DOI: 10.23729/e6ae1e0b-33e3-4e95-bb60-49bb69aafdd2
title: Research data of rust diseases in project ’Bio-based products to protect Scots pine against damage due to rust fungi’

description: Description of rust files (1-8):

#### Data 1: Chemical composition of leaves of eight alternate hosts of Cronartium spp.

The aim of the study was to investigate the chemical variation of leaves of susceptible and resistant alternate hosts of Cronartium spp. It was aimed to identify chemicals that distinguish susceptible and resistant closely related species from one another. The studied plant species were Melampyrum sylvaticum, M. pratense, Veronica longifolia, V. chamaedrys, Impatiens balsamina, I. glandulifera, Ribes nigrum and R. rubrum. Twenty plants per species were investigated.

The data (four accurate mass files) consisted of automatic chemical data given by the LCMS masspectrometer device. Each peak shown as peak area in relation to retention time indicated a chemical compound. The chemical data from samples were measured in separate runs in positive and negative mode. Chromatography and MS parameters were identical, while only some source settings were optimized individually for each polarity. The fifth file contained concentrations of 12 selective chemicals in the 160 leaf samples. 

The plant leaves were collected in the Oulu city area, air dried, cold stored and extracted in methanol in Luke's (Oulu) Lynet laboratory. Twenty plants from eight plants species each were used. The chemical analysis was done in Biocenter of the University of Oulu using a LCMS mass spectrometer. The device measured automatically from the samples peak areas representing chemical compounds. Based on this data, chemicals that separated significantly susceptible and resistant alternate host pairs from each other were identified. In addition, based on literature, concentrations of 12 selected compounds were calculated and compared between susceptible and resistant species. The used protocols were presented in detail in Piispanen et al. 2023, Eur. J. Pl. Pathol., 165, 677-682. The data contains features generated by the Compound Discoverer software as specified in Piispanen et al. 2023, Eur. J. Pl. Pathol., 165, 677-682. Each feature represents a compound characterized by accurate mass, chromatographic retention time, and the peak area. The fifth file contained results for targeted (absolute) quantification of 12 selected chemicals in the 160 leaf samples.
 
 Four Excel-data of negative and positive accurate mass data from LCMS were connected to the data. The data includes from each compound calculated automatically by the device Molecular weight, Retention time, Maximun area, and Raw areas of every peak. The data have been shown in two positive and negative accurate mass files. Based on these files, the compounds significantly differing between plant species pairs were calculated. In addition, 12 selected compounds were picked up from the data in all samples and compared between plant species. The variables in the Excel data are Sample/Plant species, Sample size mg, Extraction 1, Extraction 2, Extraction total and callculation of the amount of 12 compounds. The Plant species were: Melampyrum sylvaticum, M. pratense, Veronica chamaedrys, V. longifolia, Impatiens glandulifera, I. balsamina, Ribes nigrum and R. rubrum. The chemical compounds were chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, rutin, hyperoside, ferulic acid, quercitrin, myricetin, luteolin, apigenin and chamepherol. The results were presented in Piispanen et al. 2023, Eur. J. Pl. Pathol.,165,677-692.

 In Materials 1-3, the LCMS-data was collected by Biocenter, University of Oulu, where the responsible person was Dr. Ulrich Bergmann. The extractions and other calculations were done in Luke.

#### Data 2: Temporal, spatial and leaf age variation of 12 chemicals of leaves of Melampyrum pratense and M. sylvaticum. 

The aim of this study was to investigate the chemical variation of 12 compounds between susceptible and resistant alternate hosts, temporal variation within growing season, spatial variation between locations and variation between young and old plant leaves.

The data consisted of results from 440 leaf samples of M. pratense and M. sylvaticum collected in 1-3 locations in Oulu city either in late June, mid-July or late August. Samples represented either young or old leaves. The results represented concentrations of 12 selective compounds. The analyses were done using LCMS. 

