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.     

Temporal metabolic profiling of the Quercus suber–Phytophthora cinnamomi system by middle‐infrared spectroscopy

The oomycete Phytophthora cinnamomi is an aggressive plant pathogen, detrimental to many ecosystems including cork oak (Quercus suber) stands, and can inflict great losses in one of the greatest ‘hotspots’ for biodiversity in the world. Here, we applied Fourier transform‐infrared (FT‐IR) spectroscopy combined with chemometrics to disclose the metabolic patterns of cork oak roots and P. cinnamomi mycelium during the early hours of the interaction. As early as 2 h post‐inoculation (hpi), cork oak roots showed altered metabolic patterns with significant variations for regions associated with carbohydrate, glycoconjugate and lipid groups when compared to mock‐inoculated plants. These variations were further extended at 8 hpi. Surprisingly, at 16 hpi, the metabolic changes in inoculated and mock‐inoculated plants were similar, and at 24 hpi, the metabolic patterns of the regions mentioned above were inverted when compared to samples collected at 8 hpi. Principal component analysis of the FT‐IR spectra confirmed that the metabolic patterns of inoculated cork oak roots could be readily distinguished from those of mock‐inoculated plants at 2, 8 and 24 hpi, but not at 16 hpi. FT‐IR spectral analysis from mycelium of P. cinnamomi exposed to cork oak root exudates revealed contrasting variations for regions associated with protein groups at 16 and 24 h post‐exposure (hpe), whereas carbohydrate and glycoconjugate groups varied mainly at 24 hpe. Our results revealed early alterations in the metabolic patterns of the host plant when interacting with the biotrophic pathogen. In addition, the FT‐IR technique can be successfully applied to discriminate infected cork oak plants from mock‐inoculated plants, although these differences were dynamic with time. To a lesser extent, the metabolic patterns of P. cinnamomi were also altered when exposed to cork oak root exudates.     

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’.     

Pre‐ and post‐inoculation water stress affects Sphaeropsis sapinea canker length in Pinus halepensis seedlings

In order to assess the influence of water stress on the development of Sphaeropsis sapinea cankers in Pinus halepensis, the stems of 4‐ to 5‐year‐old potted seedlings were artificially inoculated with the fungus before and after being kept at controlled water regimes from April 1997 to March 1998. In the pre‐water‐stress inoculation experiment, the canker length, measured 5 months after inoculation (September 1997), was greater in seedlings predisposed to extreme water deficit (midday needle water potential between ?4.5 and ?5.5 MPa). In the post‐water‐stress inoculation experiment, the fungus was inoculated in April 1998, after irrigation had enabled the seedlings to resume normal needle water potential. In this case also, at 5 months after inoculation, longer cankers were visible in seedlings that had been subjected to extreme water deficit. These findings suggest that the occurrence of marked water stress, although apparently tolerated by Aleppo pine, can enhance the development of S. sapinea cankers in this species, regardless of whether the stress occurs before or after infection by the fungus.     

Effects of associated fungi Sclerophoma pythiophila and Cenangium ferruginosum on Gremmeniella abietina dieback in Spain

The interactions between Gremmeniella abietina and either Sclerophoma pythiophila or Cenangium ferruginosum, fungi frequently isolated from diseased twigs along with G. abietina, were studied under laboratory (dual cultures) and greenhouse conditions (double‐inoculations). Virulence of each species was also evaluated in greenhouse experiments by means of single‐inoculations. In vitro interactions were assessed on Petri dishes containing malt agar with pine needle extract, and greenhouse experiments were performed on 1‐year‐old Pinus halepensis seedlings. In vitro growth of G. abietina was inhibited by both fungi when grown in dual culture. In single‐inoculations, G. abietina caused the greatest necrosis length on P. halepensis seedlings, followed by S. pythiophila, whereas C. ferruginosum did not cause significant necrosis. In double‐inoculations, C. ferruginosum was able to reduce the length of necrosis caused by G. abietina on the P. halepensis seedlings. In contrast, necrosis length was greater in seedlings inoculated with both S. pythiophila and G. abietina than in those inoculated with G. abietina alone. Therefore, S. pythiophila seems to play a role in disease expression caused by G. abietina on P. halepensis in Spain.     

