Asymptomatic Trees Research Articles

The role of tyloses in crown hydraulic failure of mature walnut trees afflicted by apoplexy disorder

In the Central Valley of California, mature walnut trees afflicted with apoplexy disorder exhibit rapid and complete crown defoliation within a few weeks of symptom initiation. Symptoms are typically found throughout the entire crown and are initially expressed as wilting and chlorosis followed by scorching of leaves. Since the cause of apoplexy disorder is unknown, we set out to elucidate the water relations physiology underlying this condition. Stem water potential (Psi(s)) of healthy, asymptomatic trees remained high throughout the 2007 growing season, while that of apoplexy-afflicted trees decreased significantly with the onset of symptoms for both healthy-appearing and symptomatic portions of these trees. Psi(s)s were significantly reduced by at least 0.7 MPa in the lower, middle and upper portions of the symptomatic canopies compared with those from asymptomatic trees. Heat pulse velocities measured in the main trunk at three radial depths consistently decreased prior to the onset of symptoms and eventually reached zero with complete crown defoliation. Comparison of sap flow with predicted water use based on walnut evapotranspiration suggests that stomata of symptomatic trees were closing at higher evaporative demand prior to symptom formation. Specific hydraulic conductivity (K(s)) of symptomatic stems was significantly lower than that of asymptomatic stems, and no detectable K(s) could be measured on several of the symptomatic stem samples. However, shallow root K(s) did not significantly differ between symptomatic and asymptomatic trees, suggesting that hydraulic failure was isolated to the crown of these grafted trees. Light and scanning electron microscopy of stem and trunk sapwood revealed extensive tylose development in vessels throughout the crown of symptomatic trees. Analysis of the formation of tyloses on multiple dates revealed rapid development of these vessel occlusions in conjunction with visual symptom formation and dramatic decreases in sap flow. In 2008, tylose development was associated with elevated ethylene production in the active sapwood of symptomatic trees. The cause of elevated ethylene associated with tylose production and symptom formation is yet to be determined.

Austrocedrus chilensis growth decline in relation to drought events in northern Patagonia, Argentina

The significant mortality of the Austrocedrus chilensis (D. Don) Pic. Serm. et Bizarri forests, locally known as “Mal del Ciprés”, has been reported since 1945 for most sites across its distribution in Argentina. However, the cause of this decline is still a topic of discussion. In this study, radial growth patterns from symptomatic and asymptomatic A. chilensis trees were analyzed to determine the influence of drought events on tree growth. Fifty pairs of symptomatic and asymptomatic trees with similar DBH, competition, and microsite conditions were cored at five pure A. chilensis stands near El Bolsón, Río Negro, Argentina. A reference chronology from nonaffected trees was used to cross-date all cores and to determine the relationship between A. chilensis radial growth and climate. The growth of A. chilensis is favored by above average precipitation in late spring–early summer (November and December). A strong relationship was also observed between radial growth patterns and the Palmer drought severity index, a measure of the regional water deficit. Significant differences in growth patterns were recorded between symptomatic and asymptomatic trees. Following extreme drought events, the growth of symptomatic trees is consistently lower than in asymptomatic trees. Based on the larger number of droughts recorded during the past decades and on future climatic predictions suggesting increasing trends in the frequency and intensity of drought events in northern Patagonia, a gradual increase in the number of trees affected by “Mal del Ciprés” along the twenty-first century is likely expected.

First Report of the Huanglongbing Bacterium 'Candidatus Liberibacter asiaticus' Infecting Satkara (Citrus macroptera) in India.

