The dieback of forests is currently an important point of discussion of the world scientific community (Sano et al. 1995). The prevailing assumption is that the dieback is closely associated with the ongoing climate change (Dale et al. 2001; Lebourgeois et al. 2010; Pinto et al. 2007; Stoyko 2009; Jactel et al. 2012; Yavorovs’kyy 2015; Shvydenko et al. 2018; Zhang et al. 2018), the development of pathogenic organisms of different systematic and functional affiliations (Gvozdyak et al. 2012; Goychuk et al. 2020b), the natural changes in the biotopes (Levanic 1997; Schelhaas et al. 2003; Pinto et al. 2008; Kobal et al. 2015), forest management practices (Kalutsky 2008; Kulbanska 2015; Meshkova et al. 2018) or complex causes (Gašperšić 1967; Manko and Gladkova 2001; Elling et al. 2009).
In the Spanish Pyrenees, the dieback is associated with the spread of fungal white rot caused by
The dieback may also be caused by acid rain, parasites or bacterial pathogens, bark beetle, and so on (Cherpakov 2012; Goychuk et al. 2020c). Deterioration of the health condition of common silver fir stands in Bulgaria is associated with mistletoe infestation (Rosnev and Petkov 1994).
A serious problem of Croatian forestry (since the 19th century) was the mass mortality of common silver fir (so-called ‘dieback of silver fir’), which is manifested by mass dieback, loss of vital energy and reduced growth rates of trees. The phenomenon of fir extinction is associated with a number of causes of abiotic and biotic origin (Seletković et al. 2008; Ugarković et al. 2009).
An extensive syntaxonomic research of forest communities involving
The studies on phytobacteriological aspects and their relation to the dieback of fir forests are scanty. The first information about the ‘wetwood disease’ of coniferous species dates back to 1934 (Lagerberg 1934), and the first report of bacterial wetwood of deciduous species of the family Ulmaceae dates back to 1945 (Carter 1945). Typical symptoms of bacterial wetwood of some species of the genus
In 1963, A.L. Shcherbin-Parfenenko described a bacteriosis called ‘bacterial wetwood’ caused by the gram-negative bacterium
In contrast,
The aim of the research is to study the symptoms of bacterial wetwood disease of
The research was carried out in the forests of the state enterprise Kutske forestry and adjacent territories of the Ivano-Frankivsk region. According to geobotanical zoning, the research region is located within the Pokut-Bukovyna spruce–fir–beech and spruce–beech–fir forests, the sub-district of dark coniferous–beech watershed forests, the district of beech forests of the Ukrainian Carpathians (Golubec 2003; Plikhtyak 2019). According to forest zoning, the objects of research were within the forest region of the Outer Carpathians with beech and dark coniferous–beech forests, mountain Carpathian district, forestry region of the Ukrainian Carpathians (Hensiruk 1964).
Geobotanical and forestry descriptions of vegetation and flora inventory, sampling of wood, forest floor, bark, cones and seeds, mycothalluses of pathogenic and mycorrhizal macromycetes for laboratory studies were performed. In the process of research, 17 model trees of
The phytocenotic features of groups and macroscopic features of individual fir plants, which could pre-diagnose the pathogen without complex laboratory tests, were outlined based on the ecological–floristic classification of vegetation and the method of J. Braun-Blanquet (1964).
