Experimental testing of rust fungus‐mediated herbivory resistance in Betula pendula

Correlative evidence suggests that pathogenic leaf rust fungus, Melampsoridium betulinum, in late summer may negatively affect folivorous insects in the following summer. Correlative association does not necessarily, however, reveal causality. Alternatively, other interconnected plant characters may determine rust densities and herbivore performance. In this study, we used birch clones and rust fungus inoculations to manipulatively test the effects of birch rust and birch genotypes on the growth performance of folivorous moth larvae of Epirrita autumnata (Lep. Geometridae) in the subsequent year. The inoculation treatment increased rust densities (three‐ to 60‐fold) compared with natural infection levels. E. autumnata performance varied among birch clones and showed 4% lower growth performance on rust‐inoculated trees. However, the larval performance did not differ between rust‐treated shoots and untreated control shoots and the use of tree‐specific rust densities as a covariant in statistical analyses failed to reveal any negative association between rust fungus and larval performance. As the slight difference in larval growth performance also levelled until pupation, we propose that rust infection has biologically insignificant importance to the performance of E. autumnata.     

Variation in Melampsoridium resistance among European white-birch clones grown in different fertilization treatments

Resistance of 2-year-old plantlets of seven European white birch, Betula pendula, clones to birch rust, Melampsoridium betulinum, was studied in field experiments and in a leaf-disc bioassay. In addition, rust resistance of plantlets growing in a nursery under three fertilization treatments was tested. The birch clones clearly varied in their levels of resistance. One of the clones was consistently the most resistant, and two were very susceptible. Plantlets growing in the lowest fertility treatment were the most resistant. Clone × fertilization interaction was small. The plantlets grown in the lowest fertilization treatment, in particular, deviated from the other treatments. Generally good rust-resistance correlations were obtained between different experiments. The leaf-disc bioassay was an effective way of determining the field rust resistance of birch clones. The possibility of trade-offs between rust resistance and tree growth is discussed.     

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.     

Genetic variation in dieback resistance in Fraxinus excelsior confirmed by progeny inoculation assay

Ash dieback caused by the pathogenic fungus Hymenoscyphus fraxineus [previously known as H. pseudoalbidus (sexual stage) and Chalara fraxinea (asexual stage)] is a widespread problem in Europe. Here, we assess crown damage from natural infection and necrosis development following artificial controlled inoculations on full‐sib and half‐sib progeny from Danish Fraxinus excelsior clones with contrasting and well‐characterized levels of susceptibility to the disease. The inoculation assay was performed on a total of 123 offspring, and necrosis development monitored over two years. The offspring from low susceptible mother clones developed smaller necroses when compared to offspring from susceptible clones. Their crown damage due to natural infections was also significantly less. The correlation coefficient between average crown damages of mother clones and the average of their progeny was 0.85 (natural infections), while the correlation between crown damage of mother clones and the average necrosis development in their progeny after controlled inoculation was 0.73. The correlation between resistance of parent trees and crown damage/necrosis development on their offspring confirms the presence of heritable resistance and indicates that a bioassay based on controlled inoculations has the potential of becoming a fast and cost‐effective tool for estimation of dieback susceptibility in breeding programmes for resistance in ash trees.     

Rust severity in bioenergy willow plantations treated with additional nutrients

A 3‐year field study was carried out to determine the effect of wastewater irrigation and previous differences in mineral fertilization on the occurrence of willow leaf rust (Melampsora epitea). The experiment was conducted in two energy forest plantations: one designed for wastewater purification and the other as a mineral fertilization experiment. The severity of leaf rust on different clones and sites with different treatments was assessed by counting the number of uredinia per leaf unit area. Generally, plants irrigated with wastewater consistently had more leaf rust, irrespective of the study years or willow clones. Previous mineral fertilization had mixed effects on different clones 2 years after the last application. Three years after the last fertilizer application, however, no impact of the treatment on rust disease development was detected. In general, the rust levels differed from year to year probably due to climate. In this study, no correlation was detected between shoot age and rust severity, whereas climate and treatments strongly influenced leaf rust levels on some willow clones.     

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.     

