Individual resistance of Fraxinus angustifolia clones to ash dieback

The presumed resistance of individual ash trees to ash dieback caused by invasive pathogen Hymenoscyphus fraxineus is an important issue for the maintenance of ash in European forests. All known studies regarding the resistance of ash trees to ash dieback were conducted in plantations and stands of F. excelsior; however, no such data exist for F. angustifolia. Crown damage assessments were performed over four consecutive years between 2009 and 2012 at a F. angustifolia clonal plantation in Hra??ica, Slovenia. Inoculation of H. fraxineus into the branches of the most and least damaged clones of F. angustifolia and leaf phenology assessments was performed to verify the presence of defence mechanisms that limit fungal growth or promote disease escape. Additionally, root collars of selected clones were inspected for fungal infections. The crown damage assessments showed considerable differences among F. angustifolia clones, indicating genetic variability in susceptibility to ash dieback. Crown dieback progressed significantly over the 4‐year time period; the mean crown damage of individual clones in 2012 varied between 16.7% and 83.8%. Significant differences among F. angustifolia clones were found in the inoculation trials and leaf phenology assessments. However, defence mechanisms such as early leaf flushing, early leaf shedding and the ability to inhibit pathogen growth in host tissues were not confirmed. High frequency of Armillaria spp. and H. fraxineus root collar infection demonstrated the need for whole tree inspection to determine causal agent of damages on individual ash trees. Armillaria spp. may be highly associated with ash decline epidemiology.     

Methods of inoculation and evaluation of Erwinia psidii in eucalypt

The dieback and wilting caused by Erwinia psidii are emerging eucalypt diseases that have been observed since 2014 in the south and central‐south regions of Brazil. Field observations have shown variability in disease severity resistance among Eucalyptus spp. clones and species. It is hypothesized that this variability is due to genetic resistance. To confirm this hypothesis, inoculations in genetically distinct eucalypt plants are necessary. However, lack of an inoculation method and disease assessment makes difficult to select resistant genotypes for use in commercial plantations or genetic breeding programmes. Three inoculation methods were tested on eight clones of Eucalyptus spp. Among them, inoculum deposition with bacteria‐impregnated toothpick on the axillary buds was the simplest and most effective, capable to reproduce the disease symptoms observed under conditions of natural infection. We also developed a rating scale for disease assessment. Among eight clones tested, only Clone 1 (Eucalyptus saligna) and Clone 2 (Eucalyptus urophylla) were resistant.     

Effect of Betula pendula clone and leaf age on Melampsoridium betulinum rust infection in a field trial

Ten 5‐year‐old Betula pendula clones were studied for their rust resistance in the field. The trees were treated by inoculating 10 leaves on a shoot with Melampsoridium betulinum urediniospore suspension or spraying the control leaves with water. The birch clones differed significantly in their resistance to M. betulinum leaf rust fungus and the clones also varied in their responses to the local rust strain and the inoculated rust strains. However, natural rust infections and inoculation treatment were positively correlated. The older leaves had fewer infections than the younger ones on the tip of the shoot in the control trees, but in the inoculation treatment no significant correlation was found between the leaf ages and rust infection. The factors behind the different leaf susceptibilities are discussed.     

Genetic resistance to Hymenoscyphus pseudoalbidus limits fungal growth and symptom occurrence in Fraxinus excelsior

The European common ash (Fraxinus excelsior) is currently threatened by a pathogenic fungus, Hymenoscyphus pseudoalbidus, which seems to enter the trees through the leaves. Continuous assessments of 39 clones in Danish field trials have shown that there are significant differences in the susceptibility of clones to the new disease. Interestingly, clones that showed early leaf senescence in the autumn were in general less susceptible to the disease than late‐senescing clones. Thus, variation in susceptibility could be owing to phenological differences associated with the infection biology. To test whether differences in susceptibility are driven by genetically based factors other than phenology, we compared inoculations with H. pseudoalbidus on four highly susceptible clones with those of four less susceptible clones. Development of necrosis was hereafter followed regularly. The growth of the fungus in the inner bark was further detected with species‐specific PCR primers. The severity of the response to infection shows significant differentiation among clones and significant correlation with clone susceptibility, as assessed from natural infections in field trials. The fungus was detected in tissues immediately surrounding the necrosis but showed some signs of endophytic growth. The results suggest that healthier clones are able to limit the growth and spread of the fungus and thereby minimize the occurrence of symptoms. This gives hope for the future preservation of F. excelsior in Europe through selection and breeding.     