Effect of uninucleate Rhizoctonia on Scots pine and Norway spruce seedlings

One‐year‐old container‐grown seedlings were planted in spring on clear cut areas: the Norway spruce (Picea abies) on a moist upland site (Myrtillus‐type) and Scots pine (Pinus sylvestris) on a dryish upland site (Vaccinium‐type). While still in the nursery, half of the seedlings of each species had been inoculated during the previous summer, with a uninucleate Rhizoctonia sp., a root dieback fungus. At outplanting all the seedlings appeared healthy and had a normal apical bud, although the height of the inoculated seedlings was less than that of the uninoculated control seedlings. At the end of the first growing season after planting, the mortality of inoculated Scots pine and Norway spruce seedlings was 25 and 69%, respectively. After two growing seasons the mortality of inoculated seedlings had increased to 38% for Scots pine and 93% for Norway spruce. The mortality of control seedlings after two growing seasons in the forest was 2% for Scots pine and 13% for Norway spruce. After outplanting the annual growth of inoculated seedlings was poor compared with the growth of control seedlings. These results show that, although Rhizoctonia‐affected seedlings are alive and green in the nursery, the disease subsequently affects both their survival and growth in the forest.     

Effects of nickel and copper on growth and mycorrhiza of Scots pine seedlings inoculated with Gremmeniella abietina

Scots pine (Pinus sylvestris) seedlings were planted in soil originating from two localities with different background levels of nickel and copper. In addition, some of the seedlings were exposed to additional nickel (20.5 mg Ni/l of soil) or copper (63.5 mg Cu/l of soil), or a combination of both Ni and Cu, via soil without direct shoot exposure during their second growing period. The seedlings were either irrigated with spring water (pH 6) or got only natural rain during the whole field experiment. All seedlings were inoculated with conidia of a shoot‐pathogen Gremmeniella abietina during their third growing season, and harvested the following spring. Lengths of shoots of different year‐classes were used as growth estimates. In roots, the proportion of fungal (assumedly mycorrhizal) biomass was estimated by measuring ergosterol concentration. Guajacol peroxidase activity was measured. Short roots were classified into two groups according to their condition and the composition of the mycorrhizal community was expressed as a proportion of morphotypes in the roots. The seedlings exposed to additional Ni had higher shoot growth than the seedlings in the other treatments. The mean Ni concentration in the roots of seedlings exposed to additional Ni was 79 p.p.m. and in other seedlings 16 p.p.m. Additional Ni also decreased the frequency of clearly senescent short roots and the proportion of the mycorrhizal morphotype with the thinnest mantle. These results indicate that the Ni exposure levels used in this experiment had some positive effects on the seedlings. The relative fungal biomass was about 6% lower (p = 0.0981) in the fine roots of seedlings treated with additional Cu. The mean Cu concentration in the roots of seedlings exposed to additional Cu was 256 p.p.m. and in other seedlings 29 p.p.m. Peroxidase activity, which was used as a general stress indicator in this study, was not affected by any of the treatments. The shoot growth and the relative biomass of fungi in the fine roots were positively correlated in all seedlings, and this correlation was stronger in seedlings exposed to additional Ni that were not irrigated compared with seedlings not exposed to additional Ni that were irrigated. The frequency of asymptomatic infections of G. abietina was positively correlated with the proportion of senescent short roots in the irrigated seedlings but not in not‐irrigated seedlings. The general condition of seedlings may be an important factor for infection by G. abietina when moisture is high enough for the fungi to infect seedlings by conidia.     

