Effects of combined drought and pathogen stress on growth, resistance and gene expression in young Norway spruce trees.

Drought-induced mortality is a major direct effect of climate change on tree health, but drought can also affect trees indirectly by altering their susceptibility to pathogens. Here, we report how a combination of mild or severe drought and pathogen infection affected the growth, pathogen resistance and gene expression in potted 5-year-old Norway spruce trees [Picea abies (L.) Karst.]. After 5 weeks of drought, trees were inoculated with the fungal pathogen Endoconidiophora polonica. Combined drought-pathogen stress over the next 8 weeks led to significant reductions in the growth of drought-treated trees relative to well-watered trees and more so in trees subjected to severe drought. Belowground, growth of the smallest fine roots was most affected. Aboveground, shoot diameter change was most sensitive to the combined stress, followed by shoot length growth and twig biomass. Both drought-related and some resistance-related genes were upregulated in bark samples collected after 5 weeks of drought (but before pathogen infection), and gene expression levels scaled with the intensity of drought stress. Trees subjected to severe drought were much more susceptible to pathogen infection than well-watered trees or trees subjected to mild drought. Overall, our results show that mild drought stress may increase the tree resistance to pathogen infection by upregulating resistance-related genes. Severe drought stress, on the other hand, decreased tree resistance. Because drought episodes are expected to become more frequent with climate change, combined effects of drought and pathogen stress should be studied in more detail to understand how these stressors interactively influence tree susceptibility to pests and pathogens.

Long-lasting memory of jasmonic acid-dependent immunity requires DNA demethylation and ARGONAUTE1.

Stress can have long-lasting impacts on plants. Here we report the long-term effects of the stress hormone jasmonic acid (JA) on the defence phenotype, transcriptome and DNA methylome of Arabidopsis. Three weeks after transient JA signalling, 5-week-old plants retained induced resistance (IR) against herbivory but showed increased susceptibility to pathogens. Transcriptome analysis revealed long-term priming and/or upregulation of JA-dependent defence genes but repression of ethylene- and salicylic acid-dependent genes. Long-term JA-IR was associated with shifts in glucosinolate composition and required MYC2/3/4 transcription factors, RNA-directed DNA methylation, the DNA demethylase ROS1 and the small RNA (sRNA)-binding protein AGO1. Although methylome analysis did not reveal consistent changes in DNA methylation near MYC2/3/4-controlled genes, JA-treated plants were specifically enriched with hypomethylated ATREP2 transposable elements (TEs). Epigenomic characterization of mutants and transgenic lines revealed that ATREP2 TEs are regulated by RdDM and ROS1 and produce 21 nt sRNAs that bind to nuclear AGO1. Since ATREP2 TEs are enriched with sequences from IR-related defence genes, our results suggest that AGO1-associated sRNAs from hypomethylated ATREP2 TEs trans-regulate long-lasting memory of JA-dependent immunity.

A model for peak and width of signaling windows: Ips duplicatus and Chilo partellus pheromone component proportions--does response have a wider window than production?

Pheromone communication systems have a reliable signal with a restricted window of amounts and ratios released and perceived. We propose a model based on a Gaussian response profile that allows a quantification of the response peak (location of optimum) and a measure of the peak width (response window). Interpreting the Gaussian curve, fitted by nonlinear regression (NLR), as a standard normal distribution, the peak location equals the mean (it) and the window width equals 2 x the standard deviation (2sigma). The NLR procedure can provide an objective measure for both peak location and width for a wide range of data sets. Four empirical data sets as well as 10 literature data sets were analyzed. The double-spined spruce engraver, Ips duplicatus, was field tested in four populations to find the optimum proportion for attraction to the two male aggregation pheromone components, ipsdienol (Id) and (E)-myrcenol(EM), ranging from 0 to 100% of Id. Tests in Norway and the Czech Republic confirmed the preference of western populations for a blend between 50 and 90% Id. A population in Inner Mongolia showed a preference for traps with the 10 and 50% Id baits. The NLR fitted values for response peak and width (mu; 2sigma) were: Norway 0.64, 0.73; Czech Republic 0.53, 0.73; NE China 0.77, 0.29; and Inner Mongolia 0.33, 0.50. The signal produced by Norwegian field-collected males had a narrower window width (2sigma = 0.12). Males of the maize stem borer, Chilo partellus, were tested in a flight tunnel for their response to variation in the two major female sex pheromone gland components, (Z)- l1-hexadecenal and the corresponding alcohol (OH). Variation of the alcohol in seven levels from 2 to 29% OH showed the highest male response for 17% OH. For all behavioral steps, the peak of male response was near mu = 0.14, while the window width fell from 2sigma = 0.5 to 0.2 for eight sequential behavioral steps from take-off to copulation. Female production had a similar peak location (mu = 0.13) but a narrower width, 2sigma = 0.14. Literature data from other moth species showed similar patterns, with a wider male response relative to the female production windows. Literature data on response to enantiomer ratios in a hymenopteran and to pheromone amounts in a dipteran were also described by our model. In a bark beetle population (Ips pini), with two hybridizing enantiomeric strains, the production peaks were narrower (0.1) than the response peaks (0.5). Thus, it in general, seems that in the pheromone systems analyzed, the width of the response window (2sigma = 0.1 to 0.8) is larger than that of the production window (2sigma = 0.03 to 0.14), irrespective of the sex of the sender.