Leaf samples of M. pratense and M. sylvaticum were collected from 5 locations in the Oulu city area. Samples were collected also in early, mid- and late season. Leaves were analysed also from young and old leaves of the plants. Ten plants per species were collected in each collection. The samples were analysed similar to samples of material 1 as well as also the 12 selected chemicals were using LCMS. The used protocols were presented in Piispanen et al. 2023, Eur. J. Pl. Pathol., 165, 677-692. A manuscript Piispanen et al. 2024 from the results is under process.

The Excel-data consisted of the amounts of 12 compounds found in the samples. The weights were based on LCMS-analysis. The variables in the Excel.-file Material 2.xlsx were Extraction date, Collection date, Plant species (M. sylvaticum or M. pratense), No. of plants (1-10), young/old leaves (n=young leaves, v=old leaves), No. of extraction, Weight (mg), Sample number (401-840), Amounts of compounds (ng/mg): chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, rutin, hyperoside, ferulic acid, quercitrin, myricetin, luteolin, apigenin and caempherol. NF=Not found=0 in calculations. Minus values=0 in calculations. . = Missing data. A paper of the results Piispanen et al. 2024 is under process.

#### Data 3: Effect of Cronartium spp. infection on 12 chemicals in leaves of susceptible alternate hosts of Cronartium spp. 

The aim of this study was to investigate the chemical changes in 12 compounds after Cronartium spp. inoculation just after infection before fruitbody formation.

The data consisted of concentrations of 12 selected chemicals from inoculated and control leaves either 3 or 6 days after inoculation. The inoculations with Cronartium spp. were done in the greenhouse for three susceptible alternate hosts. The analyses of compounds were done using LCMS. 

All leaves of 20 susceptible plants of C. pini, P. lactiflora and I. balsamina, were inoculated with C. pini in the greenhouse by dusting spores on leaves and incubating the plants for 24 h with a moistened plastic bag. The inoculation protocol has been presented in detail in Kaitera et al. 2015, Pl. Pathol. 64, 738-747. In addition, leaves of one branch per plant of 20 R. nigrum were inoculated similarly with C. ribicola. Twenty plants were left uninoculated as controls for each species. Ten inoculated leaves were collected from all plants 3 and 6 days after inoculation. The leaves were prepared and 12 selected chemicals analysed from the samples using LCMS as in material 2. For the protocols, see Piispanen et al. 2023, Eur. J. Pl. Pathol.,165, 677-692. A manuscript Piispanen et al. 2024 from the results is under process.

The Excel-data consisted of the amounts of 12 compounds found in the samples. The weights were based on LCMS-analysis. The variables in the Excel-file Material 3.xlsx were: Plant species (Ribes nigrum, Paeonia lactiflora, Impatiens balsamina), No. of plants (1-20), Inoculation (Cronartium pini, C. ribicola)/Control, Incubation days (3 or 6 days), Sample number (1-240), compounds (ng/mg): chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, rutin, hyperoside, ferulic acid, quercitrin, myricetin, luteolin, apigenin, caempherol.NF=Not found=0 in calculations. Minus values=0 in calculations. . = Missing data. A paper of the results is in process.

#### Data 4: Temporal and spatial variation of endophytes in leaves of eight alternate hosts of Cronartium. 

The aim of this study was to investigate spatial variation between locations and temporal variation within growing season in endophyte composition of leaves of seven alternate hosts of Cronartium spp.

The data consisted of frequencies of isolates representing 37 morphotypes of endophytes in leaves of eight alternate hosts of Cronartium spp. The morphotypes were divided among collection locations and divided into two times of collections, early and late season, which were divided in two files. Also the genetic identification of representative endophytes of different morphotypes was included. 

Five healthy leaves per plant from five plants of eight susceptible and resistant alternate hosts of Cronartium spp. were collected from one to three locations in Oulu city area in late June and early September. Five small pieces were cut sterily from each leaf, sterilized and inserted on agar. Endophytes emerging on the agar were transferred to pure cultures and grown at 18 C for one month after which the morphotype of the endophyte was characterised and grouped into 37 groups based on their morphology on agar. Endophytes representing the most common morphotypes and alternate hosts were identified genetically. The isolation procedure, genetic analysis and the results have been presented in Piispanen et al. 2024, Eur. J. Pl. Pathol. (under review). 

