Induction of disease resistance in Norway spruce (Picea abies) by necrotizing fungi

Norway spruce trees ( Picea abies ) preinoculated with the root rot fungus Heterobasidion annosum , Nectria fuckeliana or a pathogenic strain of the blue-stain fungus Ceratocystis polonica were more efficiently protected against a subsequent massive inoculation with pathogenic C. polonica than trees pretreated with nonpathogenic C. polonica or sterile malt agar. Control trees that received no pretreatment were extensively colonized by the mass inoculation. There was a strong negative correlation between the length of the phloem necroses induced by the pretreatment inoculations and the extent of host symptoms caused by mass inoculation with pathogenic C. polonica . The degree of induced resistance in Norway spruce thus depended on the amount of host tissue destroyed by the pretreatment.     

Xylem sap flow of Norway spruce after inoculation with the blue-stain fungus Ceratocystis polonica

In a field experiment, the sap flow of Norway spruce ( Picea abies ) was measured in response to inoculation with Ceratocystis polonica , a virulent fungal associate of the spruce bark beetle Ips typographus. Four vigorous, mature Norway spruce trees were mass-inoculated with C. polonica at a density of four inoculations per dm² within a 120-cm-wide band on the lower bole. Two other trees were inoculated with sterile agar and served as controls. Sap flow of the six trees was measured by the thermal dissipation method from three weeks prior to inoculation until five months after inoculation. In three trees that had been inoculated with the fungus, sap flow density decreased dramatically within the first weeks after inoculation until no sap flow was measurable 4–6 weeks after inoculation. No marked changes in sap flow density were recorded in the fourth tree that had been inoculated with C. polonica , and this tree survived the inoculation. Inoculation with sterile agar did not have any apparent influence on the sap flow of the control trees. The results confirm the ability of C. polonica to kill mature Norway spruce trees. Sap flow measurements by the thermal dissipation method provided a direct and nondestructive method for monitoring the effects of fungal infection on sapwood functionality. Its application for further studies on vascular wilt and vascular stain diseases of trees is therefore recommended.     

Assessing the virulence of four bark beetle-associated bluestain fungi using Norway spruce seedlings

The virulence of two isolates of each of four different bark beetle-associated bluestain fungi was evaluated by wound-inoculating 2- and 4-year-old Norway spruce seedlings. One isolate of Ceratocystis polonica killed 40% of the 2-year-old plants and 20% of the 4-year-old plants, whereas the other fungi ( Ophiostoma piceae , Ophiostoma sp., an unidentified fungus with dark sterile mycelium) and the control treatments (inoculation with sterile agar and unwounded plants) did not kill any plants during the 11-week incubation period. Only C. polonica and the Ophiostoma sp. caused any bluestain of the sapwood. The two C. polonica isolates caused significantly deeper bluestain penetration into the sapwood and longer necrotic lesions on the sapwood surface than all other isolates. The symptoms caused by the other fungi were similar to those on the sterile inoculated control plants. The virulence of the fungal isolates tested in this study agrees largely with results from a previous mass inoculation study using the same isolates inoculated into 40-year-old Norway spruce trees. Thus, inoculation of seedlings seems to be a reliable, inexpensive and convenient bioassay for determining the virulence of bark beetle-associated bluestain fungi.     

Dieback and death of larch caused by Ceratocystis laricicola sp. nov. following attack by Ips cembrae

Circumstantial and experimental evidence suggests that the bark beetle Ips cembrae acts as a vector for Ceratocystis laricicola sp. nov. which it introduces into larches during breeding attacks. The fungus kills bark and cambium, and invades the sapwood causing the formation of areas of 'blue stain' surrounded by dry wood. Multiple inoculations resulting from numerous beetle attacks may totally disrupt conduction causing dieback and death of whole trees. Attacks seem frequently to be associated with drought and other forms of stress. Trees damaged by I. cembrae and C. laricicola may be attacked subsequently by the woodwasp Urocerus gigas which introduces a sapwood decay fungus, Amylostereum chailletii.
This is the first record of an association between I. cembrae and a species of Ceratocystis and the first record of dieback in larch caused by I. cembrae and C. laricicola. I. cembrae was introduced into Britain from mainland Europe and a similar association may occur there.     