In India, satkara (Citrus macroptera) is found in the northeastern regions of Shella and Dowki near Cherrapunji in Meghalaya, the Jampui Hills of Tripura and Mizoram, and the Chendel District of Manipur. Locally, it is called satkara because 'sat' refers to multiples of seven and the fruit generally contains fourteen segments. The fruit is used in the preparation of pickles and its oil is used in the perfume industry. In January 2007, in Behliangchhip, Jampui Hills, Tripura, India, we noticed seven satkara trees of 26 showing leaf mottling and yellowing in young shoots, symptoms typical of huanglongbing (HLB). HLB is a destructive citrus disease caused by a nonculturable, phloem-limited alpha-proteobacterium of the genus 'Candidatus Liberibacter'. Among the three known 'Ca. Liberibacter' species (Ca. Liberibacter asiaticus, Ca. L. africanus, and Ca. L. americanus), only Ca. L. asiaticus has been reported in India (1) but not from satkara. To identify the pathogen, total DNA was extracted and purified from 200 mg of leaf midribs from five symptomatic and five symptomless trees with the DNeasy Plant Mini Kit (Qiagen, Gmbh, Hilden, Germany) as per the manufacturer's instructions. Samples were tested for 'Ca. L. asiaticus' by PCR with primer sets OI1/OI2c targeting the 16S rDNA locus (3) and A2/J5 targeting the beta-operon locus of ribosomal proteins (2). DNA samples from all symptomatic leaves were positive with both primer sets. The OI1/OI2c primers generated an amplicon of approximately 1,160 bp and digestion of the amplicon by XbaI yielded two DNA fragments of approximately 640 bp and 520 bp, suggesting the presence of 'Ca. L. asiaticus'(3). The A2/J5 primer set generated an ~703-bp amplicon, indicating 'Ca. L. africanus' was not present. No DNA was amplified from the asymptomatic satkara trees. One each of the OI1/OI2c and A2/J5 amplicons was purified, cloned (pGEM-T Easy vector, Promega, Hampshire, UK) and sequenced (Bangalore Genei, Bengaluru, Karnataka, India). The derived sequences, 1167 bp for the OI1/OI2c amplicon and 703 bp for the A2/J5 amplicon, have been deposited in the GenBank database under Accession Nos. GQ369792 and GU074017, respectively. Sequence comparison revealed that GQ369792 and GU074017 shared >99% identity to the corresponding regions of 'Ca. L. asiaticus' in the GenBank database. No DNA was amplified by PCR with primer set GB1/GB2 (4) specific for 'Ca. L. americanus'. To our knowledge, this is the first report of molecular identification of 'Ca. L. asiaticus' infecting satkara in India.

Using hyperspectral remote sensing to detect and quantify southeastern pine senescence effects in red-cockaded woodpecker ( Picoides borealis) habitat

Conservation of threatened and endangered species requires maintenance of critical habitat. The red-cockaded woodpecker Picoides borealis (RCW) is a threatened bird species, endemic to the mixed conifer forests of the southeastern United States. RCW nests and forages preferentially in mature longleaf pine Pinus palustris, but also uses mature loblolly pine Pinus taeda and shortleaf pine Pinus echinata forests. In the last century, the extent of mature pine forests has been greatly reduced by logging. The RCW, in contrast to other woodpeckers, excavates nest cavities in living trees and senescence symptoms (year round leaf chlorosis and leaf mortality) have been observed in mature pine stands across the southeast. Widespread mortality of the mature pine forests would threaten the long-term survival of the RCW. We used airborne hyperspectral data across a portion of Ft. Benning Military Installation, Georgia, U.S.A., to determine if senescent trees can be identified and mapped and assess the likely persistence of mature pines in the RCW habitat. Univariate analysis of variance showed good separation between asymptomatic, senesced and dead physiological conditions with asymptomatic trees having significantly higher reflectance for all bands in the wavelength range between 0.719 and 1.1676 µm, senescent trees having significantly lower reflectance for bands in the range between 1.1927 and 1.3122 µm, and dead trees having significantly higher reflectance for bands in the range between 1.8151 and 1.9471 µm. Classification and Regression Tree (CART) models achieved correct classification rates and kappa statistics above 70%. CART models selected information from wavelength regions similar to those identified from the ANOVA, which likely explains their performance. Our aggregated CART model of tree senescence estimated that 141.4 ha (3%) of the study area is affected. RCW nests occurred in areas with significantly higher tree cover, and trees within foraging and home ranges did not have significantly more senescence than areas without RCW. These results indicate that although tree senescence is widespread, mortality is yet to significantly affect RCW habitat. Results and analysis of critical habitat similar to those exemplified in this study can extend our knowledge about the stressors of these important and imperiled components of biodiversity.