The Excel-data of file Morphotypes_early season.xlsx and Morphotypes_late season.xlsg contain frequencies of endophytes from eight plants species collected in late June or early September from 1-3 locations and classified in different morphotypes. The variables were Morphotype (1-37), Plant species: Melampyrun sylvaticum, M. pratense, Veronica chamaedrys, V. longifolia, Impatiens glandulifera, I. balsamina, Ribes nigrum, R. rubrum/Areas (1-3), Total. The results of the DNA-analysis are included in Excel-file DNA-analyses.xlsg containing the variables: Sample, Isolate, Plant species (Melampyrun sylvaticum, M. pratense, Veronica chamaedrys, V. longifolia, Impatiens glandulifera, I. balsamina, Ribes nigrum, R. rubrum, Morphotype, Sequence tag, Fungal species, Match (to GenBank), Sequence.

#### Data 5: Effect of leaf extracts on germination of Cronartium spores. 

The aim of this study was to investigate the effect of leaf extracts of susceptible and resistant alternate hosts of Cronartium and some commercial extracts added to water agar at three concentrations on germination of aeciospores of Cronartium spp. 

The data consisted of germination results of Cronartium spp. on eight leaf extracts from alternate hosts, two commercial extracts and two control treatments on water agar after 24 h incubation at 21,5ºC. Three concentrations, 50ppm, 100ppm and 500 ppm, were used from each extract. For the water agar control, the concentration was 0. Germination of the spores was counted as percentage of germinating aeciospores under stereo microscope in 10 random fields on agar plates.

Plant extracts from eight alternate hosts, chlorogenic acid and quercitrin were extracted as in materials 1 and 2 and added on agar at three concentrations, 50ppm, 100ppm and 500ppm. Methanol and pure malt agar were used as controls. Concentration for the malt agar was 0. Aeciospores of C. pini and C. ribicola were dusted on the agar plates and germination of the spores was counted under light microscope after 24 h incubation at 21,5ºC. Germination was counted from 10 random fields on each plate. A manuscript Piispanen et al. 2024 from the results is under process.

The Excel-data Material 5.xlsx contains variables Extract (eight plant extracts from Melampyrum sylvaticum, M. pratense, Veronica chamaedrys, V. longifolia, Impatiens glandulifera, I. balsamina, Ribes nigrum and R. rubrum, chlorogenic acid, quercitrin, methanol and pure malt agar control), Dose (500ppm, 100ppm, 50ppm for the extracts and 0ppm for the malt agar control), Plate, Rust (inoculated rust species: Cronartium pini or C. ribicola), sample 1-10 (germination % counted from 10 areas on the agar).

#### Data 6: Effect of leaf extracts on growth of endophytes of alternate hosts of Cronartium spp. 

The aim of this study was to investigate the effect of leaf extracts from susceptible and resistant alternate hosts of Cronartium spp. on the growth of common endophytes of the alternate hosts of these Cronartium rusts.

The data consisted of growth measurements of seven selected endophytes of different alternate hosts. The most common endophyte of each plant was used. Extracts were spread on agar and the growth of each endophyte was measured. Three concentrations were used for each extract.

Seven endophytes that represented the most common endophytes of the alternate hosts of Cronartium, were transferred to agar plates added with leaf extracts and methanol of eight alternate hosts, two commercial chemicals and two controls, methanol and agar without any extract. The extracts were added at concentrations of 50ppm, 100ppm and 500ppm. The malt agar control had 0ppm extract. Growth of the endophytes were first drawn on plastic film after 6-20 days of incubation depending on the growth rate of each endophyte. The same incubation time was used among extracts of each endophyte. The growth area was measured from the films using a planimeter. A manuscript Piispanen et al. 2024 from the results is under process.

