Johanna Witzell

Phenolic metabolites in the resistance of northern forest trees to pathogens — past experiences and future prospects

Phenolic metabolites are frequently implicated in chemical defense mechanisms against pathogens in woody plants. However, tree breeding programmes for resistance to pathogens and practical tree-protection applications based on these compounds seem to be scarce. To identify gaps in our current knowledge of this subject, we explored some of the recent literature on the involvement of phenolic metabolites in the resistance of northern forest trees (Pinus, Picea, Betula, Populus, and Salix spp.) to pathogens. Although it is evident that the phenolic metabolism of trees is often activated by pathogen attacks, few studies have convincingly established that this induction is due to a specific defense response that is capable of stopping the invading pathogen. The role of constitutive phenolics in the resistance of trees to pathogens has also remained unclear. In future studies, the importance of phenolics in oxidative stress, cell homeostasis and tolerance, and the spatial and temporal localization of phenolics in relation to invading pathogens should be more carefully acknowl- edged. Possibilities for future studies using advanced methods (e.g., metabolic profiling, confocal laser scanning micro- scopy, and use of modified tree genotypes) are discussed. Resume ´ : Les metabolites phenoliques sont souvent impliques dans les mecanismes de defense chimiques contre les agents pathogenes chez les plantes ligneuses. Cependant, il semble y avoir peu de programmes damelioration des arbres pour la resistance aux agents pathogenes ou de mesures pratiques de protection des arbres qui sont bases sur ces composes. Dans le but didentifier les lacunes dans nos connaissances actuelles sur ce sujet, nous avons exploreune partie de la litterature recente traitant du role des metabolites phenoliques dans la resistance des especes forestieres nordiques (Pinus, Picea, Be- tula, Populus et Salix spp.) aux agents pathogenes. Bien quil soit evident que le metabolisme des composes phenoliques chez les arbres est souvent declenchepar les attaques des agents pathogenes, il a rarement eteetabli de facon convaincante que cette induction est due aune reaction de defense specifique capable denrayer linvasion de lagent pathogene. De plus, on nest toujours pas certain du role que jouent les composes phenoliques constitutifs dans la resistance des arbres face aux agents pathogenes. Dans les etudes ulterieures, on devrait accorder plus dattention a limportance des composes phenoliques en lien avec le stress oxydatif, lhomeostasie et la tolerance des cellules ainsi quala localisation spatiale et temporelle des composes phenoliques en relation avec linvasion des agents pathogenes. La discussion porte sur la possibi- liteque les etudes futures utilisent des methodes de pointes telles que le profilage metabolique, le microscope confocal a balayage laser et aient recours a des arbres dont le genotype a etemodifie ´. (Traduit par la Redaction)

Nutritional niche overlap potentiates the use of endophytes in biocontrol of a tree disease

Asymptomatic endophytic fungi are often regarded as potent biocontrol agents in plants, but the competitive interactions between endophytes and other microbes within the same host plant are poorly understood. We tested a hypothesis that as compared to asymptomatic endophytes, an aggressive pathogen inhabiting the same host is able to utilize carbon substrates more efficiently. Using phenotype microarray, we determined the carbon utilization profiles of the highly virulent Dutch elm disease (DED) pathogen Ophiostoma novo-ulmi, and four asymptomatic elm (Ulmus spp.) endophyte isolates that were selected based on their differential association to the DED-susceptibility pattern of the host elms. The competitive interactions between isolates were evaluated using a niche overlap index. In contrast to our hypothesis, the studied endophytes exhibited extensive niche overlap with the pathogen, suggesting that some endophyte strains might protect elms against DED-pathogen through competition for substrates and provide new tools for biocontrol of DED.

Strong in vitro antagonism by elm xylem endophytes is not accompanied by temporally stable in planta protection against a vascular pathogen under field conditions

Some endophytic fungi provide their host plants with protection against abiotic and biotic stressors, including pathogens. Endophyte-mediated mechanisms might be behind the environmental resistance shown in the field by some adult Ulmus minor trees to the Dutch elm disease (DED) pathogen, Ophiostoma novo-ulmi. We isolated and characterized seven endophyte fungi from the xylem of three adult U. minor trees that have survived the DED epidemics within areas in Spain ravaged by the disease. The antagonism of the isolated endophytes towards O. novo-ulmi was evaluated in vitro by means of dual culture assays. Six of the studied endophytes hindered the pathogen growth through antibiosis, competition for the substrate, or a combination of both mechanisms. Four of these endophytes were selected for in vivo tests where their protective effect was evaluated in field experiments during three successive years (2011–2013). The conditioning inoculation of two endophytes (Monographella nivalis and Alternaria tennuissima) reduced DED symptoms in 2011 and 2012, respectively. However, the same isolates did not show any prophylactic effect in 2013, which suggests that the repeatability of the treatments is low. A significant treatmentu2009×u2009clone interaction was found, showing that the effectiveness of the treatments depended on the tree clone. The future use of endophytes in biocontrol strategies might be oriented towards taking into consideration the whole fungal microbiome in forest breeding programs rather than the external application of particular endophyte strains.