Phloem parenchyma cells are involved in local and distant defense responses to fungal inoculation or bark-beetle attack in Norway spruce (Pinaceae).

The anatomical response of Norway spruce bark polyphenolic parenchyma cells (PP cells) to inoculation with the phytopathogenic fungus Ceratocystis polonica and attack by its bark-beetle vector Ips typographus was examined. Fungal inoculation on the periderm surface had no effect, while inoculation just below the periderm or halfway into the phloem (mid-phloem) generated detectable responses within 3 wk. The responses included increase in PP cell size and in periodic acid-Schiff's staining of PP cell phenolics, wound periderm initiation from PP cells, and cambial zone traumatic resin duct formation. Fungi were not seen in samples 3 wk after subperiderm or mid-phloem inoculation, but were found in some samples 6 and 9 wk after mid-phloem inoculation. In contrast, inoculations into the cambium resulted in partial (3 wk) or complete (6 and 9 wk) fungal colonization and death of tissue in the infected area. This indicates that PP cells have defenses capable of inhibiting fungal growth. Samples taken near bark-beetle galleries had similar anatomical responses as inoculated samples, validating the inoculation approach to studying defense responses in spruce. These results show that PP cells represent not only a constitutive defense system, but are also involved in local and remote inducible defenses against fungal and beetle attack.

Induced resistance to pathogenic fungi in norway spruce

Norway spruce (Picea abies) trees (approximately 16 m high) of a single clone were used to study the effects of fungal infection and wounding on induction of resistance to the bark beetle-associated bluestain fungus Ceratocystis polonica. A dose-response experiment was designed involving three different dosages of fungal (fungus and wound) and sterile agar (wound) pretreatment inoculations (10, 50, or 100 inoculations/m(2) on the stem between 0.8 and 2.0 m high). Three weeks after pretreatment, trees were challenged with a massive C. polonica inoculation (400 inoculations/m(2)). Control trees that received no pretreatment were heavily colonized and killed by the challenge inoculation. The high and medium fungal pretreatments reduced subsequent fungal colonization success by 76% to 97% relative to the control, and fungal pretreatments protected the trees much more efficiently than sterile agar pretreatments. The protection was demonstrated to be local and not systemic in a subsequent experiment, where trees were pretreated with the medium fungal dosage on the lower bole and challenge inoculated further up the stem. Protection was also demonstrated to be pathogen nonspecific, as trees that had been pretreated with a medium dosage of the root rot fungus Heterobasidion annosum showed enhanced resistance to challenge inoculation with C. polonica.

Mechanical injury and fungal infection induce acquired resistance in Norway spruce.

Norway spruce trees (Picea abies (L.) Karst.) pretreated by wounding and fungal infection showed highly enhanced resistance to a subsequent challenge inoculation with the pathogenic bluestain fungus Ceratocystis polonica (Siem.) C. Moreau. This is the first time the effectiveness of the constitutive and inducible defenses has been shown to depend on prior wounding and infection in conifers, although such acquired resistance has previously been found in several angiosperms. Trees that were pretreated with a combination of 12 bark wounds (1.6 x 10 cm), four fungal inoculations and four sterile inoculations 1-15 days before mass inoculation with C. polonica at 400 inoculations per square meter over a 0.8 m stem section had significantly shorter necroses in the phloem, less bluestained sapwood, and less dead cambium than untreated control trees. Pretreatment with four fungal or sterile inoculations alone did not lead to enhanced resistance. Pretreatment by bark wounding alone seemed to provide an intermediate degree of resistance compared to bark wounding, fungal inoculations and sterile inoculations combined. All trees had a marked increase in the number of resin ducts in the year of inoculation compared with previous years, suggesting that formation of traumatic resin ducts play an important role in the development and maintainance of enhanced resistance.

Pathogenicity of four blue-stain fungi associated with aggressive and nonaggressive bark beetles.

ABSTRACT The pathogenicity of two isolates of each of four bark beetle-associated blue-stain fungi was evaluated after mass inoculation of about 40-year-old Norway spruce trees (Picea abies). Trees were inoculated with a different isolate of each fungus in 1995 and 1996 at a density of 400 inoculations per m(2) in a 1.2-m-wide band on the lower bole (about 270 inoculations per tree). Trees were felled 15 weeks after inoculation. In 1995, Ceratocystis polonica was the only fungus that had stained the sapwood (56.3% of cross-sectional sapwood area). It induced five times longer phloem necroses, 21 times more dead cambium, and 11 times more dead phloem than any other fungus. In 1996, C. polonica induced less extensive host symptoms and an unidentified Ambrosiella sp. induced comparable symptoms to C. polonica in the phloem and cambium. No trees showed any foliar symptoms 15 weeks after inoculation, but six out of eight trees inoculated with C. polonica in 1995 had only 0 to 25% functional sapwood and probably would have died if felling had been delayed. This study confirms that C. polonica, an associate of the aggressive bark beetle Ips typographus, is pathogenic to Norway spruce. The pathogenicity of the Ambrosiella sp., which is associated with a nonaggressive bark beetle, seems moderate and varies between isolates. The two remaining fungi included in this study (Ophiostoma piceae and a dark fungus with sterile mycelium), which are associated with nonaggressive bark beetles, were nonpathogenic in both experiments. These results are consistent with the hypothesis that aggressive bark beetle species vector virulent fungi that may help them kill trees, but the results also show that some nonaggressive bark beetles may vector phytopathogenic fungi.