The Excel-data Material 6.xlsx contains variables: Extract (8 plant extracts of M. sylvaticum, M. pratense, V. chamaedrys, V. longifolia, I. glandulifera, I. balsamina, R. nigrum and R. rubrum, and two controls of methanol and pure malt agar), concentration (500ppm, 100ppm, 50 ppm and 0ppm), Endophyte (7 endophytes from M. sylvaticum, M. pratense, V. chamaedrys, I. glandulifera, I. balsamina, R. nigrum and R. rubrum), plate (1-5 repeats), Area, cm2 (growth rate after incubation of 6-20 days), Days (No. of incubation days). . = Missing data.

#### Data 7: Effect of leaf extracts on fruiting and sporulation of Cronartium spp. on susceptible alternate hosts. 

The aim of this study was to clarify, if extracts from resistant alternate hosts of Cronartium spp. can protect susceptible alternate hosts to Cronartium infection, fruiting and sporulation.

The data consisted of frequencies of uredinia and telia formed on susceptible alternate hosts after inoculation with Cronartium spp. and 2-6 weeks of incubation prior which extracts from resistant alternate hosts and commercial extracts had been spread on the leaves as protection. In the laboratory experiment, leaf extracts from 4 resistant plant species, two commercial chemicals and three controls were sprayed at 100ppm on plant leaves floating on water plates. Two leaves per plate with 5 replicats for each plant and extract were used. Four susceptible test plants were used. One plant species was inoculated with C. ribicola and three species with C. pini after extract spreading. The coverage of uredinia and telia were estimated visually in seven coverage classes from 0-100% after 2-, 4 and 6-weeks of incubation. In the greenhouse, the same four leaf extracts from resistant alternate hosts, two commercial chemicals and two controls were sprayed at 100ppm concentration each on all leaves of two susceptible plant species, I. balsamina and P. lactiflora. Spraying of the extracts was also done for one branch of R. nigrum. After that the leaves were inoculated either with C. pini (for I. balsamina and P. lactiflora) or C. ribicola (for R. nigrum). The number of leaves with either uredinia or telia per plant was counted after 2-, 4 and 6-weeks of incubation.

Leaf extracts from four rust-resistant alternate hosts, M. pratense, V. chamaedrys, I. glandulifera and R. rubrum, two commercial compounds, chlorogenic acid and quercitrin, and methanol and water controls, were sprayed on leaves of rust-susceptible alternate hosts, M. sylvaticum, I. balsamina, P. lactiflora and R. nigrum, floating on water plates in Petri dishes in the laboratory. Two leaves were layed on each plate and five plates per treatment were used. After spread of the extracts, aeciospores of C. pini were dusted on leaves of M. sylvaticum, I. balsamina and P. lactiflora, and those of C. ribicola on R. nigrum. Formation of uredinia and telia on inoculated leaves was estimated after 2-, 4- and 6-weeks of incubation. The fruiting stages have been described in Kaitera et al. 2015, Pl. Pathol. 64,738-747. The coverage of uredinia and telia on the leaves were estimated in seven classes: 0%, 1%-20%, 20%-40%, 40%-60%, 60-80%, 80-99%, 100%. In the greenhouse, eight plants of P. lactiflora and I. balsamina each were inoculated with C. pini, and eight plants of R. nigrum were inoculated with C. ribicola. Prior to inoculation, leaf extracts of four resistant alternate hosts, M. pratense, I. glandulifera, V. chamaedrys and R. rubrum, two commercial compounds, chlorogenic acid and quercitrin, and two controls, methanol and water, were sprayed on the leaves for protection. The inoculation was done by dusting spores on the leaves and incubating the plants for 24 h with a moistened plastic bag. Number of leaves with either uredinia or telia were counted after 2-, 4- and 6-weeks of incubation. A manuscript Piispanen et al. 2024 from the results is under process. 