The opportunistic pathogen,Neofusicoccum australe, is responsible for crown dieback of peppermint (Agonis flexuosa) in Western Australia

Recently, severe dieback symptoms have been observed in Agonis flexuosa, a tree native to the south-west of Western Australia. It was suspected that these symptoms were caused by an opportunistic pathogen and a sampling strategy was designed to investigate the causal agent of the cankers in symptomatic trees and the presence (or absence) of this pathogen as an endophyte of asymptomatic trees. Neofusicoccum australe was the most commonly isolated species from cankers of A. flexuosa. It was also the only species isolated which was a known canker pathogen. A greater diversity of fungi was isolated as wood endophytes of asymptomatic A. flexuosa, but N. australe was again the most common associate. In several cases the same vegetative compatibility group of N. australe was found in cankered and asymptomatic trees. The pathogenicity of isolates of N. australe from cankered and asymptomatic trees towards seedlings of A. flexosa was similar. These data support the hypothesis that the recent dieback of A. flexuosa is caused by a common fungal endophyte, which is capable of causing disease in a stressed host. The disease is not caused by an introduced pathogen. The inciting factors leading to the decline are still unknown.

First Report of Chalara fraxinea on Common Ash in Italy.

In many European countries, the anamorphic Chalara fraxinea Kowalski (teleomorph Hymenoscyphus albidus [Roberge ex Desm.] Phillips; 1-3) is responsible for a severe and rapidly spreading dieback of common ash (Fraxinus excelsior L.) since it was first reported in Poland. Recently, this disease was added to the EPPO Alert List and the NAPPO Phytosanitary Alert System. Symptomatic trees were observed in a 1.8-ha ash-maple forest in northeastern Italy (Fusine, UD; 46°30'N, 13°37'E; 782 m above sea level) along the Italo-Slovenian border in July 2009. Symptoms were found on approximately 10% of mature common ash and 70% of seedlings. Main symptoms were shoot, twig, and branch dieback, wilting, and bark cankers (1). Fungal fruiting bodies were not found on or near the canker surface. Furthermore, longitudinal and radial sections through the cankers revealed gray-to-brown xylem discoloration. One symptomatic 3-year-old plant was randomly selected and from the necrotic margin of one canker previously surface-sterilized with 3% sodium hypochlorite and rinsed, four 2-mm-wide chips were placed on malt extract agar (MEA) and incubated at 21 ± 1°C in the dark. Among a variety of microorganisms, after 19 days, slow-growing colonies (mean radius of 12 mm) appeared that were effuse, cottony, and often fulvous brown but sometimes dull white with occasional gray-to-dark gray patches. The purified isolate was then transferred to the same medium at 4 ± 1°C in the dark, and after 11 days, hyaline-to-dark gray phialides were observed producing numerous conidia in slimy droplets and sometimes in chains. Phialophores measured 8.6 to 21.0 (15.1) μm long (n = 20), 4.2 to 13.4 (8.8) × 3.6 to 5.5 (4.7) μm at the base, and 5.2 to 8.7 (6.5) × 2.5 to 3.1 (2.8) μm at the collarette; conidia measured 2.8 to 4.2 (3.4) × 1.9 to 2.5 (2.2) μm (n = 40); and first formed conidia measured 5.5 to 6.5 (5.9) × 1.8 to 2.5 (2.1) μm (n = 20). These morphological characteristics matched Kowalski's (1) description of C. fraxinea. In August of 2009, the fungal isolate was used to test pathogenicity with current year shoots of 25 6-year-old (150 to 210 cm high) asymptomatic common ash trees under quarantine conditions (Slovenian Forestry Institute's experimental plots). For every plant, the bark of the main shoot (10 to 13 mm in diameter) was wounded with a 6-mm-diameter cork borer. Twenty saplings were inoculated with one 6-mm-diameter mycelial plug obtained from the margin of a 26-day-old culture (MEA), while five saplings were inoculated with sterile MEA plugs. All wounds were sealed with Parafilm and aluminum foil. After 28 days, all plants inoculated with the C. fraxinea showed bark lesions (2 to 39 mm long, mean 7 mm) and wood discoloration (6 to 85 mm long, mean 22 mm) from which the pathogen was reisolated. These symptoms were absent from controls and the pathogen was never reisolated. To our knowledge, this is the first report of C. fraxinea in Italy. Investigations on its presence in all Fraxinus species naturally growing in the investigated area and in the nearest regions are in progress. The obtained isolate is preserved in both Padova and Ljubljana herbaria as CFIT01.

