Cellulolytic activity and virulence of Ophiostoma ulmi and O. novo‐ulmi isolates

The cellulolytic activity (exoglucanase, endoglucanase and β‐glucosidase) of Ophiostoma ulmi (four isolates), O. novo‐ulmi (19 isolates) and ‘fast‐waxy’ (five isolates) was determined in growth media containing carboxymethylcellulose (CMC) and cellulose powder. Differences in enzyme activities were observed among isolates, irrespective of the species and substrate used. Inoculation experiments on Ulmus minor with randomly selected isolates of O. ulmi (two isolates), O. novo‐ulmi (five isolates) and ‘fast‐waxy’ (two isolates) were also performed. Disease was assessed as the percentage of leaves showing yellowing and browning. Ophiostoma novo‐ulmi and ‘fast‐waxy’ isolates exhibited a great variability in their capacities to cause the disease. In the presence of CMC, a significant correlation between the activity of exoglucanase and β‐glucosidase in vitro and virulence was found.     

Starch‐like exopolysaccharide produced by the filamentous fungi Ophiostoma ulmi and O. novo‐ulmi

This paper describes the chemical and biochemical properties of exopolysaccharides (EPS) produced by Ophiostoma ulmi and O. novo‐ulmi isolates, the Dutch elm disease (DED) fungi. Some of EPS have been considered as pathogenicity factor in the DED complex. The selected isolates grow well and produce EPS in a medium containing various types of carbon and nitrogen sources. EPS obtained from potato dextrose broth (PDB) medium appeared to be opaque, firm and stained purple blue with iodine‐potassium iodide solution, whereas those from yeast extract (YE) medium were less opaque, jelly‐like and remained unchanged in iodine solution. The selected fungal isolates produced much higher molecular weight EPS from the medium containing YE than from PDB. The results of this study suggest that high molecular weight compounds produced by O. ulmi (W9) and O. novo‐ulmi (R136) are not involved in DED pathogenesis. Spectrometric analysis of acid‐digested EPS obtained from PDB and YE revealed the presence of a monomer similar to glucose used as a standard. Thin layer chromatography indicated that glucan‐1,4‐α‐glucosidase (glucoamylase) only hydrolyses EPS from PDB media and releases glucose. The results strongly indicate that isolates of O. ulmi and O. novo‐ulmi produce starch‐like EPS from PDB medium. The EPS obtained from YE medium lacked this characteristic. The biological significance and the potential use of these EPS are discussed.     

Variation among Italian and French elm clones in their response to Ophiostoma novo‐ulmi inoculation

In 1995 a clone bank of elms from across Italy was created at Antella (Florence, I) under a European Union project. In 1998 an experimental planting of some clones from this source was established in Antella together with material from a similar clone bank in France and two reference clones: Commelin (relatively susceptible) and Lobel (relatively resistant). In May 2001, the elms were inoculated with Ophiostoma novo‐ulmi and disease development was assessed throughout that summer and the next. Significant differences in susceptibility were found between taxonomic groups, with putative natural hybrids between Ulmus minor and Ulmus pumila being the most resistant and Ulmus glabra the most susceptible. Significant differences were also found within U. minor, some clones being as resistant as Lobel. For U. minor, a statistically significant relation was found with latitude, southerly clones showing less disease than more northerly ones. Relationships were also found between disease severity and times to bud burst, these relationships applying both to the different taxonomic groups and within U. minor. Relationships between growth increment and disease severity were inconsistent. These results are discussed in relation to the intrinsic genetically‐controlled differences that exist between elms and to differences in susceptibility that occur as the growing season proceeds and as a consequence of environmental influences.     

First report on the Dutch elm disease pathogen Ophiostoma novo‐ulmi from Latvia

During 2017, Dutch elm disease was investigated on 67 Ulmus glabra and 29 Ulmus laevis samples from 28 sites in Latvia. The presence of Ophiostoma novo‐ulmi was detected in 61.2% of the samples from U. glabra and in 34.5% of the samples from U. laevis. In 11% of cases, hybrids between subsp. novo‐ulmi and subsp. americana were identified, particularly in the sites in the southern part of Latvia. This study presents the first verified record of Ophiostoma novo‐ulmi and its subspecies in Latvia.     