The Excel-data Laboratory experiment.xlsx contains variables: Plant species (R. nigrum, M. sylvaticum, P. lactiflora, I. balsamina), Extract (leaf extract of M. pratense, I. glandulifera, V. chamaedrys and R. rubrum, chlorogenic acid, quercitrin and three controls, water-methanol, dry control without watering and control without inoculum), Plate (nos. 1-5), Leaf (no. 1 or 2), coverage of uredinia after 2 weeks of incubation, coverage of telia after 2 weeks of incubation, coverage of uredinia after 4 weeks of incubation, coverage of telia after 4 weeks of incubation, coverage of uredinia after 6 weeks of incubation and coverage of telia after 6 weeks of incubation. The %-coverages were estimated visually in 7 classes (1-7) with coverages 0, 1-20%, 21-40%, 41-60%, 61-80%, 80-99% and 100%. 

The Excel data Greenhouse experiment.xlsx contains variables: Plant species (R. nigrum, P. lactiflora, I. balsamina), Extract (leaf extract of M. pratense, I. glandulifera, V. chamaedrys and R. rubrum, chlorogenic acid, quercitrin and two controls, water-methanol and dry control without watering), Number of leaves with uredinia after incubation of 2 weeks, Number of leaves with telia after incubation of 2 weeks, Number of leaves with uredinia after incubation of 4 weeks, Number of leaves with telia after incubation of 4 weeks, Number of leaves with uredinia after incubation of 6 weeks, Number of leaves with telia after incubation of 6 weeks, Number of inoculated leaves and Infection-% (0-100%).

#### Data 8: Effect of leaf extract on fruiting and sporulation of C. pini on Scots pine. 

The aim of this study was to clarify, if plant extract from a rust-resistant M. pratense can reduce C. pini fruiting and sporulation on Scots pine.

The data consisted of fruiting variables of C. pini on Scots pine branches either sprayed with M. pratense extract or without treatment as control at the spraying year (2022) and one year after the treatment (2023). Another data consists of annual C. pini inventory in a experimental field in the campus area of University of Oulu. Survival and rust sporulation was estimated in 2021-23. The seedlings were inoculated either with the autoecious or heteroecious life-cycle form of C. pini.

Scots pine seedlings were first planted in the field around the campus of University of Oulu. The plants were inoculated with either aeciospores of C. pini (for the inoculation protocol, see Kaitera & Nuorteva. 2008, For. Ecol. Manag., 255, 973-981) or basidiospores. The basidiospores were harvested from inoculated leaves of P. anomala and P. lactiflora carrying C. pini telia to water after incubating telia on moistened filter paper on Petri dishes. Two years after inoculation the disease establishment as aecia formation on the seedlings was estimated. The inoculations resulted in only three infected seedlings in which the aecia were sprayed with leaf extract from M. pratense. Aeciospores were collected before and after spraying to morphological assessment. In addition, 20 sporulating lesions on young pines were marked in a Scots pine stand heavily infected by C. pini in Pudasjärvi. Ten of these lesions were sprayed with extract of M. pratense, while 10 lesions were left unsprayed as controls. Fruiting and sporulation of the lesions were estimated the next year after spraying. Spores were collected from aecia prior to and after spraying for microscopical morphology assessment in the laboratory. A manuscript Piispanen et al. 2024 from the results is under process.

The Excel-data Scots pine shoots_Pudasjärvi.xlsx contains information of sporulation and growth of C. pini in sporulating lesions a year after spraying of the lesions and in control lesions. The variables are: Shoot (1-10), Treatment (Control or spraying), Dead top (no/yes), Spreading (spreading along the lesion: no/yes), Sporulation (sporulation in the lesion: no/yes), Alive top (living leader of the shoot carrying the lesion: no/yes), No new sporulation (no/yes). The three Excel-data, Survival of pine seedlings_Oulu University field_2021.xlsx, Survival of pine seedlings_Oulu University field_2022.xlsx and Survival of pine seedlings_Oulu University field_2023.xlsx contain annual survival and rust infection on seedlings. The survival assesments were: Seedling and bud are alive (=OK), seedling is dead and removed (=X) and seedling carries aecia of the rust, C. pini (=R).


### Project and funding
RustChem project ’Bio-based products to protect Scots pine against damage due to rust fungi (41007-00195501)’, which was funded by Finnish Academy of Science and Natural Resources Institute Finland.