Detection and characterization of Citrus tristeza virus stem pitting isolates in Jamaica

An island wide survey for Citrus tristeza virus (CTV) in citrus orchards across Jamaica (13 regions) was conducted over 2 years. Trees (1, 885) showing virus-like symptoms as well as asymptomatic trees were randomly sampled for testing by ELISA and 55 samples from the 6 major citrus growing regions were graft inoculated on indicator plants. Most samples (74%) reacted to polyclonal antibodies against CTV in ELISA, while 20% were positive in tests using monoclonal antibodies specific to severe CTV strains. Samples collected from the 6 major citrus growing regions produced vein clearing and stem pitting symptoms on Mexican lime indicator plants (87%). In addition, stem pitting symptoms were induced on Duncan grapefruit, sweet orange, sour orange or sweet orange grafted on sour orange. Nucleotide sequencing of the coat protein gene sequences isolated from these samples indicated high identities (88 to 95.5%) among the Jamaican isolates and previously reported stem pitting strains from Central and North America and Eurasia (88 to 100%). The results suggest a shared ancestry with isolates from other geographical locations, rather than geographical speciation, and presumably separate CTV introductions into Jamaica.

Genetic Diversity of Prunus necrotic ringspot virus Isolates Within a Cherry Orchard in New York.

A survey for Prunus necrotic ringspot virus (PNRSV) in an orchard of Prunus cerasus cv. Montmorency and Prunus avium cv. Hedelfingen in New York by enzyme-linked immunosorbent assay indicated an eightfold higher infection rate in sour cherry (33%, 32 of 96) than in sweet cherry (4%, 6 of 136) trees. The presence of PNRSV was confirmed by reverse transcription-polymerase chain reaction and amplification of the coat protein (CP) gene in total RNA from infected leaf tissue. Latent infection was prevalent in the majority of trees infected (87%, 33 of 38), while a few of them exhibited shock symptoms or had severely reduced growth (13%, 5 of 38). Asymptomatic PNRSV-infected trees clustered in spatial proximity to symptomatic trees. Sequence analysis of the CP gene (675 bp) indicated a population structure consisting of one predominant molecular variant for 10 isolates and six minor molecular variants for seven isolates. A high sequence identity was found between the CP gene of PNRSV isolates from cherry trees and other isolates from diverse hosts and various geographic origins at the nucleotide and amino acid levels (88 to 100%). Phylogenetic analyses showed a clustering of PNRSV isolates from cherry trees in New York in the predominant group PV-96.

Leaf Chlorophyll Fluorescence Parameters and Huanglongbing

Chlorophyll fluorescence and photochemical and nonphotochemical quenching parameters were measured in 20 genotypes of Citrus spp. or relatives grown in the greenhouse and commercial ‘Valencia’ sweet orange (Citrus sinensis) trees at two Florida locations. The purpose was to determine the utility of measurements for early huanglongbing [HLB (Candidatus Liberibacter asiaticus)] detection in asymptomatic trees and to examine the leaf response to HLB infection. Polymerase chain reaction (PCR)-negative healthy and PCR-positive symptomatic, asymptomatic, and distant asymptomatic leaves were used for fluorescence analysis using a portable chlorophyll fluorometer. Greenhouse-grown genotypes were separated into mild, moderate, and severe symptom groups based on leaf mottling, color, and size. In general, mild symptom genotypes were characterized by increased photosystem II (PSII) excitation pressure and unregulated heat dissipation and decreased regulated heat dissipation, whereas moderate and severe symptom genotypes increased loss of photosynthetic efficiency and increased unregulated and regulated heat dissipation. Distant asymptomatic leaves could be distinguished from healthy ones in moderate and severe symptom genotypes by increased total and regulated heat dissipation measurements. In the field, overall photosynthetic efficiency and total regulated heat dissipation measurements could distinguish between healthy and asymptomatic ‘Valencia’ sweet orange leaves at the location with slow or more recent infection, but not at the location where infection appeared to progress faster or was of longer duration. Starch content followed a similar pattern. The results indicate that no single measurement uniquely described the relationship between HLB and the host in asymptomatic and healthy leaves, but accuracy of field-based detection could be strengthened by a combination of total nonphotochemical quenching, overall photosynthetic efficiency, starch content, and PCR analyses. Chlorophyll fluorescence and quenching measurements suggest a PSII-based explanation for, and temperature dependency of, leaf symptom development.