Phenotype MicroArrays as a complementary tool to next generation sequencing for characterization of tree endophytes

There is an increasing need to calibrate microbial community profiles obtained through next generation sequencing (NGS) with relevant taxonomic identities of the microbes, and to further associate these identities with phenotypic attributes. Phenotype MicroArray (PM) techniques provide a semi-high throughput assay for characterization and monitoring the microbial cellular phenotypes. Here, we present detailed descriptions of two different PM protocols used in our recent studies on fungal endophytes of forest trees, and highlight the benefits and limitations of this technique. We found that the PM approach enables effective screening of substrate utilization by endophytes. However, the technical limitations are multifaceted and the interpretation of the PM data challenging. For the best result, we recommend that the growth conditions for the fungi are carefully standardized. In addition, rigorous replication and control strategies should be employed whether using pre-configured, commercial microwell-plates or in-house designed PM plates for targeted substrate analyses. With these precautions, the PM technique is a valuable tool to characterize the metabolic capabilities of individual endophyte isolates, or successional endophyte communities identified by NGS, allowing a functional interpretation of the taxonomic data. Thus, PM approaches can provide valuable complementary information for NGS studies of fungal endophytes in forest trees.

Feeding by Scolytus bark beetles to test for differently susceptible elm varieties

Dutch elm disease (DED), caused by the fungi Ophiostoma ulmi and O. novo‐ulmi, has reduced elm populations severely in Europe and North America. Breeding programmes are in action to find less susceptible elm varieties suitable for re‐establishing elm stands. Bark beetles, mainly Scolytus spp., are the only known natural vectors of DED. During twig feeding, beetles transfer Ophiostoma spores to healthy elms. Thus, less palatable elms should run a lower risk of DED infections. In feeding preference bioassays, we offered twigs from elms exhibiting different degree of susceptibility to O. novo‐ulmi, together with non‐host trees to Scolytus beetles. Scolytus multistriatus preferred wych elm, Ulmus glabra, to 100% in two‐choice tests, whereas S. laevis did not discriminate between a tolerant and a susceptible variety of field elm, U. minor. We suggest that the feeding assay is useful as a low‐tech method in breeding programmes for evaluating the suitability of promising elm genotypes to vector insects.

Antagonistic activity of fungal endophyte filtrates against Gremmeniella abietina infections on Aleppo pine seedlings

Biological control agents (BCAs), and among them some species of fungal endophytes, are potential substitutes for chemical pesticides in the control of plant diseases due to their non-toxicity to human beings and their surrounding environment. One mode of action of fungal BCAs is through their bioactive, extracellular products, which can inhibit the growth of pathogens. In this study, the effect of fungal filtrates from four endophyte isolates (Trichoderma viride, Aureobasidium pullulans, Aureobasidium sp. and the unknown endophyte 20.1) on the advance of the pathogen Gremmeniella abietina on 2-year Pinus halepensis seedlings was evaluated. Both preventive and therapeutic treatments of the filtrates were studied by applying the filtrates either before or after the pathogen inoculation, respectively. Since G. abietina is a necrotrophic fungus, the length of the necrosis produced by the pathogen was used as response variable in our experiment. In order to explore the chemical composition of the fungal filtrates, a simple HPLC screening of UV-absorbing components was conducted. The results of the study showed that all fungal filtrates were able to reduce the advance of G. abietina when compared to the control seedlings, regardless of the time of inoculation and the treatment. Low-molecular weight phenolic compounds could be detected in some but not all filtrates, warranting further studies on the possible role of these compounds in fungal filtrates.

