Title of the project: Investigation of the pathology of the genus Armillaria and the possibility of biological control of the forest pest

Project ID number: GINOP 2.3.2-15-00052

Beneficiary: University of Sopron (9400 Sopron,

Bajcsy-Zsilinszky u. 4

Amount of support: 269 883 102, - Ft

Project Management

Professional Leader: Dr. György Sipos

Financial Manager: Márta Brazsil

Availability:

email: sipos.gyorgy@uni-sopron.hu

Intermediate Organization: Ministry of National Economy

Start of project implementation: 2017.01.01.

Completion of the project: 2020.12.31

Summary of the project:

A)

The Armillaria species are the natural soil co-creators of forests, some of their plants are the largest and the longest living creatures of the Earth's surface. They include white rotting fungi, which can break down all the major molecular components of the plant cell wall so they play a fundamental role in ensuring the natural circulation of nutrients necessary for inland life (Hetala: Forests, 6,3304,2015). Some Armillaria species, however, are among the most serious forest-damaging fungi. They attack and destroy trees that are weak in their resistance, and after the host plant dies, they completely break down the wood's tissues (Baumgartner: Mol. Plant Pathol., 12,515,2011). Last year, the role of the Armillaria species also occurred in the destruction of the forests of the Keszthely Mountains.

The defense against their damage is difficult to accomplish because the pathogen can survive in woody residues in the soil for a long time. Chemical protection in forestry has a number of adverse effects, so there is an increasing demand for environmentally-friendly, biological defense methods. The aim of the project is to assess the forestry impact of the Armillaria species, to examine their genetic characteristics and to develop an effective forest protection strategy based on biological protection. Armillaria species cause significant forest degradation worldwide. The explosive growth of the total number of available genomic sequences has now made it possible to investigate the genomic level of this serious economic damage problem, and thus the project is extremely timely. The topic is based on the genomic approach and the modern molecular biology methods (RNA-Seq and dual RNA Seq , interaction with single cell transcriptomics) are among the top-ranked researches. The comparative genomic analysis designed to achieve this is the first, large-scale, systematic study on the subject, the results of which are highly anticipated in the scientific community, so the planned research creates a scientific breakthrough. The academic part of the project is based on the use of colonizing virulent and saprotrophic Armillaria isolates, already existing in the strain collection.

1-second year: functional annotation and comparative genomics of rats, rhizome and trunk test, in vitro invasion experiments 2-3. year: plant-mushroom interaction studies Project area: 1.-2. year: identification of the Armillaria species and their unique plants from the damaged forests, sequencing of the haploid genome of "PacBio" of two species of colonizing species, determination of genetic diversity by Illumina sequencing, RNA-Seq and functional annotation of the genomes concerned. Year: Collection of associated microbial samples, genomic isolation, metagenomics Part of the project biochemistry: 1st year: testing of potential biochemical agents on virulent isolates 2nd year: Molecular identification of microbial isolates from associated microbes, assessment of their biochemical control year 3: Optimization of fermentation of biochemical agents 4 years : field testing.