In vitro antagonism of Trichoderma and naturally occurring fungi from elms against Ophiostoma novo‐ulmi

The antagonist ability of four fungal endophytes isolated from elms (Trichoderma atroviride GF13, Diaporthe eres GF2, Diplodia ulmi GF5 and Alternaria alternata GF6) and two fungi from a collection (Trichoderma harzianum and Trichoderma virens) was evaluated against six strains of Ophiostoma novo‐ulmi (1, 10, 19, 32, 43 and O‐Va), the causal agent of Dutch elm disease. Dual culture tests were established in PDA and MA culture media to test the interactions between pathogenic fungi and potential antagonist ones. The fungi showed varying levels of antagonist capacity against O. novo‐ulmi. Among them, T. atroviride and T. harzianum had the greatest inhibiting effect (around 50%). Culture filtrates and previously inoculated cellophane discs from T. harzianum and T. atroviride also caused growth inhibition of O. novo‐ulmi, with less intensity in strains 10 and 19. The control mechanisms involved in the process were competition, mycoparasitism and the production of soluble and diffusible antifungal metabolites. The new isolate T. atroviride GF13 was efficient as antagonist. It showed antagonist ability against non‐elm‐derived fungi such as Verticillium, Fusarium, Aspergillus, Rhizopus and Penicillium. These results suggest interesting application possibilities in the biological control of Dutch elm disease.     

Rapid differentiation of Ophiostoma ulmi and Ophiostoma novo‐ulmi isolates by matrix‐assisted‐laser‐desorption/ionization time‐of‐flight/time‐of‐flight mass spectrometry

The intact yeast phase spores of Ophiostoma ulmi and O. novo‐ulmi isolates were analysed by matrix‐assisted‐laser‐desorption/ionization time‐of‐flight/time‐of‐flight mass spectrometry. The results clearly showed that isolates of O. ulmi and O. novo‐ulmi have different mass peak profiles. The specific mass in m/z may vary slightly. One of the peaks from m/z 7623 to 7630 dominated (in high intensity) mass spectra for all isolates of O. novo‐ulmi tested in this study. This specific peak is believed to be CU (cerato‐ulmi) protein. However, no specific m/z peak can be found to distinguish between the isolates of ssp. novo‐ulmi and ssp. americana. On the contrary, all isolates of O. ulmi tested possessed one of a dominate peak at m/z 7337 to 7342, but none around m/z 7626 as shown in O. novo‐ulmi isolates.     

American elm cultivars: Variation in compartmentalization of infection by Ophiostoma novo‐ulmi and its effects on hydraulic conductivity

Five cultivars and two populations of wild‐type seedlings of American elm (Ulmus americana), 3 and 4 years old, were examined for differences in their abilities to compartmentalize and resist infection by artificially inoculating with Ophiostoma novo‐ulmi. Morphological characteristics of tree defence, often referred to as the compartmentalization of decay in trees model, were used as a conceptual framework, with particular emphasis on the limiting of tangential spread of infection within the xylem and barriers that limit spread outwards to cells formed after infection. To investigate the change in functional xylem over time, 3‐year‐old trees were assessed at multiple time points following inoculation for hydraulic conductivity. Three and four‐year‐old cut trees were placed in 0.1% w/v safranin O for 18 to 24 hr to indicate functional xylem. Transverse sections of the stained stems were used to calculate per cent of sap‐conducting xylem area and the per cent of circumference conducting of first formed cells and later formed cells. At each collection time, trees were assessed for disease severity on a 1–12 scale, based on the percentage of permanent wilt in the crown. There was considerable variation between cultivars in disease severity and their capacity to localize and resist infection. “Prairie Expedition,” which had the lowest disease severity rating in 2015 and the second lowest in 2016, consistently limited the spread of infection into newly formed xylem and had functional xylem around the entire circumference of the stem at 90 days post‐inoculation. “Valley Forge” in 2016 had the lowest overall disease severity rating and was the only cultivar to consistently limit the tangential spread of infection within extant xylem. This research identifies key characteristics that some cultivars have to resist and limit infection and provides new information that can be used in disease screening programmes to evaluate other cultivars and older plant material.     