Pedunculate Oaks (Quercus robur L.) Differing in Vitality as Reservoirs for Fungal Biodiversity

Ecological significance of trees growing in urban and peri-urban settings is likely to increase in future land-use regimes, calling for better understanding of their role as potential reservoirs or stepping stones for associated biodiversity. We studied the diversity of fungal endophytes in woody tissues of asymptomatic even aged pedunculate oak trees, growing as amenity trees in a peri-urban setting. The trees were classified into three groups according to their phenotypic vitality (high, medium, and low). Endophytes were cultured on potato dextrose media from surface sterilized twigs and DNA sequencing was performed to reveal the taxonomic identity of the morphotypes. In xylem tissues, the frequency and diversity of endophytes was highest in oak trees showing reduced vitality. This difference was not found for bark samples, in which the endophyte infections were more frequent and communities more diverse than in xylem. In general, most taxa were shared across the samples with few morphotypes being recovered in unique samples. Leaf phenolic profiles were found to accurately classify the trees according to their phenotypic vitality. Our results confirm that xylem is more selective substrate for endophytes than bark and that endophyte assemblages in xylem are correlated to the degree of host vitality. Thus, high vitality of trees may be associated with reduced habitat quality to wood-associated endophytes.

Enhancement of Populus alba tolerance to Venturia tremulae upon inoculation with endophytes showing in vitro biocontrol potential

Several studies have provided evidence that endophytes of forest trees can provide the trees with protection against pests and pathogens, but more experimental evidence is still needed to better understand if endophytes could be utilized in biocontrol of forest diseases. Here, we tested the hypothesis that fungal endophytes of Populus alba L. contribute to tree tolerance to Venturia tremulae Aderh., causal agent of shoot dieback in trees within Populus section. Fungal endophytes were isolated from twigs of two healthy P. alba trees, and classified according to their morphology and ITS sequence. Pleosporales, Dothideales and Eurotiales were the main fungal orders represented. Twelve isolates were challenged by the pathogen in in vitro dual assays and 10 of them reduced the growth rate of the pathogen. A mixed cell suspension from these endophytes was applied as a preventive treatment to P. alba seedlings growing in the greenhouse with the exception of positive control plants. Fifteen days later, plants were treated with a V. tremulae spore suspension. An additional control group of Populus x canescens (Aiton) Sm. seedlings were also inoculated with V. tremulae. Although the overall incidence of symptoms was low, both the incidence and severity of damages were lower in P. alba plants pretreated with endophytes than in controls (43.18 and 12.16% of incidence and severity reduction, respectively). Incidence of symptoms was higher in P. x canescens than in P. alba seedlings, evidencing the higher tolerance of P. alba to Venturia shoot blight. The results suggest that P. alba endophyte community may reinforce the host’s tolerance to the pathogen.

Risk of False Positives during Sampling for Heterobasidion annosum s.l.

A standard method to detect infection by Heterobasidion annosum sensu lato (s.l.) in stumps or stems is to cut a disc and examine it under a microscope. Concerns have been raised that spores can be transferred from the bark to the cut surface, thus contaminating the sample. The aims of this study were to test whether viable basidiospores of H. annosum s.l. can be transferred from the bark onto disc surfaces by a chainsaw and to investigate the impacts of different sampling procedures on the extent of contaminations. Logs were cut with or without adding basidiospores to the bark prior to the cut. Infection measurements were significantly greater for discs with treated bark (100% infected, infection coverage 40 cm2 dm-2 of disc area) compared with control discs (47% infected, infection coverage 0.2 to 0.3 cm2 dm-2). In addition, trees were sampled under authentic field conditions using different procedures. The infection measurements differed significantly depending on the procedure; sampling involving debarking or disinfection of the bark with 70% ethanol prior to cutting had lower measurements (6 to 19% and 13% infected, respectively) compared with leaving the bark on untreated (63 to 75% infected). Consideration of the contamination risk is warranted when evaluating the results of earlier studies and when planning new experiments.

Phenolic profiles of two Melampyrum species differing in susceptibility to Cronartium rust

Cronartium flaccidum is an important pathogen in boreal forestry where it causes pine stem rust. The hemiparasite Melampyrum sylvaticum serves as a telial host for this heteroecious fungus while the congeneric M. pratense is highly resistant. We hypothesized that the variation in susceptibility is due to differences in the composition of secondary phenolic compounds in host plant tissues. To test this hypothesis, phenolic compounds of leaf extracts taken from both species of Melampyrum were analyzed with HPLC. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used for data processing. PCA indicated difference between the species, with the first two components explaining more than 70xa0% of the total variance of data that was separated into two distinct groups in a scatter plot. Two clusters, formed by the two species, were also suggested by HCA. Phenolic acids (such as chlorogenic acid), flavanones and apigenin flavonoids dominated the profiles of rust-resistant M. pratense, while kaempferol and luteolin flavonoids occurred in high relative concentrations in rust-susceptible M. sylvaticum. Our results suggest that phenolics may contribute to resistance or susceptibility of Melampyrum to Cronartium rusts as one factor. Future studies on the mode of action of phenolics in this interaction are thus warranted.