobacterium-Induced Endophyte Community Changes Correspond with Protection of Plants against Pathogen Attack

Plant inoculation with endophytic bacteria that normally live inside the plant without harming the host is a highly promising approach for biological disease control. The mechanism of resistance induction by beneficial bacteria is poorly understood, because pathways are only partly known and systemic responses are typically not seen. The innate endophytic community structures change in response to external factors such as inoculation, and bacterial endophytes can exhibit direct or indirect antagonism towards pathogens. Earlier we showed that resistance induction by an endophytic Methylobacterium sp. in potato towards Pectobacterium atrosepticum was dependent on the density of the inoculum, whereas the bacterium itself had no antagonistic activity. To elucidate the role of innate endophyte communities in plant responses, we studied community changes in both in vitro and greenhouse experiments using various combinations of plants, endophyte inoculants, and pathogens. Induction of resistance was studied in several potato (Solanum tuberosum L.) cultivars by Methylobacterium sp. IMBG290 against the pathogens P. atrosepticum, Phytophthora infestans and Pseudomonas syringae pv. tomato DC3000, and in pine (Pinus sylvestris L.) by M. extorquens DSM13060 against Gremmeniella abietina. The capacities of the inoculated endophytic Methylobacterium spp. strains to induce resistance were dependent on the plant cultivar, pathogen, and on the density of Methylobacterium spp. inoculum. Composition of the endophyte community changed in response to inoculation in shoot tissues and correlated with resistance or susceptibility to the disease. Our results demonstrate that endophytic Methylobacterium spp. strains have varying effects on plant disease resistance, which can be modulated through the endophyte community of the host.