Juan A. Martín

Resistance to Dutch Elm Disease Reduces Presence of Xylem Endophytic Fungi in Elms (Ulmus spp.)

Efforts to introduce pathogen resistance into landscape tree species by breeding may have unintended consequences for fungal diversity. To address this issue, we compared the frequency and diversity of endophytic fungi and defensive phenolic metabolites in elm (Ulmus spp.) trees with genotypes known to differ in resistance to Dutch elm disease. Our results indicate that resistant U. minor and U. pumila genotypes exhibit a lower frequency and diversity of fungal endophytes in the xylem than susceptible U. minor genotypes. However, resistant and susceptible genotypes showed a similar frequency and diversity of endophytes in the leaves and bark. The resistant and susceptible genotypes could be discriminated on the basis of the phenolic profile of the xylem, but not on basis of phenolics in the leaves or bark. As the Dutch elm disease pathogen develops within xylem tissues, the defensive chemistry of resistant elm genotypes thus appears to be one of the factors that may limit colonization by both the pathogen and endophytes. We discuss a potential trade-off between the benefits of breeding resistance into tree species, versus concomitant losses of fungal endophytes and the ecosystem services they provide.

Influence of Plant Age on Symptom Development in Ulmus minor Following Inoculation by Ophiostoma novo-ulmi

In American and European breeding programs, numerous elm trees from many species (Ulmus spp.) and hybrids have been inoculated annually with the fungus Ophiostoma novo-ulmi (the Dutch elm disease pathogen) in screening tests for resistance. Because trees were inoculated at different ages, it appeared necessary to study the influence of host age on the symptoms shown. Four Ulmus minor trees and one U. minor × U. pumila tree were cloned annually from 1994 to 1999. The replicates obtained (usually n = 6) were inoculated on 17 May 2000 with an O. novoulmi strain. At the end of the season, 2-year-old U. minor trees showed average wilting of 8 ± 3% (ES), significantly less than that of 3-year-old trees (34 ± 5%) (P ≤ 0.01). Elms that were 4, 5, 6, and 7 years old showed wilting values of around 50%. A positive relationship between the symptoms shown and the diameter of the elm xylem vessels was observed, and the implications for elm resistance and breeding are discussed. Breeders and pathologists should use trees of the same age and physiological phase when determining the relative resistance among elm clones. It is concluded that under the growing conditions of this experiment, the optimal age for U. minor screening was 4-year-old plants.

Antifungal effect and reduction of Ulmus minor symptoms to Ophiostoma novo-ulmi by carvacrol and salicylic acid

There are still no effective means to control Dutch elm disease (DED), caused by the vascular fungi Ophiostoma ulmi and O. novo-ulmi. Plant phenolics may provide a new strategy for DED control, given their known antifungal activity against pathogens and their involvement in plant defence mechanisms. The in vitro antifungal activity of salicylic acid, carvacrol, thymol, phenol, o-cresol, m-cresol, p-cresol, and 2,5-xylenol against the DED pathogens was tested. Also, the protective effect of watering Ulmus minor seedlings with these compounds was tested against O. novo-ulmi. Salicylic acid, carvacrol, and thymol showed the strongest antifungal in vitro activity, while carvacrol and salicylic acid provided the strongest in vivo protection against O. novo-ulmi (63 and 46% reduction of leaf wilting symptoms with respect to controls, respectively). The effect of the treatments on tree phenology was low, and a significant negative relation was observed between the number of days to bud burst and the leaf wilting symptoms after inoculation, probably determined by genetic differences among the elm tree progenies used. The treatments with salicylic acid, carvacrol and thymol induced the highest shift in phenolic metabolite profile with respect to control trees. The protective effect of carvacrol and salicylic acid is discussed in terms of their combined activity as antifungal compounds and as inductors of tree defence responses.

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.

Ecological Aspects of Endophyte-Based Biocontrol of Forest Diseases

Recent studies have shown that the asymptomatic fungal endophytes may influence the outcome of forest trees’ interactions with pathogens and herbivores, raising a promise that endophytes might be utilized as biocontrol agents in integrated pest and disease management. However, practical applications for forest protection based on endophytes are still rare, in particular in the case of the economically and ecologically important large trees and their diseases. A better understanding of the ecological and biological background of the protection provided by endophytes may help to design new forest protection strategies that utilize endophytes in control of tree diseases. More information is also needed regarding the effects of silvicultural methods on endophyte communities at the level of single trees and forest stands. In this chapter, we discuss the motivation for continued research on endophyte-based biocontrol of forest tree diseases and some ecological aspects related to the topic.

Chemical changes in Ulmus minor xylem tissue after salicylic acid or carvacrol treatments are associated with enhanced resistance to Ophiostoma novo-ulmi

Application of endogenous plant hormone salicylic acid (SA) or essential oil component carvacrol (CA) in elms enhances tree resistance to the Dutch elm disease pathogen, although the effect of these compounds on tree metabolism is unknown. The chemical changes induced by SA or CA treatments in Ulmus minor were studied through gas chromatography-mass spectrometry (GC-MS) analysis of xylem tissues. Treatments consisted of fortnightly irrigating seedlings with water, SA or CA at 600 mg L⁻¹. The chemical composition of the xylem tissues sampled from treated trees was significantly altered depending on the treatment type. SA treatment induced an accumulation of the sinapyl alcohol, a precursor of lignin and other phenylpropanoid-derived products. CA treatment induced an accumulation of the methyl esters of palmitic, linoleic and stearic acids. Both treatments resulted in early bud burst and SA significantly reduced sapwood radial growth, possibly as a consequence of a trade-off between tree growth and tree defence. The enhanced resistance provided by these treatments is discussed.

Influence of region of provenance and climate factors on wood anatomical traits of Pinus nigra Arn. subsp. salzmannii

Region of provenance is defined as an area with uniform ecological conditions where stands with similar phenotypic or genetic features are found. This study assesses the effect of differing climate conditions of eight Spanish regions of provenance of Pinus nigra Arn. subsp. salzmannii on earlywood anatomical traits measured in samples from basal discs from mature trees. Results showed that variation in wood biometry between provenances was high and more pronounced than intrapopulation variation. When comparing P. nigra with other Mediterranean pines, high intertracheid wall strength values are associated with better adaptation of pines to arid conditions. However, the intraspecific variations of this parameter in P. nigra did not follow the same pattern, due to the influence of mechanical support requirements. Trees subject to greater aridity were characterised by short tracheids, apparently resulting from their poorer growth, and high frequency of rays and ray parenchyma cells, which would allow trees to store greater amounts of starch, which is the source of metabolites invested in minimising the limitations imposed by water stress. Severe winter cold spells were strongly associated with high axial resin canal frequency and large radial resin canals, creating a powerful, preformed defence system. Increased tracheid lumen involved an increase in the size of bordered pits, favouring sap flow between tracheids, in addition to an increase in the maximum diameter of cross-field pits, favouring the flow of water and metabolites between the axial and radial systems. The high influence of region of provenance on structural variation in P. nigra shows the importance of provenance in the selection of seed origin for reforestation.

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 treatment × clone 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.

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.

Large Reynolds number streak description using RNS

We use the Reduced Navier-Stokes (RNS) equations for the simulation of the nonlinear evolution of streaks in a flat plate boundary layer. The RNS are asymptotically derived from the Navier Stokes equations for Re » 1, and they are appropriated for flow configurations with one slow scale and two short scales. We derive the RNS with the appropriate boundary conditions, comment the details of the numerical method used, and compare our 3D streak simulations with the results present in the literature (computed using DNS and PSE).