For bacteriological analysis, common silver fir wood samples with typical signs of bacterial wetwood disease were used. In particular, the material of the affected wood (on the border with externally healthy tissue) was selected for bacteriological analysis. Bacteriological analysis was performed by homogenising the plant material, followed by a culture test in Petri dishes on agar nutrient media and growing under thermostat conditions at 28°C for 4–5 days. In the process of conducting experimental studies (for the purpose of a detailed analysis of the characteristics of bacteria), microbiological inoculation was carried out in test tubes on agar media. Bacteria were Gram stained and tested for oxidase and protopectinase. Oxidase-negative isolated bacteria were investigated for their identification; their properties were studied and compared with the collection strain
The pathogenic properties of the isolates were tested
Latin names of higher plant species are given by The Plant List (
Massive dieback of
The next visual examination of the affected fir stands revealed the second group of evidence – macroscopic signs of bacterial wetwood disease, which was confirmed by laboratory studies. The symptomatology of bacterial wetwood disease in fir is similar to its manifestation at other woody plants (Cherpakov 2012, 2017; Goychuk et al. 2020b; Kulbanska et al. 2021). These signs include the following morphological and anatomical signs and structural modifications of the affected dieback develops from the upper part of the crown, which may indicate the spread of the inoculum, including wind; cracks and ulcers form on the trunks with exfoliated rhytidome, abundant protrusions of exudate appear, the primary bark and phloem are exposed, and in 2 years, the wound meristem appears (Fig. 1A, 1B); in the middle of hot summer, the bases of the trunks remain wet, which indicates a blockage of the xylem flow in the trunk (Fig. 1C); trees have a characteristic ‘hedgehog appearance’ due to the massive development of epicormic shoots that die during several vegetative periods (Fig. 1D); in the later stages of the disease, secondary pathogens – on the cross-section of the trunks, noticeable changes in anatomical structures appear: watery xylem and phloem, areas of wet rot with a characteristic odour of fermentation, pathological nucleus; the wood of the affected trees is very heavy and practically impossible to process due to tracheal obstruction and very high humidity.
Macro-signs of bacterial wetwood disease in
It is known that a whole complex of microorganisms from the genera
For bacteriological analysis, common silver fir wood samples with typical signs of bacterial wetwood disease were used (Fig. 2).
Wet ulcer on the trunk, under the bark of which dark brown affected areas of wood were found against the background of healthy light-coloured wood – A and affected (as if burned) vessels of the sapwood part of the knot – B
Bacteria that formed grey, translucent colonies with a slightly wavy edge were isolated from the affected tissues of common silver fir and used in further experiments (Fig. 3).
Bacterial colonies on potato agar isolated from common silver fir
The selected colonies of bacteria were similar to the colonies of the collection strain of the causative agent of bacterial wetwood disease
Colonies of the collection strain
In the course of the research, it was found that common silver fir isolates, as well as the collection strain
Cells of the causative agent of bacterial wetwood disease of common silver fir under a light microscope
The studied bacteria were facultative anaerobes, using glucose in both aerobic and anaerobic conditions. All studied bacterial isolates did not form gelatinase and pectinase (not able to cause a rot of potato pieces).
Physiological and biochemical properties of isolates fromwooden samples of fir (affection of type a) by using the TNEFERMtest24 MikroLaTEST®, ErbaLachema test system
Symbol | Test | Isolates from fir (4) |
---|---|---|
OXI | Oxidase | + |
URE | Urease | + |
ARG | Arginine | + |
ORN | Ornithine | + |
LYS | Lysine | − |
AAM | Acetamide | + |
βGL | β-Glucosidase | + |
NAG | N-acetyl-β- |
− |
SCI | Simpson citrate | + |
LAC | Lactose | − |
MAN | Mannitol | + |
TRE | Trehalose | + |
XYL | Xylose | + |
ARA | Arabinose | + |
αGA | α-Galactosidase | − |
βGA | β-Galactosidase | − |
MAL | Malonate | + |
GAL | Galactose | + |
MLT | Maltose | − |
CEL | Cellobiose | + |
SUC | Saccharose | + |
INO | Inositol | − |
γGT | γ-Glutamyl transferase | + |
PHS | Phosphatase | + |
ESL | Esculin | + |
Glucose (anaerobic) | − |
Studies of the biological properties of the isolated bacterial samples were performed using two test systems – NEFERMtest24 MikroLaTEST (Figs 4 and 5, Tab. 1) and ARI 20 E test system (Fig. 6, Fig. 7, Tab. 2) – in order to determine more characteristics of bacteria required for their identification.