Detection of differential changes in lignin composition of elm xylem tissues inoculated with Ophiostoma novo‐ulmi using Fourier transform‐infrared spectroscopy

Differences in elm susceptibility against Ophiostoma novo‐ulmi have been related with differences in timing and degree of the tree defence responses. In this study, we used Fourier transform‐infrared spectroscopy, coupled with chemometric methods, to detect progressive changes in the metabolic profile of Ulmus minor and U. minor × U. pumila xylem tissues after inoculation with O. novo‐ulmi. Differences between control and inoculated trees were detected at 30 and 60 days post‐inoculation (dpi) in U. minor, and at 15, 30 and 60 dpi in U. minor × U. pumila. These differences were related with increased levels of lignin in the xylem tissues, suggesting an earlier defence response to the infection in the hybrids.     

Ophiostoma ulmi metabolites and elm cell membrane permeability. Possible use in early tests of resistance

Electrolyte leakage from leaf discs of three Ulmus species when challenged with cultural filtrate of an NAN aggressive isolate of Ophiostoma ulmi paralleled their susceptibility to the disease in the field. But the correlation was not working with some hybrids derived from the common field elm showing intermediate resistance. The possible explanation will be checked in further tests which are in progress to assess the possibility of using this technique in early screening for resistance.     

Presence and accumulation of fungitoxic substances against Ceratocystis ulmi in Ulmus americana: possible relation to induced resistance1

This paper reports the differential accumulation of inhibitory compounds in American elm seedlings after inoculation with a non-aggressive and an aggressive strain of C, ulmi.     

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.     

Diminished vessel diameter as a possible factor in the decline of European ash (Fraxinus excelsior L.)

? The aim of this work was to examine the anatomy and functioning of secondary xylem in stems in relation to the decline of European ash (Fraxinus excelsior L.).

? We tested the hypothesis that declining trees show changes in the structure of wood, which result in impaired water transport.

? Anatomical analyses were carried out on wood samples (comprising all annual rings formed during the 30 years life of the analysed trees) collected at breast height from the main stem of healthy, weakened and dead ash trees. The width of annual wood increments, the diameter and density of earlywood vessels were measured and the theoretical hydraulic conductivity index through the secondary xylem calculated by application of Hagen-Poisseuille’s formula.

? Anatomical characteristics changed both with the age of trees and in response to unfavorable factors. The largest vessels were observed in healthy trees, which implied that they had the highest hydraulic conductivity index, whereas trees considered to be in decline produce smaller vessels and hence had reduced conductivity.

     

Effects of various densities of Ophiostoma ips inoculations on Pinus sylvestris in north‐western Spain

The aim was to determine the inoculation density above which Scots pine (Pinus sylvestris) is overcome by the blue‐stain fungus Ophiostoma ips that is associated with the bark beetle Ips sexdentatus. In north‐western Spain, stems of 16 Scots pines were inoculated at various densities (0, 400, 800 or 1600 inoculi/m²) along circumferential 100 or 150 cm wide inoculation belts. Each inoculum consisted of a 5 mm diameter cylinder of malt extract agar colonized by the fungus. Three months later, all trees were harvested and trunk resinosis and foliage colour were visually assessed. The percentage of healthy, desiccated, resin soaked, and blue‐stained sapwood, as well as growth productivity indices, were calculated from stem disks cut from within the inoculated zone of each tree. Sapwood‐specific hydraulic conductivity (Ks) of each tree was measured in the middle of the inoculated zone. All parameters of tree vigour changed dramatically to the worse when inoculation densities were above 400 inoculi/m², and foliage changed from green to yellow‐green or yellow when an inoculation density of 800 instead of 400 was used. The percentage loss of sapwood‐specific conductivity (PLC) increased from 30 to 90% and the percentage of healthy, conductive sapwood dropped from 85 to 35% at 800 inoculi/m². No effect of the width of the inoculation belt was observed, and there was no relationship between tree productivity indices and the level of resistance. A non‐linear negative relationship was found between PLC and the percentage of healthy sapwood. It is concluded that tree resistance was overcome and that trees were going to die when the inoculation density was ≥800 inoculi/m².     