Inoculation with the ectomycorrhizal fungi <Emphasis Type="Italic">Pisolithus arhizus</Emphasis> and <Emphasis Type="Italic">Scleroderma</Emphasis> sp. improves early growth of <Emphasis Type="Italic">Shorea pinanga</Emphasis> nursery seedlings

Trees of the family Dipterocarpaceae are the dominant trees in Southeast Asian tropical forests where they play an important ecological role and are also important commercially. An experiment was conducted to determine the effect of ectomycorrhizal fungi on the growth of dipterocarp species in peat soils. Seedlings of Shorea pinanga were inoculated with spores of two ectomycorrhizal fungi, Pisolithus arhizus and Scleroderma sp. were grown in pots containing sterilized peat soil for 7 months. The percentage of ectomycorrhizal colonization on S. pinanga exceeded 86%. Colonization of S. pinanga roots by ectomycorrhizal fungi resulted in increased shoot height, stem diameter, number of leaves, and shoot fresh and dry weight. Survival rates of S. pinanga were greater for inoculated seedlings than control seedlings. These results suggest that inoculation of ectomycorrhizal fungi can improve the early growth of S. pinanga grown in tropical forests and that this technique will accelerate the rehabilitation of degraded dipterocarp forests.     

蓝桉和尾叶桉混合菌根研究 Ⅱ.混合菌根的接种效应

接种苗木在生长量和生物量上均表现出极其显著的生长优势,ＥＣＭ真菌蜡蘑菌单接种及其与４种ＶＡ菌根菌剂混合接种,对苗木生长的促进作用尤为显著。在接种后１６周时,与对照苗相比,蓝桉和尾叶桉接种苗木的高生长量最大增幅分别为２８．８６％（ＬＳ）和８６．６５％（ＬＧ）;两种桉树地上部分平均干质量最大增幅分别为１２９．９３％（ＬＳ）和１３３．３４％（Ｌ）,地下部分分别为１１９．９３％（ＬＦ）和１７４．８３％（Ｌ     

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.     

Inoculation of ectomycorrhizal fungi contributes to the survival of tree seedlings in a copper mine tailing

To advance our understanding of the effects of inoculation with ectomycorrhizal fungi (EMF) on seedling colonization in mine wastelands, we conducted a field experiment in a copper tailing. Six-month-old seedlings of Japanese red pine (Pinus densiflora) and oak (Quercus variabilis) separately inoculated with three EMF species (Pisolithus sp., Cenococcum geophilum, Laccaria laccata) were transplanted to the copper tailing. The survival rates of tree seedlings were monitored monthly, and growth (biomass and height), contents of nutrients and heavy metals (K, P, Ca, Mg, Cu, Zn), and mycorrhizal infection rates of seedlings were determined 6 months after planting. Oak seedlings exhibited higher survival rates than pine seedlings after 6 months of growth on the tailing. EMF inoculations of pine seedlings significantly enhanced their survival, growth, and nutrient uptake. In contrast, EMF inoculations of oak seedlings improved growth only in terms of biomass. Additionally, EMF inoculation caused pine seedlings to accumulate more Cu and Zn in roots compared to non-inoculated seedlings, whereas inoculation inhibited the accumulation of heavy metals in shoots. However, similar results were not observed in oak seedlings. Observations of roots indicated that the rates of mycorrhizal infection of both tree species had dramatically declined at harvest time. In conclusion, ectomycorrhizal symbioses can improve the survival and performance of pine seedlings in mine tailings. The present study provided direct evidence of the importance of EMF inoculation of seedlings to the reforestation of mine wastelands.     

Rhizomorph production and stump colonization by co‐occurring Armillaria cepistipes and Armillaria ostoyae: an experimental study