Effectiveness of a detached‐leaf assay as a proxy for stem inoculations in backcrossed chestnut (Castanea) blight resistance breeding populations

A recently developed detached‐leaf blight resistance assay has generated interest because it could reduce the amount of time needed to evaluate backcrossed hybrid trees in the American chestnut blight resistance breeding programme. We evaluated the leaf inoculation technique on a sample of advanced progeny from the Indiana state chapter American Chestnut Foundation breeding programme, along with susceptible American chestnut (Castanea dentata), the recurrent parent, and resistant Chinese chestnut (Castanea mollissima), the donor parent for blight resistance. In experiments over 2 years using two pathogen isolates, we found no biologically meaningful relationship between leaf lesion size and the size (length and width) or severity (1–5 canker severity rating) of stem cankers on 5‐year‐old trees. Chinese chestnuts did develop significantly smaller leaf lesions than American or backcrossed chestnuts. We conclude that while the detached‐leaf assay may have utility in some chestnut breeding applications, it is not a suitable proxy for the established practice of stem inoculations.     

Xyleborus glabratus attacks and systemic colonization by Raffaelea lauricola associated with dieback of Cinnamomum camphora in the southeastern United States

Laurel wilt, caused by Raffaelea lauricola, is responsible for extensive mortality of redbay and other American members of the Lauraceae in the southeastern United States. Raffaelea lauricola is a mycangial symbiont of the redbay ambrosia beetle (Xyleborus glabratus), and the beetle and fungus were accidentally introduced from Asia. Branch dieback of camphortree (Cinnamomum camphora), an Asian member of the Lauraceae, has been occasionally observed in areas where laurel wilt has decimated redbay populations, and R. lauricola was isolated from such camphortrees. However, the role of X. glabratus and R. lauricola in this branch dieback remains unclear. Examination of camphortrees on Jekyll Island, Georgia showed that healthy‐appearing trees and those with branch dieback had been attacked by X. glabratus, but the trees with branch dieback had four times as many beetle attacks. Raffaelea lauricola was routinely isolated from discoloured xylem near beetle tunnels in healthy trees and those with dieback. Single‐point inoculations with R. lauricola on stems of mature, healthy camphortree trees failed to induce wilt‐like symptoms or branch dieback, although areas of discoloration were scattered throughout the xylem, and R. lauricola was reisolated irregularly at various heights in some inoculated trees. In growth chamber experiments, single‐point inoculations with R. lauricola resulted in systemic colonization but no wilt symptoms or branch dieback in camphortree saplings. In contrast, inoculations at multiple points along the stem (simulating multiple attacks by the vector) caused branch dieback and wilt‐like symptoms, including a brownish, diffuse discoloration of the xylem. Camphortree appears to be more resistant than American species of Lauraceae to the vascular wilt caused by R. lauricola. The fungus does colonize camphortrees systemically, however, and can apparently cause branch dieback. This suggests that the fungus may provide brood material for X. glabratus in Asia as it does in the southeastern United States.     

Competition as a predisposing factor of crown dieback in a secondary forest of <Emphasis Type="Italic">Betula maximowicziana</Emphasis> in Hokkaido,northern Japan

We analyzed the probability that Betula maximowicziana Regel (monarch birch) would suffer crown dieback (crown-dieback probability) and the basal area growth rate (GB), which was found to be a predisposing stress factor making birch trees susceptible to crown dieback. First, we analyzed the relationship between the probability that birch trees would suffer from crown dieback in 1999 and GB from a period prior to the occurrence of crown dieback (1985–1987), using a data set of repeated measurements on 217 trees. Logistic regression analysis revealed that monarch birch had a larger crown-dieback probability when GB was low in the preceding period. Hence, there were predisposing stress factors that reduced GB and continued to affect trees for at least a decade. Next, we analyzed GB in the same period in relation to symmetrical and asymmetrical competition between trees and found that GB was reduced by symmetrical competition, suggesting that this was one of the predisposing factors for crown dieback. Based on these results, we used selected models for crown-dieback probability and GB to calculate crown-dieback probabilities for individuals with different initial basal areas and experiencing different intensities of symmetrical competition. The predicted crown-dieback probability decreased with decreasing symmetrical competition between trees. We discuss a possible process of crown dieback to death for monarch birch and the use of thinning as a method to reduce the risk of crown dieback.     