Loss of pathogenicity in the blue-stain fungus Ceratocystis polonica

The pathogenicity and growth of six cultures of the bark-beetle-associated blue-stain fungus Ceratocystis polonica were compared. Four were subcultures, established at different times, of the same isolate (80–53/7), and at least one of these was suspected to have lost its pathogenicity to Norway spruce ( Picea abies ) during subculturing. Two other cultures (93–208/115 and 94–169/13) were pathogenic. The pathogenicity of all cultures to Norway spruce was compared in three inoculation experiments, using (i) massive inoculation (≈144 inoculations per tree), (ii) low-density inoculation (14 inoculations per tree) of larger trees and (iii) single inoculation of 2-year-old seedlings. Fungal growth rates were tested in vitro on malt agar, and on malt agar under oxygen-deficient conditions. Isolates 93–208/115 and 94–169/13 and one of the subcultures of isolate 80–53/7 were pathogenic in all three inoculation experiments. Two other subcultures were nonpathogenic, while the last subculture was intermediate. The pathogenic cultures had higher growth rates on malt agar than the nonpathogenic and intermediate cultures, and they also grew better under oxygen-deficient conditions. These results demonstrate that blue-stain fungi may lose their pathogenicity after serial vegetative transfers on artificial substrates. The results are also consistent with the hypothesis that rapid growth and the ability to grow in oxygen-poor environments are important pathogenicity factors in blue-stain fungi.     

Death of endemic Virgilia oroboides trees in South Africa caused by Diaporthe virgiliae sp. nov.

Numerous dead and dying individuals of the Western Cape endemic tree Virgilia oroboides (Fabaceae) were recently observed within a South African national botanical garden. Root‐rot fungi and fungi symbiotic with bark beetles (Curculionidae; Scolytinae) from diseased trees were assessed for their respective roles in V. oroboides mortality. Disease progression was also monitored over 1 year. Fungi were isolated from surface sterilized bark and root samples from diseased trees and provisionally identified using data from the internal transcribed spacer regions (ITS1, ITS2), including the 5·8S rRNA gene (ITS). Pathogenicity of selected fungi towards V. oroboides was tested under field conditions. The pathogenicity of various bark beetle‐associated Geosmithia (Hypocreales: Hypocreomycetidae) spp. from V. oroboides were similarly assessed. The only fungus consistently isolated from lesions on the roots and bark of declining V. oroboides, and never from healthy individuals, represented an undescribed Diaporthe (Diaporthales, Diaporthaceae) species that was characterized using molecular (using data from the ITS marker and part of the β‐tubulin gene, TUB), cultural and morphological characters. It is an aggressive pathogen of V. oroboides, newly described here as Diaporthe virgiliae sp. nov. Trees of all ages are susceptible to this pathogen with subsequent bark beetle attack of mature trees only. All Geosmithia spp. from beetles and/or infected trees were nonpathogenic towards V. oroboides. Diaporthe virgiliae caused a severe decline in the health of the monitored V. oroboides population over a period of only 1 year and should be considered as a significant threat to these trees.     

Bark beetle (Coleoptera, Scolytidae) outbreak and system of IPM measures in an area affected by intensive forest decline connected with honey fungus (Armillaria sp.)

The character of a bark beetle outbreak, planning system, and implementation of IPM measures in a forest affected by intensive decline connected with honey fungus (Armillaria sp.) is described. It is possible to distinguish two levels of outbreak in the study area. The first level is characterized by spruce mortality connected with yellowing of spruce and presence of plant pathogens, mostly Armillaria sp. Forest stands have disperse infestations of bark beetles. Identification of infested trees and salvage cutting in time are problematic. In case of late processing of infested trees, the second level of outbreak could become manifest. The populations of bark beetle increase. Beetles could attack relatively healthy trees. The classical outbreak with forest edges and spot infestations starts. The identification of infested trees is easier. The application of pheromone trap barriers is effective. Thus, the second level of outbreak could be easily managed. The planning of IPM measures is concerned with localization of various pheromone trap systems in particular forest stands. It is based on field survey, data acquisition, estimation of swarming bark beetle population, and information of pheromone system effectiveness.     