Characteristics of F5 isolate which were determined by using the NEFERMtest24 MikroLaTE test system
Growth of bacterial isolates on glucose medium (anaerobic) using the NEFERMtest24 MikroLaTEST®, ErbaLachema test system
Physiological and biochemical properties of fir isolates (affection of type a, Fig. 2) (test system API 20 E)
Tests | Bacterial isolates |
---|---|
Yellow pigment | − |
Reduction of nitrates | + |
Formation of H2S | − |
Formation of indole | − |
β-galactosidase | − |
Arginine dehydrolase | + |
Lysine decarboxylase | + |
Ornithine decarboxylase | + |
The use of citrate | + |
Urease | − |
Tryptophan deaminase | + |
Voges–Proskauer reaction | + |
Gelatinase | + |
The use of glucose (anaerobic) | − |
Mannitol, inositol, sorbitol, rhamnose, saccharose | − |
Melibiose, amygdalin, arabinose | − |
Vials 1, 4, 7 – control, growth of bacteria in aerobic conditions; vials 2, 3, 5, 8 – growth of bacteria under a layer of oil (anaerobic conditions)
It was found that the isolated bacteria do not use lactose and glucose (anaerobically), like bacteria of the genus
Thus, bacteria isolated from the internal tissues of fir are not pathogens of bacterial wetwood, but are saprotrophic bacteria.
Identification of two isolates was performed by comparing their characteristics with the collection strain
Morphological and biochemical properties of isolates from fir
Tests | Bacterial isolates | |
---|---|---|
1 | 2 | 3 |
Gram's staining | − | − |
Yellow pigment | − | − |
Oxidase | − | − |
Reduction of nitrates | + | + |
Formation of H2S | − | − |
Formation of indole | − | − |
β-Galactosidase | + | + |
Arginine dehydrolase | − | − |
Lysine decarboxylase | − | − |
Ornithine decarboxylase | + | + |
The use of citrate | + | + |
Urease | − | − |
Tryptophan deaminase | + | + |
Voges–Proskauer reaction | + | + |
Gelatinase | − | − |
Pectinase activity | − | − |
The use of: Glucose (anaerobic) | + | + |
Mannitol | + | + |
Inositol | − | − |
Sorbitol | − | − |
Rhamnose | + | + |
Melibiose | + | + |
Amygdalin | + | + |
Arabinose | + | + |
Note: + presence of properties, − absence of properties, n/d − no data.
Characteristics of isolate 11 using API 20E test system
The bacteria do not use inositol and sorbitol and do not form indole and hydrogen sulphide (H2S), but are able to reduce nitrates. They lack arginine dehydrolase and lysine decarboxylase, but β-galactosidase and ornithine decarboxylase are present
According to the set of studied characteristics, bacterial isolates isolated from common silver fir are similar to the collection strain
Therefore, isolates of bacteria from the affected samples of common silver fir, identified based on morphological, physiological and biochemical properties as
A number of assumptions have been made about the causes of this phenomenon, including climate change and hydrothermal stress, invasive infectious agents and pests, but currently, each of the theories needs scientific confirmation (Gvozdyak and Yakovleva 1979; Patyka and Pasichnik 2014; Goychuk et al. 2020c).
Based on the studies of white fir stands within the Ukrainian Carpathians, we claim that the disease we have identified is systemic, vascular–parenchymal bacteriosis, known as bacterial wetwood disease of fir, which affects all tissues of plants and generative organs at all stages of ontogenesis.
The common manifestations of bacterial wetwood disease of common silver fir include the formation of separate foci of lesions, characterised by defoliation and death of annual shoots. Other symptoms may include bark cracks and deformation, dark mucous with an odour of butyric acid fermentation, necrotic wet wounds, epicormic shoots on the trunks and so on.
Summarising the obtained results, we can conclude that the most common and harmful component of the pathogenic microflora isolated from the lesion of common silver fir is bacteria, which, according to morphological, physiological and biochemical properties, are identified by us as
In the course of experimental studies during artificial infection with
Other research methods must be involved in the search for the root causes of the disease, particularly, phytocenotic, which allows revealing both the degree of alteration of forest coenosis and the direction of destructive and regenerative processes in it.