In managed spruce forests, Armillaria cepistipes and A. ostoyae are efficient stump colonizers and may compete for these resources when they co‐occur at the same site. The aim of this experiment was to quantify the mutual competitive ability of the two Armillaria species in producing rhizomorphs and in colonizing Norway spruce (Picea abies) stumps. Five isolates of A. cepistipes and two isolates of A. ostoyae were simultaneously inoculated pair‐wise into pots containing a 4‐year‐old spruce seedling. For comparison, each isolate was also inoculated alone. One year after inoculation, stumps were created by cutting down the seedlings. Six months after creation of the stumps, rhizomorph production and stump colonization were assessed. Armillaria spp. were identified from 347 rhizomorphs and 48 colonized stumps. Armillaria cepistipes dominated both as rhizomorphs in the soil and on the stumps. Nevertheless, A. ostoyae was relatively more frequent on the stumps than in the soil and A. cepistipes was relatively more frequent in the soil than on the stumps. In both species, the ability to colonize the stumps in simultaneous inoculations was significantly reduced compared with single inoculations. In respect to rhizomorph production, simultaneous co‐inoculations had a slightly stimulatory effect on A. cepistipes and no significant effect on A. ostoyae. Our study suggests a rather neutralistic co‐existence of A. cepistipes and A. ostoyae as rhizomorphs in the soil. Concerning the ability to colonize stumps, the two species experience a mutual negative effect from the interaction, probably because of interspecific competition.     

Traumatic resin canals as markers of infection events in Douglas‐fir roots infected with Armillaria root disease

In response to an infection, traumatic resin canals (TRCs) are formed in the roots of many conifers, which may be used to determine the timing and sequence of infection events essential for epidemiological studies of root diseases. Juvenile Douglas‐fir (Pseudotsuga menziesii) tree roots at coastal and interior sites in British Columbia were wounded at various times of the year or were inoculated with an isolate of Armillaria ostoyae, and root sections were taken to determine the timing and extent of TRC formation. Naturally infected Douglas‐fir were also examined to determine the extent of the TRCs in infected and uninfected roots on infected trees and in the lower stem. Wounds made in March and October had poor or no TRC formation while the summer wounds responded strongly and were associated with resin soaking. Roots wounded in October did not respond until the following year in all trees except one. Trees produced TRCs and resin soaked tracheids at all times in response to the fungal inoculations. The most striking difference between wounding or fungal inoculation was the multiple bands of TRCs produced in response to the fungus. TRCs at natural A. ostoyae infections were found 92% of time in roots at the stem junction and 74% of the time in the stem at soil line. TRCs were produced in uninfected roots on infected trees but disappeared with increasing distance from the initiating lesion. TRCs can be used to time yearly and seasonal root infections when they can be traced from an identified lesion.     

Virulence of two Finnish Gremmeniella abietina types (A and B)

Thirteen Finnish Gremmeniella abietina isolates of types A and B were compared for differences in their virulence. Three kinds of inoculations were made: one with conidia on young Scots pine (Pinus sylvestris) seedlings and two with mycelia on the stems and shoots of young Scots pine trees. Inoculations with conidia were carried out in August 1992 and inoculations with mycelia were carried out six times between 11 August and 20 October, 1992. The experiments were evaluated in the late spring and early summer of 1993. The results showed that there was a difference in virulence between the two types. In the conidial inoculations type A infected 34.7% and type B infected 11.0% of the inoculated seedlings. For mycelial inoculations with type A the mean canker (stems) and necrosis (shoots) lengths were 19.3 mm and 8.6 mm longer, respectively, than with type B inoculations. In shoot inoculations there also was a clear difference between the two types in the number of such inoculations where no symptoms were observed. For type B shoot inoculations there was no fungal growth in 21.5% whereas for type A inoculations the figure was only 3.7%.     

Induction of hairy and normal roots on Picea abies,Pinus sylvestris and Pinus cortorta by agrobacterium rhizogenes

Two‐ to three‐week‐old seedlings of Picea abies, Pinus sylvestris and Pinus contorta were inoculated with the supervirulent strain Agrobacterium rhizogenes R1600 2cm above the base of the hypocotyl; other treatments included inoculation with Agrobacterium tumefaciens strain A281 (pTVK291) or mock inoculation. Hairy or highly branched roots formed on 2–3% of seedlings of all 3 species that were infected by R1600 but on none of the other seedlings. Normal adventitious roots were also induced on 5–10% of Agrobacterium‐treated seedlings but none appeared on the other seedlings. Opine assays and growth rates of hairy and normal roots indicated that only roots with the hairy morphology were transformed.     

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.     