Genetic variation in dieback resistance: growth and survival of Fraxinus excelsior under the influence of Hymenoscyphus pseudoalbidus

The invasive fungal pathogen, Hymenoscyphus pseudoalbidus V. Queloz, has decimated stands of Fraxinus excelsior L. over most of the species' natural distribution area. We assessed crown damage from 2009 to 2014 (at ages 8, 11 and 13) in 43 open-pollinated ash families planted in north-eastern Zealand, Denmark, and confirmed the presence of substantial genetic variation in ash dieback susceptibility. The average crown damage increased in the trial from 61% in 2009 to 66% in 2012 and 72% in 2014, while the estimated heritability was 0.42 in both 2009 and 2012 but increased to 0.53 in 2014. Genetic correlation between assessments was 0.88 between 2009 and 2012 and 0.91 between 2009 and 2014, suggesting fairly good possibilities for early selection of superior genotypes in the presence of high infection levels in the trial. The level of crown damage had strong negative effect on growth and survival. Only 34% of the trees with high levels of damage in 2009 were still alive in 2014, emphasising that high susceptibility is associated with low fitness.     

Induced resistance of Norway spruce,variation of phenolic compounds and their effects on fungal pathogens

Three clones of Norway spruce (Picea abies) were studied for their response to mass‐inoculation with the blue‐stain fungus Ceratocystis polonica. The effect of different pretreatments (fungal inoculation and wounding) before mass‐inoculation was investigated for their possible role in an acquired resistance reaction. Pretreated trees showed enhanced resistance to the subsequent mass‐inoculation relative to control trees that received no pretreatment. Furthermore, the fungal colonization of inoculated trees was less than that of wounded trees. The phenolic content of the bark, analysed by RP‐HPLC, was compared in trees receiving different treatments. Trees inoculated with C. polonica had higher average concentration of (+)‐catechin, taxifolin and trans‐resveratrol than wounded trees. Both inoculated and wounded trees had higher average concentrations of these compounds than control trees. The effect of the phenolic extract of Norway spruce bark on the growth of the root rot fungus Heterobasidion annosum and the blue‐stain fungi C. polonica and Ophiostoma penicillatum were investigated in vitro. Heterobasidion annosum was not negatively affected, and the extracts had fungistatic effects on the blue‐stain fungi. The growth of O. penicillatum was more inhibited than the growth of the more aggressive C. polonica.     

Physiological and biochemical differences among Ulmus minor genotypes showing a gradient of resistance to Dutch elm disease

Dutch elm disease (DED) spread across Europe and North America in the 20th century killing most natural elm populations. Today, breeding programmes aim at identifying, propagating and studying elm clones resistant to DED. Here, we have compared the physiology and biochemistry of six genotypes of Ulmus minor of variable DED resistance. Leaf gas exchange, water potential, stem hydraulic conductivity and biochemical status were studied in 5‐year‐old trees of AB‐AM2.4, M‐DV2.3, M‐DV2 × M‐CC1.5 and M‐DV1 and 6‐year‐old trees of VA‐AP38 and BU‐FL7 before and after inoculation with Ophiostoma novo‐ulmi. Leaf water potential and net photosynthesis rates declined, while the percentage loss of hydraulic conductivity (PLC) increased after the inoculation in susceptible trees. By the 21st day, leaf predawn and midday water potential, stomatal conductance to water vapour and net photosynthesis rates were lower, and PLC was higher in trees of susceptible (S) genotypes inoculated with the pathogen than in control trees inoculated with water, whereas no significant treatment effect was observed on these variables in the resistant (R) genotypes. Fourier transform infrared spectroscopy analyses revealed a different biochemical profile for branches of R and S clones. R clones showed higher absorption peaks that could be assigned to phenolic compounds, saturated hydrocarbons, cellulose and hemicellulose than S clones. The differences were more marked at the end of the experiment than at the beginning, suggesting that R and S clones responded differently to the inevitable wounding from inoculation and repeated sampling over the experimental course. We hypothesize that a weak activation of the defence system in response to experimental wounding can contribute to the susceptibility of some genotypes to O. novo‐ulmi. In turn, the decline in shoot hydraulic conductivity and leaf carbon uptake caused by the infection further exacerbates tree susceptibility to the fungus.     