High‐resolution melting analysis allows rapid and accurate differentiation of the three Melampsoridium species occurring on Alnus

Alder (Alnus) leaves can be infected by three species of Melampsoridium rust: M. alni, M. betulinum and M. hiratsukanum. The latter is an invasive species in Europe that can be differentiated from the other two on the basis of morphological and molecular characters. This study presents a rapid, one‐step, closed‐tube and inexpensive method to differentiate species of Melampsoridium using two primer sets and high‐resolution melting (HRM) analysis of alder leaf material.     

Screening of clones of Betula pendula and B. pubescens against two forms of Melampsoridium betulinum leaf rust fungus

Leaf discs from seedlings of six clones of B. pendula Roth and two clones of B. pubescens Ehrh. were inoculated with two urediniospore isolates of M. betulinum Kleb. The field collection iso-lates were obtained from B. pendula and B. pubescens growing in the field. The B. pendula rust was more specialized than the B. pubescens rust. The clones of B. pubescens showed partial resis-tance against the B. pendula rust, while the B. pubescens isolate was compatible to both birch species. Some interclonal variation was also found in both birch species. The results support an earlier suggestion by Klebahn that M. betulinum has two formae speciales.     

Pre-penetration behaviour of the urediniospores of birch rust,Melampsoridium betulinum,on greenhouse-grown and microcultured leaves of Betula spp.

In two different experiments the urediniospores of Melampsoridium betulinum from Betula pendula and Betula pubescens germinated both on mature leaves of greenhouse-grown plants and on sterile in vitro leaves of micropropagated plantlets, which were cloned from the same B. pendula and B. pubescens genotypes. The urediniospores and germ tubes were more easily detached from the leaf surfaces of in vitro leaves. If germination took place on a leaf vein, the growth continued across the veinal ridges; otherwise, no determined growth towards the stomata could be observed with either of the leaf types studied. In both experiments on the in vitro leaves of B. pubescens clone V5944, the germ tubes of the rust isolate from B. pendula mislocated appressoria significantly more often than the germ tubes of the rust isolate from B. pubescens. On the mature leaves of B. pendula clone E4214 and B. pubescens clone V5940 there were also significant differences in appressorial locations between the two rust isolates but the clonal responses were inconsistent. The results of the inoculations suggest that the incompatibility of the rust isolates from B. pendula on the leaves of B. pubescens is not related to the significantly higher ratio of failures in locating appressoria in this host-rust combination.     

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.     

Alder rust,Melampsoridium hiratsukanum Ito,identified from Wales,UK and British Columbia,Canada

An epidemic rust, Melampsoridium hiratsukanum, originating from East Asia has spread to Europe over the last two decades. We report the first observation of this rust in the British Isles on leaves of Alnus incana and A. glutinosa. In addition to M. hiratsukanum, a native M. betulinum–like rust co‐occurred on the same leaves as a mixed infection. We also confirm that a rust on A. rubra in Western Canada is identical to M. hiratsukanum present in East Asia and Europe.     

Variation in leaf phenolics of field‐cultivated willow (Salix myrsinifolia) clones in relation to occurrence of Melampsora rust

Concentrations of potential antifungal phenolics in field‐cultivated willow (Salix myrsinifolia) clones were analysed during three growing seasons, and correlated to the occurrence of Melampsora rust. Consistent relationships between phenolics and rust were not found across the experimental years. There was significant clonal and temporal variation in phenolic content and rust frequency. Levels of some phenolics varied considerably within a sequence of four full‐grown leaves, but the variation in rust occurrence within the same leaf sequence was nonsignificant. The results suggest that the possible association between willow phenolics and rusts is not straightforward, and emphasize the importance of long‐term studies to investigate the chemical basis of willow rust interactions.     

Rust resistance in Salix induced by inoculations with avirulent and virulent isolates of Melampsora larici‐epitea

Six Salix clones and four Melampsora larici‐epitea isolates were used in two leaf‐disc inoculation experiments to determine whether disease severity was affected by the presence of both virulent and avirulent rust genotypes. In the first experiment, an equal amount of urediniospores of a virulent isolate and an avirulent isolate was applied simultaneously using four levels of spore suspension. In the second, the willows were inoculated with one isolate first and then, after 3 days, with another using two spore concentrations. In the first experiment, overall rust spore production was reduced by 48.6% in inoculations with mixed inocula compared with that in the inoculations with single virulent isolates. In the second experiment, 20 of the 36 combinations involving pre‐inoculations with avirulent isolates showed significant reduction in spore production. The suppressive effects on rust sporulation were more obvious at the higher inoculum densities and on the clones S. × calodendron and S. × mollissima‘Q83’.     