Effect of season and different fungi on phenolics in response to xylem wounding and inoculation in Eucalyptus nitens

Pot‐grown and plantation‐grown Eucalyptus nitens trees (approximately 2 and 3 years old, respect‐ively) were experimentally wounded and inoculated with different fungi and in different seasons. Decay lesion development and defence zones were assessed. Two zones were described, a narrow brown decay interface (interface reaction zone, IRZ) and a diffuse zone beyond this being either pale brown or purple (reaction zone, RZ). The total phenol levels in the reaction zone were determined. Selected phenolics (pedunculagin, tellimagrandin 1, tetragalloylglucose, pentagalloylglucose and catechin) were quantified by liquid chromatography–mass spectrometry (LC–MS). A range of fungi (mainly decay‐causing) were used to inoculate wounds and the results indicated that more extensive decay lesions were generally associated with greater production of soluble phenols in response. Sterile inoculations and weakly aggressive fungi were associated with no or little xylem discoloration, whereas aggressive fungi elicited more discoloration and phenolic accumulation in advance of infection. This indicates that phenol accumulation is not a generalized response to wounding, but a variable response due to the interaction between microorganisms and sapwood. In plantation‐grown trees examined 6 months after wounding, purple reaction zones were commonly associated with large decay lesions. Seasonal differences in decay column area caused by Ganoderma applanatum were not significant 1 month after wounding and inoculation.     

Effects of benomyl and captan on growth and mycorrhizal colonization of Sitka‐spruce (Picea sitchensis) and ash (Fraxinus excelsior) in Irish nursery soil

The effects of two fungicides (benomyl and captan ‐ at recommended doses and up to three soil drenches) on root development and mycorrhizal colonization of Sitka‐spruce (Picea sitchensis) and ash (Fraxinus excelsior) were examined after 20 weeks growth, under environmentally controlled conditions, in soil from an Irish tree nursery. Although four mycorrhizal types have been found on Sitka‐spruce at the nursery, only one ectomycorrhizal type (Piceirhiza horti‐inflata) was identified on the short roots in this study. An inoculant (Vaminoc: MicroBio Ltd, Hemel Hempstead, UK) was used to inoculate ash and 20‐week‐old seedlings had a higher arbuscular mycorrhizal (AM) colonization in comparison with uninoculated controls. Multiple applications (2–3) of benomyl reduced the length of root and shoot and shoot dry mass of Sitka‐spruce, whereas in ash, it only depressed root length. Benomyl decreased the numbers of ectomycorrhizas of Sitka‐spruce and arbuscular mycorrhizal colonization of Vaminoc‐inoculated ash. A single application of captan stimulated root length and ectomycorrhizal colonization of Sitka‐spruce and root dry mass in ash compared with uninoculated controls. Applications of captan reduced arbuscular mycorrhizal colonization of Vaminoc‐inoculated ash to levels near to those of uninoculated controls. Of the two fungicides used, benomyl had the most deleterious effect on root length and mycorrhizal colonization of Sitka‐spruce and ash.     

Conifer seedling root fungi and root dieback in Finnish nurseries

Fungi were isolated from the roots and growth substrate of bare‐rooted and containerized Pinus sylvestris and Picea abies nursery seedlings displaying a root dieback. Isolations were also made from visually healthy seedlings. The potential pathogenicity of all isolated species was determined in laboratory trials.

Cylindrocarpon spp., Fusarium spp. and Trichoderma viride were frequently isolated. The isolation frequency of a uninucleate Rhizoctonia‐like fungus, Pythium spp. and Phytophthora imdulatum from diseased containerized seedlings and their pathogenicity in tests suggest that these fungi are likely involved in the root dieback disease in containers. The pathogenic Rhizoctonia‐like fungus in addition to Pythium spp. was also isolated from bare‐rooted seedlings. In greenhouse tests Pythium spp. were more pathogenic to 4‐week‐old Scots pine seedlings grown before transplantation in unsterile substrate than to those seedlings grown axenically in agar. External factors are considered to have some role in the expression of disease.     

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.