Ozone sensitivity and ethylenediurea protection in ash trees assessed by JIP chlorophyll a fluorescence transient analysis

The effect of ethylenediurea (EDU) was tested using the chlorophyll (Chl) a fluorescence transient analysis, performed with JIP-test, to assess ambient ozone (O3) effects on photosynthesis of adult trees under natural conditions. Twelve adult European ash (Fraxinus excelsior L.) trees, known to be sensitive or tolerant to O3, determined by presence symptomatic (S) or absence asymptomatic (AS) trees of foliar symptoms in previous years, were treated either with distilled water containing 450 g m-3 EDU or with distilled water. Once a month across the growing season [the accumulated exposure over a threshold of 40 nmol(O3) mol-1 was 32.49 µmol mol-1 h-1], Chl a fluorescence transients were measured in vivo on dark-adapted leaves of 1-year-old labeled shoots, from the lower crown part. Twenty-five parameters were calculated. The maximum quantum yield of primary photochemistry (ϕPo or Fv/Fm) did not differentiate between S-and AS-trees, while increased Chl content and de-excitation rates suggested compensation of O3 injury in S-trees. Seasonal reductions in absorbing fluxes and increase in heat and fluorescence dissipation processes was due to leaf ageing and drought, the latter suggesting water deficit influenced Chl a fluorescence stronger than ambient O3 exposure. AS-trees showed elevated probability of connectivity among photosystem 2 units, a mechanism to stimulate energy dissipation and reduce photo-oxidative injury. EDU prevented the inactivation of reaction centers. This slight effect does not warrant EDU as a tool to assess O3 effects on photosynthesis, while the JIP-test is suggested for a quantitative assessment in adult trees.

Root recovery rates for Phytophthora cinnamomi and rate of symptom development from root rot on Abies fraseri trees over 7 years

SummaryPhytophthora root rot on Abies fraseri trees was monitored from 2001 to 2007 within the disease front of a 12‐year‐old Virginia plantation where trees had been dying of the disease since 1994. After a slow increase in early foliage symptom development from July 2001 to September 2002, the frequency of A. fraseri trees with early symptoms accelerated for about 15 months. While the slow increase occurred during a 18.7% lower than normal rainfall period and the acceleration occurred during a 31.2% higher than normal rainfall period, the percentage of trees with early symptoms continued to increase during the mid‐winter months (December–February) when the estimated mean minimum daily soil temperature (25 cm depth) was unfavourable (<10°C) to Phytophthora cinnamomi pathogenic activity. The time required for trees to progress from early foliage symptoms to completely dead foliage, from November 2000 to October 2007, was highly variable, ranging from 4 to 35 months. Root recovery rates for P. cinnamomi, assayed on a selective medium, were 6.4 times greater for symptomatic foliage trees than for asymptomatic foliage trees in this deep, silt‐loam soil. Following an atypical cold period in February 2007, when the mean minimum daily soil temperature was 0.8°C, symptomatic roots yielded only a low level of germinable propagules of P. cinnamomi. Further, during an atypical midsummer in 2007 (June–August), when the soil water potential was at or below −9 bars for 68 of 92 days, symptomatic roots yielded no germinable propagules of P. cinnamomi. Addition of thiophanate‐methyl to the selective medium aided P. cinnamomi isolation by inhibiting many undesired pythiaceous colonies growing from symptomatic roots.