Bark beetle (Coleoptera, Scolytidae) outbreak and system of IPM measures in an area affected by intensive forest decline connected with honey fungus (Armillaria sp.)

The character of a bark beetle outbreak, planning system, and implementation of IPM measures in a forest affected by intensive decline connected with honey fungus (Armillaria sp.) is described. It is possible to distinguish two levels of outbreak in the study area. The first level is characterized by spruce mortality connected with yellowing of spruce and presence of plant pathogens, mostly Armillaria sp. Forest stands have disperse infestations of bark beetles. Identification of infested trees and salvage cutting in time are problematic. In case of late processing of infested trees, the second level of outbreak could become manifest. The populations of bark beetle increase. Beetles could attack relatively healthy trees. The classical outbreak with forest edges and spot infestations starts. The identification of infested trees is easier. The application of pheromone trap barriers is effective. Thus, the second level of outbreak could be easily managed. The planning of IPM measures is concerned with localization of various pheromone trap systems in particular forest stands. It is based on field survey, data acquisition, estimation of swarming bark beetle population, and information of pheromone system effectiveness.     

Aetiology and causal agents of mango sudden decline disease in the Sultanate of Oman

Mango sudden decline is a recently introduced, economically serious disease in Oman. Affected mango trees have wilting symptoms that usually begin on one side and later spread to involve the entire tree. Trees exude amber-coloured gum from the bark of their trunks or branches and vascular tissues are discoloured. Having entered Oman in the recent past, survey data is presented that shows the disease to have spread throughout the northern part of the country. Evidence is presented that the vascular wilt pathogen Ceratocystis fimbriata causes mango sudden decline disease in Oman, possibly in concert with Lasiodiplodia theobromae and the recently described Ceratocystis omanensis. Isolates of these fungi from affected trees, cause infection and can be recovered from inoculated seedlings. Bark beetles (Hypocryphalus mangiferae) are shown to carry C. fimbriata and L. theobromae and are presumably responsible for transmitting both pathogens to healthy mango trees. Acting as a wounding agent and vector, the bark beetle is likely to have assisted the rapid spread of the disease across Oman.     

Types of bark beetle (Coleoptera: Scolytidae) infestation in spruce forest stands affected by air pollution, bark beetle outbreak and honey fungus (Armillaria mellea)

583 spruce stands in an area affected by air pollution and bark beetle outbreak in Eastern Slovakia were studied in 1996. According to bark beetle infestation of dominant and codominant trees, stands were classified into following types of spruce stand decline:Ips typographus-A,Ips typographus-B,Polygraphus poligraphus, I. typographus/P. poligraphus—A,I. typographus/P. poligraphus—B. The presence of attacked trees in forest edges, bark beetle spots and forest interior was the key important factor for the classification. Data from forest inventory and forest management evidence together with data on types of spruce stands decline were used in further analyses. Results shows that the distribution of forest stands classified into different types or uninfested stands is related mainly to host size and site quality. The percentage of spruce, exposition of stands and stand density showed significant effects. The mechanisms of spreading of studied bark beetle outbreak could be explained by direct effects of stress of trees caused by an abrupt increase of level of solar irradiation and by weakening of trees by the honey fungus.     

Patterns of bark beetle occurrence in Norway spruce stands of national parks in Tatra Mts. in Poland and Slovakia

This paper presents the results of a research project concerning bark beetle populations in Norway spruce stands managed under a nature protection regime in national parks in Tatra Mountains in Poland and Slovakia. The preliminary data on bark beetle occurrence and related selected stand characteristics are presented. The dynamics of a bark beetle outbreak that occurred from 1994–97 in the stands localized on both sides of the state border and in various nature protection regimes is analyzed retrospectively. The data concerning the tree/stand characteristics, i.e. DBH and increment of trees are discussed in relation to its potential role in the mechanism for the spread of bark beetle populations. The patterns of the spatial distribution of trees attacked during the research period (1999–2001) are described.     