B) Research plan

1. Genetic analysis of pathogenic Armillaria species and their associated microbial communities in severely damaged and naturally occurring forest associations
1.1 Collection of Armillaria samples (rhizome, mycelium, fruit tree) from Armillaria severely damaged and naturally occurring forest associations
1.2 Identification of Armillaria species and their larger single- and somatic incompatibility tests
1.3 Selection of larger representative Armillaria individuals (virulent and non-virulent), study of site conditions, collection of samples for the microbial communities associated with the Armillaria rhomorphoras
1.4 Isolation of the bacterial and fungi components of the associated microbials, identification of microbial species amplification and sequencing of diagnostic value loci and metagenomic devices
1.5 Isolated bacteria (Szeged Microbiology Collection, szmc.hu)
2. Investigating genetic factors determining the pathogenicity and virulence of Armillaria species
2.1 Establishment of Armillaria genomes and databases
2.1.1 Two Armillaria species colonizing pine (A. ostoyae and A. cepistipes) with a full-scale annotation of the available genome of the genome with RNA-Seq data
2.1.2 Supplemental sequencing of the genomes of our Armillaria isolates (A. gallica and A. mellea), colonizing with leafy trees, "Illumina" PacBio "technology, development of genomes and databases
2.1.3 Creating computer background and storage capacity for bioinformatics work
2.2 Genome-specific determination of the unique genetic background of diploid and haploid Armillaria isolates used in pathogenicity tests
2.2.1 The available and field-isolated strains have a parallel" Illumina ", determination of genetic identity and diversity (SNP pattern)
2.2.2 Application of" PacBio "in duplicate, difficult-to-combine variable sequences
2.3 Mushroom host plant interaction ok investigation
2.3.1 In vitro cambium invasion tests Comparative transcriptional analysis of mycelial tissue of mycobacteria entering the cambium of fresh and autoclaved fractures. During the experiments we are planning to test our already existing diploid virulent and non-virulent A. ostoyae isolates and the pathogenic isolates collected from the damaged forests.
2.3.2 Developing a herbicidal-mushroom interaction test using spruce (known genome) clones (spruce-armillaria pathosystem)
2.3.2.1 Investigation of Armillaria-Spruce Interaction with Light Microscopy
2.3.2.2 Examination of Gene Expression Interaction Patterns in Armillaria-Spruce Interaction Different Phases (Dual RNA Seq )
2.3.2.3 Investigation of Armillaria-spruce interaction with the tools of metabolomics and proteomics
3. Evidence of the evolutionary origin of Armillaria pathogenicity by transcriptomic, single-cell transcriptional and comparative genomic methods
3.1 Comparative genomic assays
3.1.1 Reconstruction of genomic evolutionary events by comparative evolutionary genomic methods (COMPARE analysis)
3.1.2 Reconstruction and characterization of genetic innovations characteristic of the Armillaria genus and individual species with functional annotations, some outstanding genetic families with detailed phylogenetics and its analysis
3.1.3. Detailed genomic analysis of CAZy gene families involved in the degradation of plant cell wall biopolymers
3.2. Exploration of genetic background of rhizome tree as the unique propagation and colonization mechanism characteristic of the Armillaria genus
3.2.1. Examination of gene expression patterns in the top and tribal zones of the rhymorphs of the Armillaria ostoyae C18 strain compared to the air with rhizomorphic top and tribal zones in Armillaria ostoyae C18 strain
3.2.2 Examination of gene expression patterns in the strains of Armillaria ostoyae C18
3.2.3 Comparative genomic analysis of the genomes of the genes involved in the rhizomorphic and phytoplankton genes, conservation of their conservation and evolutionary origin
3.2.4 The role of small regulatory RNAs in the growth and root colonization of rhomomorphs
3.2.5 Investigation of Armillaria-Spruce Interaction with Single-Cell Transcriptional Methods
4. Development and Practical Testing of Biocontrol Strategy in Severely Damaged Forestry Areas
4.1 Selection of Virulens Armillaria Strains in Table 1.1.
4.2 Generation and optimization of in vitro screening methods for the detection of antagonisms of bacteria (eg Bacillus, Pseudomonas) and fungi (eg Trichoderma species) against virulent Armillaria species
4.3. interaction study of isolated bacterial and fungal strains against virulent Armillaria species in laboratory conditions with in vitro antagonism and inhibition tests carried out on substrate plates, determination of biochemical index values, selection of bacteria and fungi with good antagonistic capabilities against virulent Armillaria species
4.4 Optimization of fermentation production of selected microorganisms
4.5 In vivo experiments with the selected microorganisms in the host plant-mushroom interaction test developed in task 2.4, metatranscriptomics analysis
4.6 Compilation of microbial mixtures corresponding to Hungarian forestry challenges, creation of prototype preparation
4.7 Field test of the preparation prototype in field conditions for the remediation of the soil of the areas infected with Armillaria before new afforestation, during afforestation for the treatment of saplings, application technology optimization.