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.     

Influence of severe drought on the resistance of Pinus yunnanensis to a bark beetle‐associated fungus

Research on tree response to pest attacks under drought and heat stress is crucial for predicting the effect of climate change on forest pests and tree health. In this work, we studied the influence of severe water stress on the resistance of Pinus yunnanensis to inoculations of Grosmannia yunnanensis, a pathogenic fungus associated with the aggressive bark beetle, Tomicus yunnanensis. Experiments were performed in two plots, one located at the top of a hill and the other at the foot of a hill, in Xiaojiu, Yunnan, China, from May to September 2010 and March to April 2012. Measurements of soil and needle water content and predawn needle water potentials confirmed that severe droughts occurred during these periods. Maximum water stress intensity even occurred in 2010 during the typical rainy season. Following low‐density inoculations, the reaction zone length and fungal growth were measured in the phloem. Sapwood blue staining and total sapwood occlusion were quantified following fungal mass inoculations. Our results suggest that severe water stress has a negative impact on a tree's resistance to G. yunnanensis. By comparing our results to previous work performed from 1997 to 1999 on the same tree species, and in the same locality under mild water stress, we concluded that drought had opposite effects on tree resistance above and below a certain threshold of predawn needle water potential. The threshold, corresponded to maximum efficiency for tree defence mechanisms, was visible in our results, and was estimated to be approximately ‐1.1 MPa to ‐1.2 MPa for P. yunnanensis under local conditions. We suggest that the existence of such a threshold of water stress intensity may be viable for any conifer undergoing biotic aggressions and, specifically, bark beetle attacks.     

Wood‐rotting basidiomycetes are a minor component of fungal communities associated with Acacia hybrid trees grown for sawlogs in South Vietnam

Acacia hybrid (Acacia mangium × Acacia auriculiformis) clones are widely planted in Vietnam with a total of approximately 400,000 ha to meet the demand for pulpwood, sawn timber and wood chip exports. Silvicultural techniques such as pruning and thinning have been applied to improve productivity and sawlog quality of Acacia hybrid plantations. However, those techniques may also create opportunities for wood decay fungi to enter the Acacia hybrid stems through wounds and cause stem defects that reduce sawlog quality and the value of the plantation. The presence of fungal decay agents in Acacia hybrid trees was examined in two Vietnamese plantations. In July 2011, just prior to a second thinning, discoloured wood samples were taken from a three‐year‐old Acacia hybrid plantation at Phan Truong Hai for the isolation of fungi. In July 2012, approximately 18 months after pruning and thinning treatments, discoloured wood samples were taken from a three‐year‐old Acacia hybrid plantation at Nghia Trung for the isolation of fungi. DNA sequencing of the rDNA ITS identified the isolates. In May 2015, approximately 4 years after thinning and fertilizer treatments, discoloured and decayed wood samples were taken from the above (7‐year‐old) Acacia hybrid plantation at Phan Truong Hai for fungal identification. DNA was extracted directly from discoloured and decayed wood samples and fungal rDNA ITS amplicons sequenced on a Roche 454 sequencer. The results showed that silvicultural treatments did not affect the fungal communities associated with discoloured and decayed wood of Acacia hybrid plantation at Phan Truong Hai. A total of 135 fungal species or OTUs (operational taxonomic units) were identified, including 82 members of Ascomycota and 52 Basidiomycota.     