Lesion development in stems of rough‐ and smooth‐barked Eucalyptus nitens following artificial inoculations with canker fungi

A study of lesion development in stems of Eucalyptus nitens following artificial inoculations with canker fungi was carried out on 16‐year‐old plantation trees. In a first trial cambium bark wounds on smooth‐ and rough‐barked trees were inoculated with the mycelium of nine species of canker fungi, including Endothia gyrosa. In a second trial spores or mycelium of E. gyrosa were applied directly onto undamaged or superficially wounded bark surfaces. Infection subsequent to artificial inoculation via wounding (whatever the wounding technique or type of inoculum) resulted in significantly larger external lesions (mean lesion area up to 35.6 cm² 20 months after inoculation) on smooth bark compared with those on rough bark (up to 19.0 cm²). Microscopic studies of infected rough and smooth bark suggest that, once smooth bark is compromised by wounding and artificial inoculation, the particular anatomical structure of smooth bark may offer less mechanical resistance to post‐penetration hyphal spread in comparison with rough bark. It is suggested that at a pre‐penetration stage under natural conditions spores of E. gyrosa more easily infect rough bark via cracks associated with this type of bark but not present in smooth bark.     

湿地松改良种子园部分无性系的早期遗传评价

利用湿地松改良种子园的建园无性系生产的8个全同胞家系和20个自由授粉家系苗木共营建测定林3块。3－4年生的测定林数据分析结果表明，参试的自由授粉家系作全同胞家系的平均生长量显著地大于台山湿地松初级种子园的，20个自由授粉家系的树高、胸径、材积平均增益分别为5．54％，7．77％和23．41％，初步实现了建园目标；同一无性系的不同采种年份的自由授粉子代生长存在着差异，有随着种子园母树年龄的增大而表现渐佳的趋势；改良园中还存在着少量的误选无性系，需要继续作测定和淘汰。从无性系亲子的生长量相关分析中发现，生长较差的自由授粉家系，较大部分产自生长量缩小的母本无性系。     

Temporal evolution of collar lesions associated with ash dieback and the occurrence of Armillaria in Belgian forests

Ash dieback, caused by the fungus Hymenoscyphus fraxineus, has been observed in Europe for several years. In Belgium, the disease was first reported in 2010. Besides crown defoliation and dieback, collar lesions have sometimes been reported. To evaluate the prevalence and the progression of collar lesions and crown defoliation in ash dieback‐affected stands of various ages, a survey was conducted in 2013 and 2014 on 268 ash trees (Fraxinus excelsior) originating from 17 Walloon forest stands. The results showed that the proportion of trees with collar lesions greatly increased between June 2013 and September 2014 and that there appeared to be no significant link between a tree's diameter‐at‐breast height (DBH) and collar lesion occurrence. The mean percentage of defoliation increased in each forest stand across time, with observations conducted in September 2013 and 2014 showing a positive correlation with the mean percentage of trees with collar lesions. Molecular tests were carried out on 103 additional trees originating from 12 of the 17 stands to evaluate the occurrence of H. fraxineus and Armillaria spp. at the collar level. Most of the trees (98%) were infected by H. fraxineus. In contrast, only 41% of the samples were infected with Armillaria spp., most commonly A. gallica and A. cepistipes. This study discusses the role of Armillaria spp. and the rapid increase in the number of trees with collar lesions within the context of the evolution of ash dieback in Europe.     