Patterns of bark beetle occurrence in Norway spruce stands of national parks in Tatra Mts. in Poland and Slovakia

This paper presents the results of a research project concerning bark beetle populations in Norway spruce stands managed under a nature protection regime in national parks in Tatra Mountains in Poland and Slovakia. The preliminary data on bark beetle occurrence and related selected stand characteristics are presented. The dynamics of a bark beetle outbreak that occurred from 1994–97 in the stands localized on both sides of the state border and in various nature protection regimes is analyzed retrospectively. The data concerning the tree/stand characteristics, i.e. DBH and increment of trees are discussed in relation to its potential role in the mechanism for the spread of bark beetle populations. The patterns of the spatial distribution of trees attacked during the research period (1999–2001) are described.     

Phytophthora ramorum Colonizes Tanoak Xylem and Is Associated with Reduced Stem Water Transport

ABSTRACT Isolation, detection with diagnostic polymerase chain reaction (PCR), and microscopy demonstrated the presence of Phytophthora ramorum in the sapwood of mature, naturally infected tanoak (Lithocarpus densiflorus) trees. The pathogen was strongly associated with discolored sapwood (P < 0.001), and was recovered or detected from 83% of discolored sapwood tissue samples. Hyphae were abundant in the xylem vessels, ray parenchyma, and fiber tracheids. Chlamydospores were observed in the vessels. Studies of log inoculation indicated that P. ramorum readily colonized sapwood from inoculum placed in the bark, cambium, or sapwood. After 8 weeks, radial spread of P. ramorum in sapwood averaged 3.0 to 3.3 cm and axial spread averaged 12.4 to 18.8 cm. A field study was conducted to determine if trees with infected xylem had reduced sap flux and reduced specific conductivity relative to noninfected control trees. Sap flux was monitored with heat-diffusion sensors and tissue samples near the sensors were subsequently tested for P. ramorum. Adjacent wood sections were excised and specific conductivity measured. Both midday sap flux and specific conductivity were significantly reduced in infected trees versus noninfected control trees. Vessel diameter distributions did not differ significantly among the two treatments, but tyloses were more abundant in infected than in noninfected trees. Implications for pathogenesis, symptomology, and epidemiology are discussed.     

Fungal Infection and Mechanical Wounding Induce Disease Resistance in Scots Pine

Scots pine trees (Pinus sylvestris) recovering from a 90–100% defoliation 2–3 years previously were pretreated with small mechanical wounds or inoculations with the blue-stain fungi Leptographium wingfieldii and Ophiostoma canum. Pretreated trees were less susceptible to a subsequent massive inoculation with L. wingfieldii than untreated control trees, which were extensively colonised by the mass-inoculation. A low pretreatment dosage of L. wingfieldii was somewhat more effective in inducing disease resistance than a higher dosage. Pretreatment with L. wingfieldii, O. canum, and mechanical wounding were about equally effective inducers of resistance in Scots pine, even though L. wingfieldii is known to produce much more extensive phloem necrosis than the other pretreatments. Thus, the strength of the induced resistance response did not depend on the amount of host tissues that was destroyed by the pretreatment. Previously, induced disease resistance has been demonstrated in Norway spruce (Picea abies), and the present study shows that similar responses can be activated in Scots pine.     

A two-year study of parasitoid entomofauna associated with spruce bark beetles (Coleoptera: Curculionidae) in the altimontane belt of Slovenia (Pohorje)

Parasitoids are known to be an important factor in the population density regulation of bark beetles. In our study of the parasitoid complex of spruce bark beetles in 2011 and 2012, we collected bark samples from trees infested with I. typographus from spruce stands in the altimontane belt of Slovenia. The samples were taken at different altitudes: 450, 700 and 1230 m above sea level. A total of 9 bark beetle species and 12 parasitoid species was found. Ips typographus was most abundant, with 93% of all species of scolytids. The parasitoid species that were found belonged mainly to the Pteromalidae and Braconidae families. By far the most abundant and frequent species was Roptrocerus xylophagorum, which represented 84% of all parasitoids and was present in all study samples. Differences in species composition, parasitoid species diversity, species abundance and parasitism rate were confirmed between locations and also between both years of our study.     