Effects of acid rain treatments on the birch rust,Melampsoridium betulinum and birch,Betula pendula

Effects of acidic rain on birch rust and on the growth of Betula pendula seedlings were studied by irrigating with acidic water at pH 3, 4 and 5.6. The seedlings were inoculated with uredospores of Melampsoridium betulinum and the development of rust and the severity of rust infection was examined. The increasing acidity of the irrigation water did not affect on the development of the rust, but the nitrogen in the acidified water resulted in an extension of seedling growth, and hence the leaves became senescent at a later date in autumn and the rust grew for a longer time.     

Reliability of field,greenhouse and cut‐shoot screening procedures for evaluating susceptibility of Scots pine to Melampsora pinitorqua

The reliability of field, greenhouse and cut‐shoot screening procedures for the assessment of the susceptibility of Scots pine (Pinus sylvestris) genotypes to Melampsora pinitorqua, the causal agent of twisting rust, was evaluated. Fourteen genotypes evaluated on half‐sib progenies after natural infection of 2‐year‐old plants showed comparable rank for rust susceptibility with those evaluated after controlled inoculation of 1‐year‐old seedlings in a greenhouse. The dynamic of pycnia production was assessed at 2‐day intervals in cut‐shoot assays under controlled conditions, giving reliable rankings of Scots pine genotypes between years and being significantly correlated with rust susceptibility assessed after natural infection of 2‐year‐old progenies. These results underlined the importance of some critical factors such as inoculum load and host phenology in the reliability of susceptibility evaluations assessed under different experimental conditions. Artificial inoculation of 1‐year‐old seedlings in greenhouse experiments could provide a useful early test for the management of Scots pine breeding programmes and study of inheritance of twisting rust susceptibility. However, in specific investigations the cut‐shoot assay would constitute a reliable laboratory test for studying host–pathogen interactions and the variability in pathogenicity of Melampsora pinitorqua populations.     

Increased atmospheric carbon dioxide concentration: effects on eucalypt rust (Puccinia psidii), C:N ratio and essential oils in eucalypt clonal plantlets

Evaluation of impacts of high CO₂ atmospheric concentration is strategically important for the development of adaptation measures and sustainable crop management. The objective of this study was to evaluate the effects of increased atmospheric CO₂ concentration on eucalypt rust (Puccinia psidii), C:N ratio, yield and chemical composition of essential oils and growth of eucalypt clonal plantlets. Two clones with different levels of rust resistance were studied: a Eucalyptus urophylla × E. camaldulensis hybrid (VM 01) and an E. urophylla (clone MN 463). The experiments were performed in open‐top chambers (OTCs) with CO₂ mean concentrations (μmol mol^?1) of 399 (unenclosed control), 412 (OTCs with ambient CO₂ concentration) and 508 (OTCs with high CO₂ concentration) and in closed chambers (CCs) with CO₂ mean concentrations of 390, 405, 520 and 700. Increased atmospheric CO₂ concentrations resulted in a decrease in rust pustules per leaf, uredinia per leaf area, spores per uredinia and area under the disease progress curve in VM 01 (hybrid) clonal plantlets. The disease did not occur in MN 463 clonal plantlets, which demonstrated that high CO₂ concentrations did not change the level of rust resistance. Plant growth of the two clones was stimulated by up to 23% in height and 26% in stem diameter in OTCs and by 18% for both clones in CCs. An increased C:N ratio in leaves, stems and roots was observed only for the VM 01 clonal plantlets. Essential oils produced by VM 01 (2.8 g 100 g^?1) and MN 463 (1.4 g 100 g^?1), as well as the major essential oil compounds (80% 1.8‐cineole for VM 01; 50% 1.8‐cineole and 32% α‐pinene for MN 463), were not altered. In this study, increased concentrations of atmospheric CO₂ favourably impacted eucalypt growth and reduced rust severity, while not influencing the production of essential oils.     