Partners have decades of experience in the planning and successful implementation of similar-scale domestic and international research projects, including professional and financial / administrative tasks. The research program outlined is in line with the number of relevant researchers and other participants contributing to the implementation of the project and its professional competence. The support requested in the application will ensure the implementation of the tasks undertaken within the planned time frames. The professional management of the project and the management continuously monitor the implementation, ensuring the achievement of the milestones of the research at a planned pace. Researchers contributing to the project will coordinate the implementation of Tasks 1 and 2, and participate in the 4th task 4.7. in the implementation of its sub-task. László Nagy co-ordinates the implementation of Task 3 by the Hungarian Academy of Sciences. Researchers participating in the project from the SZTE are involved in task 1 1.4. and 1.5. and coordinate the development of a biocontrol strategy in target 4.

C)

The project creates a critical mass of high-quality R & D capacities by aligning the research activities of the Knowledge Bases of the University of West Hungary, the Department of Microbiology of the University of Szeged and the MTA SZBK Impact Mushroom Evolutionary Genomics Research Group. The project becomes a research program that affects both domestic and international scenes by studying a forestry problem causing serious economic damage worldwide and provides an opportunity to develop a methodology for forest meta-genomics and meta-transtriptomics, and to create a forest microbial bioinformatics center of excellence. The Armillaria genus embraces aggressive pathogenic species all over the world, with a large number of genetic diversity and understanding of their pathogenicity, and their potential for defending them. By analyzing the genome sequences underlying the project, the first detailed, comparative genomic analysis of the Armillaria genus is realized, which can be a milestone for future Armillaria researches. In this way, the forestry microbial center for bioinformatics in forestry can become the center of research at Armillaria internationally. The results of the project are made available to publishers of scientific public life in publications of prestigious international journals, thus a highly international recognition of research is possible and their use for educational / training purposes is provided by the opportunities offered by the academic degree of young researchers working in the field and the improved presentation of project results, in the form of extended subject-based university courses.

Research into the system of BsC / MsC and PhD training schemes is based on the opportunities offered by the academic qualifications of young researchers in the field and the improved university courses that integrate the results, and the underlying processes of R & D capacities through domestic product development co-operations with domestic SMEs. Sustainable forest management is unlimited renewable, renewable raw material and carbon neutral for unlimited time. The domestic job preservation and creation ability of the wood and furniture industry presupposes predictable raw material access, which is mainly provided by domestic forest stock. However, domestic and Central European forest stocks, which serve as the basis for the wood and furniture industry, are severely endangered by microbial, predominantly fungal diseases. Among them, Armillaria_ filaments infections are often highlighted, which often result in mass destruction in some forestry areas, which also has a major impact on the wood and furniture industry. The industrial use of logs made from wood degradation is a major logistical challenge.


The project will extend our knowledge of the severe forest pest control of Armillaria fungus with a new knowledge, both domestic and international, relative to the current level of knowledge available in genomics, transcriptomics and biological defense sciences. The project fits in with cutting-edge molecular biology and bioinformatics methods designed for use in the field of international cutting-edge research. The Strategic R & D Workshop to be established by the consortium partners in the project will provide for the strengthening of knowledge infrastructure, the adaptation of state-of-the-art international research-methodology experiences in the field of plant-fungal interaction testing and the integration of the genomic databases that will be created, collaborative, comparative genomic projects (eg 1000 Fungal Genomes Project). The comparative genomic method (COMPARE pipeline) developed by Dr. László Nagy is the first methodology that allows for the systematic identification of genes and gene scenes in the background of any phenotypic background based on complete genomic sequences. Within the framework of the project, it is possible to target targeted development of existing international scientific collaborations and to develop new collaborations. The project offers the opportunity to develop a methodology for forest meta-genomics and metatranscriptomy and to create a forest microbial center for bioinformatics. During the implementation of the project's tasks, a modern, comprehensive research methodology adapted to the protocols adopted in biology areas will be used, including microbiological, metabolomic, fermentation, molecular biology, microscopic, bioinformatics, metagenomics, comparative genomics, transcripts, new generation sequencing methods and field studies which are suitable for all purposes in achieving the goals set. The project proposal has many years of practical experience in the field of implementation of the methods that are being used.