Untersuchungen zur Fangleistung von Schlitzfallen und Fangholzhaufen bei der lokalen Dichteabsenkung von Buchdrucker (Ips typographus)-Populationen

Two kinds of pheromone-based trap systems are used in the integrated system against the European Spruce Bark Beetle: a standardized commercial slot-trap and an attract-and-kill system using insecticide-coated spruce wood. To determine the individual benefits and limits in both systems, a comparative survey has been done in 2008, respecting the slightly different requirements for deploying both systems with maximum efficiency. The observed results showed that both systems are able to attract large numbers of spruce bark beetles, however the wooden attract-and-kill system removed both larger numbers and higher percentages of the attracted individual beetles. During the survey, the wood traps were able to incapacitate between 80 and 90?% of the attracted beetles, while the slot-traps kept only 50?C60?% contained. While the use of a non-specific insecticide in a freely accessible trap system always bears the risk of attracting and killing individuals of non-targeted species, the survey gave no indication that specialised antagonists of the spruce bark beetle or other non-target-species has been overly affected. The percentage of non-target-organisms has been 0.3?% in total. It was concluded that, while both systems have the capability to reduce the local population of European Spruce Bark Beetles, the natural wood trap system using an insecticide has the ability to control a much larger population and should therefore be considered the primary choice if the conditions on the site indicating a high risk of a continuous bark beetle infestation.     

Real-time PCR-based monitoring of DNA pools in the tri-trophic interaction between Norway spruce, the rust Thekopsora areolata, and an opportunistic ascomycetous Phomopsis sp

The difficulty in subculturing biotrophic fungi complicates etiological studies related to the associated plant diseases. By employing internal transcribed spacer rDNA-targeted quantitative real-time polymerase chain reaction, we now show that the heteroecious rust Thekopsora areolata, commonly associated in natural conditions to sapling shoots and cones of Norway spruce and leaves of wild bird cherry, frequently infects nursery-grown seedlings of the conifer. A spatial sampling scheme was used to investigate seedlings and saplings of Norway spruce showing phloem necrosis: the highest concentration of DNA of T. areolata was recorded in the area with necrotic phloem. The separate analysis of bark and wood tissues suggested that the initial spread of the rust to healthy tissues neighboring the infection site takes place in the bark. A Phomopsis species found to coexist with T. areolata in several seedlings showed very high DNA levels in the upper part of the lesion, and even in the visually healthy proximal tissues above the lesions, which indicates that the ascomycete, most probably a secondary invader following primary infection by T. areolata, has a latent stage during early host colonization. We hypothesize that this hemibiotrophic mode of infection contributes to the successful coexistence of Phomopsis with a biotrophic rust.     

A method for the protection of spruce stands againstIps typographus by the use of barriers of pheromone traps in north-eastern Slovakia

A method and layout of various systems of barriers of pheromone traps is described. The rationale of the method is based on the stress of spruce and the chemical communication ofIps typographus. Various systems of pheromone trap barriers were used according to the required level of protection. The method was used in two areas affected by air pollution and bark beetle outbreak. The first area was in an unnatural spruce monoculture region while the second was in a National Park. The described method led to a significant reduction of bark beetle attacks on trees along forest edges and subsequently to the stopping of movement of the zone of attacked trees due to bark beetle damage. In the case of forest stands with no reduced canopy closure, the reduction of attacks led to a dramatic decline of bark beetle outbreak. On the other hand, this method was not able to reduce the level of bark beetle attack in the interior of forest stands with a decreased level of canopy closure. This method, as part of differentiated pest management measures in one forest district, was successful without any other protection measures in the condition of natural reserves in a spruce vegetation zone.