Oak mortality associated with crown dieback and oak borer attack in the Ozark Highlands

Oak decline and related mortality have periodically plagued upland oak–hickory forests, particularly oak species in the red oak group, across the Ozark Highlands of Missouri, Arkansas and Oklahoma since the late 1970s. Advanced tree age and periodic drought, as well as Armillaria root fungi and oak borer attack are believed to contribute to oak decline and mortality. Declining trees first show foliage wilt and browning, followed by progressive branch dieback in the middle and/or upper crown. Many trees eventually die if severe crown dieback continues. In 2002, more than 4000 living oak trees ≥11 cm dbh in the relatively undisturbed mature oak forests of the Missouri Ozark Forest Ecosystem Project (MOFEP) were randomly selected and inventoried for tree species, dbh, crown class, crown width, crown dieback condition (healthy: <5% crown dieback, slight: >5–33%, moderate: 33–66%, and severe: >66%) and number of emergence holes created by oak borers on the lower 2.4 m of the tree bole. The same trees were remeasured in 2006 to determine their status (live or dead). In 2002, about 10% of the red oak trees showed moderate or severe crown dieback; this was twice the percentage observed for white oak species. Over 70% of trees in the red oak group had evidence of oak borer damage compared to 35% of trees in the white oak group. There was significant positive correlation between crown dieback and the number of borer emergence holes (p < 0.01). Logistic regression showed oak mortality was mainly related to crown width and dieback, and failed to detect any significant link with the number of oak borer emergence holes. Declining red oak group trees had higher mortality (3 or 4 times) than white oaks. The odds ratios of mortality of slightly, moderately, and severely declining trees versus healthy trees were, respectively, 2.0, 6.5, and 29.7 for black oak; 1.8, 3.8, and 8.3 for scarlet oak; and 2.6, 6.5 and 7.1 for white oaks.     

Differences in susceptibility to ash dieback in Czech provenances of Fraxinus excelsior

The impact of ash dieback caused by Hymenoscyphus fraxineus on 17 provenances of Fraxinus excelsior and one provenance of Fraxinus angustifolia was studied in an extensive field trial established in the Czech Republic prior to the H. fraxineus invasion in 1999. A difference in the level of resistance to ash dieback between the species was found: F. angustifolia was significantly less affected by the disease than F. excelsior. Moreover, particular provenances of F. excelsior showed important differences in the level of resistance to H. fraxineus. A relationship between the impact of ash dieback and altitude was also discovered – the provenances from altitudes above 600 m a.s.l. were less affected by the pathogen than were the provenances from lower areas. No difference in the impact of the disease among provenances of F. excelsior from different ecotopes (ravine, calcareous ravine and alluvial) was found. Substantial among‐tree variability in resistance to H. fraxineus was observed throughout the trial – promising genotypes (with crown defoliation up to 5%) were identified in all 18 tested provenances. In regard to this finding, it appears that the main source of resistance to the pathogen is probably at the individual genotype level in the trial. A secondary but massive attack by Hylesinus fraxini was identified in the trees that had been greatly damaged by ash dieback, and the beetle caused their health to deteriorate significantly. A significant negative effect of the presence of collar necroses caused by H. fraxineus and browse damage was also identified.     

Identifying Fraxinus excelsior tolerant to ash dieback: Visual field monitoring versus a molecular marker

Ash dieback (ADB) caused by the pathogen Hymenoscyphus fraxineus is the cause of massive mortality of Fraxinus spp. in Europe. The aim of this work was to check for the presence of the molecular marker for ADB tolerance in mapped healthy‐looking F. excelsior trees, and to compare its occurrence in trees exhibiting severe ADB symptoms. Monitoring of 135 healthy‐looking F. excelsior on the island of Gotland, Sweden, showed that after 3–4 years 99.3% of these trees had 0%–10% crown damage, thus remaining in a similar health condition as when first mapped. After 5–6 years, 94.7% of these trees had 0%–10% crown damage. Molecular analysis of leaf tissues from 40 of those showed the presence of the molecular marker in 34 (85.0%) trees, while it was absent in 6 (15.0%) trees. Analysis of leaf tissues from 40 severely ADB‐diseased trees showed the presence of the molecular marker in 17 (42.5%) trees, but its absence in 23 (57.5%) trees (p < .0001). The results demonstrated that monitoring of healthy‐looking F. excelsior is a simple and straightforward approach for the selection of presumably ADB‐tolerant ash for future breeding. The cDNA‐based molecular marker revealed moderate capacity on its own to discriminate between presumably ADB‐tolerant and susceptible F. excelsior genotypes.     