Absence of residual effects of a defeated resistance gene in poplar

In a few plant pathosystems, defeated major genes have been shown to contribute to partial resistance to disease. This hypothesis has never been tested before in a forest tree, but pathogenic variation associated with recent hybridization in poplar rust in the Pacific northwest provided an opportunity. An F₂ progeny of 256 poplar clones in the field near Corvallis, Oregon, USA, has been monitored for rust severity and infection type since the advent of the new hybrid rust, Melampsora × columbiana, in the mid‐1990s. All 256 clones displayed a susceptible infection type in 1997 and again in 2000, and yet variation in uredinial density (i.e. partial resistance) was still observed. To determine which clones possessed a defeated resistance gene, a greenhouse inoculation was performed with an isolate of M. medusae, one of the parents of M. × columbiana. Clones that would have been resistant to M. medusae, prior to the advent of M. × columbiana, were thus identified. The inoculation resulted in a 1 : 1 segregation (χ²=0.772; p=0.38) for resistance, indicating the presence of a major gene. However, the F₂ clones possessing the defeated resistance gene displayed the same level of partial resistance in the field in both 1997 and 2000 as their full siblings lacking the gene.     

Hymenoscyphus fraxineus can directly infect intact current‐year shoots of Fraxinus excelsior and artificially exposed leaf scars

Hymenoscyphus fraxineus, the causal agent of ash dieback, was inoculated onto intact, unwounded current‐year shoots and leaf scars of 4‐year‐old, potted Fraxinus excelsior seedlings. Pieces of ash wood colonized by the fungus were used as inoculum. Three of 25 (12%) of the inoculated intact shoots and nine of 25 (36%) of the inoculated leaf scars were infected by H. fraxineus and developed typical symptoms of ash dieback, including necrotic lesions on the shoot surface and wood discoloration as well as shoot and leaf wilting distal to the inoculation site. No symptoms occurred on control seedlings, which had been inoculated in the same way but with sterile wood pieces. Visible necrotic lesions on shoots and wood discoloration were statistically significantly longer in proximal than in distal direction from the inoculation site, a pattern which resembles symptoms after natural infection. The ash dieback pathogen was re‐isolated from nine of 12 (75%) of the symptomatic seedlings. These results provide indirect supportive evidence that the fungus infects shoots via leaves and shows that it is able, under experimental conditions using a massive mycelial inoculum, to directly infect intact, unwounded current‐year shoots of its main host in Europe.     

Protocol for screening rust disease resistance in Dalbergia sissoo

A protocol has been developed for inoculation of rust disease in Dalbergia sissoo using urediniospores of Maravilia achroa. This is the first time that disease symptoms caused by M. achroa were artificially produced. The method has been successfully employed for screening 15 clones of D. sissoo against rust disease. Clone S‐167 was the most resistant remaining free from infection. Clones S‐57, S‐106 and S‐124 were also resistant. Clones S‐19, S‐24 and S‐89 were susceptible.     

Effects of pruning on the concentration of secondary metabolites in Colophospermum mopane leaves

Colophospermum mopane, commonly known as mopane, produces secondary metabolites during the growing season. However, there is still insufficient knowledge on the quantity of secondary metabolites and the effect of browsers on the concentration of secondary metabolites. A pruning experiment was conducted in the Musina Nature Reserve, Limpopo province, South Africa, to simulate the effect of browsers on the concentration of secondary metabolites in mopane leaves. The twigs from 40 selected experimental mopane trees were pruned back 50 mm from their tips using a hand shear. The total amount of leaf and shoot biomass removed from branches of selected experimental trees was less than 10%. Three independent samples composed of seven mopane leaves per 40 mopane trees were randomly collected from the canopies of experimental and control trees per sampling event. The dried leaves from each three independent samples were then mixed separately and a pooled sample of 10 g per treatment per sample cycle (55 d) was used for determination of total phenols (TP), condensed tannins (CT) and protein-precipitating tannins (PPT). Results showed that <10% pruning does not have an effect on the amount of secondary metabolites in the mopane leaves. The concentration of TP, CT and PPT increased during leaf flush in October and then declined as the leaves matured and aged. It is concluded that the amount of secondary metabolites in mopane leaves is not dependent on <10% pruning, but appeared to be associated with leaf growth stages. The ability of mopane to produce secondary metabolites has implications on the seasonal diet composition and distribution of browsers in mopane woodland.