Radial variation in sap velocity as a function of stem diameter and sapwood thickness in yellow-poplar trees

Canopy transpiration and forest water use are frequently estimated as the product of sap velocity and cross-sectional sapwood area. Few studies, however, have considered whether radial variation in sap velocity and the proportion of sapwood active in water transport are significant sources of uncertainty in the extrapolation process. Therefore, radial profiles of sap velocity were examined as a function of stem diameter and sapwood thickness for yellow-poplar (Liriodendron tulipifera L.) trees growing on two adjacent watersheds in eastern Tennessee. The compensation heat pulse velocity technique was used to quantify sap velocity at four equal-area depths in 20 trees that ranged in stem diameter from 15 to 69 cm, and in sapwood thickness from 2.1 to 14.8 cm. Sap velocity was highly dependent on the depth of probe insertion into the sapwood. Rates of sap velocity were greatest for probes located in the two outer sapwood annuli (P1 and P2) and lowest for probes in closest proximity to the heartwood (P3 and P4). Relative sap velocities averaged 0.98 at P1, 0.66 at P2, 0.41 at P3 and 0.35 at P4. Tree-specific sap velocities measured at each of the four probe positions, divided by the maximum sap velocity measured (usually at P1 or P2), indicated that the fraction of sapwood functional in water transport (f(S)) varied between 0.49 and 0.96. There was no relationship between f(S) and sapwood thickness, or between f(S) and stem diameter. The fraction of functional sapwood averaged 0.66 +/- 0.13 for trees on which radial profiles were determined. No significant depth-related differences were observed for sapwood density, which averaged 469 kg m(-3) across all four probe positions. There was, however, a significant decline in sapwood water content between the two outer probe positions (1.04 versus 0.89 kg kg(-1)). This difference was not sufficient to account for the observed radial variation in sap velocity. A Monte-Carlo analysis indicated that the standard error in estimated mean f(S) declined rapidly with increasing sample size. At n = 10, the coefficient of variation in mean f(S) was 7% and at n = 15 it was slightly less than 5%. These observations indicate that radial variation in sap velocity is an important, albeit often overlooked, source of uncertainty in the scaling process. Failure to recognize that not all sapwood is functional in water transport will introduce systematic bias into estimates of both tree and stand water use. Future studies should devise sampling strategies for assessing radial variation in sap velocity and such strategies should be used to identify the magnitude of this variation in a range of non-, diffuse- and ring-porous trees.     

Evaluation of Juglans species for resistance to Phytophthora cinnamomi: differences in isolate virulence and response to fosetyl‐Al

Phytophthora is considered as an important pathogen on walnut, and severe losses are reported in European as well as in American walnut stands. Though several Phytophthora spp. are known to attack walnut, P. cinnamomi is considered the most virulent and widespread in southern Europe. Up to now, no walnut species or hybrid is known to have a high resistance level towards P. cinnamomi. Efforts are addressed in finding rootstock material graft compatible with English walnut and resistant/tolerant to P. cinnamomi. The extension of P. cinnamomi lesions on five Juglans species was studied to find out sources of resistance/tolerance to this pathogen. Walnut species clustered into two main groups, J. hindsii, J. nigra, and J. mandshurica were the less susceptible to the colonization of P. cinnamomi, while J. regia and J. sieboldiana were the most susceptible. On this account, J. mandshurica represents the best alternative as rootstock because its employment overcomes the risk of the occurrence of black line disease, it has good level of resistance to Agrobacterium temefaciens and Brenneria nigrifluens, and it is tolerant to Xanthomonas arboricola pv. juglandis. J. mandshurica is also compatible in cross‐pollinations with J. regia and J. nigra. Differences in virulence of P. cinnamomi isolates was assessed and a marked interaction between species and isolate emerged. Treatment with fosetyl‐Al by dipping was mainly efficient in reducing the length P. cinnamomi lesions, and an interaction between species and treatment was evident with the highest efficacy on J. regia and J. sieboldiana.     

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.     

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.