Pathogenicity of ophiostomatoid fungi on Picea abies in Slovenia

Pathogenic fungi can survive and develop in living plants, often causing diseases in the host. Some theories speculate that pathogenic ophiostomatoid fungi provide benefits to its vectors – bark beetles – by overcoming the tree's defence mechanisms. This study reports the results of an experiment in south‐eastern Europe in which mature and seedling Norway spruce trees were artificially inoculated with various ophiostomatoid fungi. The aim of the experiment was to determine the relative virulence of ophiostomatoid fungi by assessing the ability of the fungi to stimulate host tree defence mechanisms through inoculation experiments. Experiments were performed by inoculation of Picea abies in seedling and mature trees. The following fungi were used in low‐density and seedling inoculations: Ophiostoma ainoae, O. brunneo‐ciliatum, Grosmannia cucullata and an unidentified Leptographium sp., O. bicolor, O. fuscum, O. piceae, G. penicillata and G. piceiperda. Endoconidiophora polonica was used in mass and seedling inoculations. Various characteristics such as host vitality, blue stain, lesion and resin outflow were measured before and after the trees were felled. E. polonica caused blue stain, induced large lesions and killed some of the mature trees and seedlings, confirming earlier reports that it is a strong wound pathogen. Only E. polonica, Leptographium sp. and O. ainoae caused blue stains in the sapwood of inoculated seedlings. In low‐density inoculations, G. piceiperda induced intense necrosis and had higher values for all the characteristics monitored. Some of the other ophiostomatoid fungi showed a moderate level of pathogenicity. Fungi with the capacity to stimulate a host defence mechanism could play a role in the establishment of bark beetle populations.     

Genetic variability of the ‘bark canker resistance’ character in several natural provenances of Cupressus sempervirens

Several Aegean (Greece) and Anatolian (Turkey) cypress provenances were studied for resistance variability to bark canker, a disease caused by the fungal pathogen Seiridium cardinale. The investigation also examined whether the low disease rate within the natural area of cypress was due to genetic or geographic‐climatic reasons. Results demonstrated strong variability for the ‘bark canker resistance’ character, in particular for trees within families. As trees from the provenances studied were not found to have genetic superiority for bark canker resistance, the above‐mentioned low disease rate could be due to geographic‐climatic barriers that inhibit the development of the fungus or its ability to infect the host. Several half‐sib progenies exhibited high resistance, suggesting that this character is totally inherited through the maternal line. Should this finding be confirmed by further research, it would facilitate the task of genetic improvement for resistance, allowing progenies of resistant trees to be obtained.     

An assessment of heterozygosity and fitness in Chir pine (<Emphasis Type="Italic">Pinus roxburghii</Emphasis> Sarg.) using isozymes

A correlation between heterozygosity of genotypes and survival (fitness) was studied in eight natural populations of Pinus roxburghii through isozyme analysis using horizontal starch gel electrophoresis. Under each population, 20 mother trees of different ages (30 to more than 100 years) were selected which were at least 50 m apart. From each tree, eight seeds were assayed for eleven enzyme systems viz., Aconitase, Aspertate aminotransferase, Glutamate dehydrogenase, Isocitrate dehydrogenase, Leucine-amino peptidase, Malate dehydrogenase, Menadione reductase, Phosphoglucose isomerase, Phosphoglucomutase, 6-Phosphogluconate dehydrogenase and Shikimic acid dehdrogenase which were found to be encoded by 18 polymorphic loci. Genetic constitution of the mother trees was determined by analysing endosperms and embryos separately. The number of alleles in the progeny (embryos) and the mother trees varied from 38 to 42 and 34 to 37, respectively, in all the populations. Distribution of degree of heterozygosity and fixation index for the progeny and the mother trees were calculated. The mother trees showed a substantial shift towards higher degree of heterozygosity as compared to the progeny. Fixation index values were significantly higher and negative for the mother trees as compared to the progeny, which revealed that heterozygosity was positively correlated with survival in P. roxburghii. Application: Heterozygous geneotypes in tree species are shown to have better survival over homozygotes under diverse environmental conditions. Every year, millions of Pinus roxburghii saplings are planted in India. However, the fact that survival is very low might possibly be due to inbred/homozygous plants that suffer from fitness disadvantage. Our study indicates that heterozygotes have a better chance of survival under environmental conditions. The use of heterozygotes should lead to effective establishments of Pinus roxburghii plantations.