Phytoplasma associated with witches’-broom disease of Ulmus minor Mill. in the Czech Republic: Electron microscopy and molecular characterization

Visual inspections of elm trees in south Moravia in 1997-2007 revealed a rare occurrence of plants with smaller and cowl-forming leaves on some twigs, i.e. a feature resembling witches'-broom disease observed on the end of twigs. The presence of phytoplasma-like bodies was observed by transmission electron microscopy of phloem tissue. On the other hand, no phytoplasmas were found in asymptomatic trees. Nucleic acids extracted from these plants were used in nested-PCR assays with primers amplifying 16S rRNA sequences specific for phytoplasmas. Sequence analyses of the 16S-23S ribosomal operon (1852 bp) allowed for the classification of the detected phytoplasmas in the elm yellows group, but its position remained on the boundary of the 16SrV-A and 16SrV-C ribosomal subgroups. Sequence analyses of the ribosomal protein of the rpl22-rps3 and secY genes lead to further classification and revealed the phytoplasmas' affiliations to the 'Candidates Phytoplasma ulmi'. Some exceptions in unique oligonucleotide sequences defined for 'Ca. Phytoplasma ulmi' were found in the Czech isolate. This is the northernmost confirmed occurrence of phytoplasma on elm trees within Europe.

Location ofXanthomonas translucensin pistachio trees

Xanthomonas translucens has been identified as the causal agent of pistachio dieback in Australia. Symptoms include decline, xylem staining, trunk and limb lesions, and excessive exudation of resin. Bacteria were previously isolated from stained wood in 2-year-old twigs but little was known about their presence in other parts of the tree. The pattern of staining and location of X. translucens were studied following felling and dissection of asymptomatic and diseased trees. Chestnut-coloured smears and specks occurred in the sapwood of diseased trees and were continuous from the trunk to 1–2-year-old twigs. X. translucens was isolated mainly from young sapwood (stained and unstained) of the main trunk, primary and younger branches and current season growth, less frequently from leaves and bunches, rarely from old, stained heartwood and not from roots and associated soil samples. Bacteria and pathogenic fungi were not found in the inner bark and cortex associated with lesions whereas the stained sapwood underlying the lesions yielded X. translucens. Scanning electron microscopy revealed bacteria in the main vessels of the xylem of stained tissue and tyloses in the proximity of colonised tissue. Information on the pattern of staining and location of the bacteria will facilitate pathogen detection, thereby improving the accuracy of disease diagnosis.

First Report of Little cherry virus 2 from Sweet Cherry in Poland.

Little cherry disease (LChD) is a serious viral disease of sweet (Prunus avium) and sour (P. cerasus) cherry trees. Infection of sensitive cultivars results in small, angular, and pointed fruits with reduced sweetness. In late summer, leaves show a characteristic red coloration or bronzing of the surfaces. One Ampelovirus species, Little cherry virus 2 (LChV-2) (2), and one unassigned species in the Closteroviridae, Little cherry virus 1 (LChV-1) (3), have been associated with LChD. Twenty-seven sour and sweet cherry trees of six varieties from orchards located in several regions of Poland were tested for LChV-1 and LChV-2. Leaf samples were taken either from trees showing fruit symptoms or from asymptomatic trees during the summer of the 2006 growing season. RNA was isolated from the leaves with an RNeasy Kit (Qiagen, Hilden, CA), and reverse transcription (RT)-PCR was performed using primer pairs LCV1U/LCV1L and LCV2UP2/LCV2LO2, which are specific for a 419-bp fragment of the LChV-1 3' nontranslated region and a 438-bp fragment of the LChV-2 methyltransferase gene, respectively (1). The primer pair L2CPF (5'-GTTCGAAAGTGTTTCTTGAT-3') and L2CPR (5'-GCAACAGAAAAACATATGACTCA-3') was designed from existing LChV-2 sequences (GenBank Accession Nos. AF416335 and NC_005065) to amplify the entire LChV-2 coat protein (CP) gene (nucleotides 13,007 to 14,134). The amplified cDNA fragments of LChV-2 genome were ligated to the bacterial vector pCR2.1-TOPO (Invitrogen, Carlsbad, CA), which was used to transform Escherichia coli TOP10 competent cells following the manufacturer's protocol. Both strands of three clones for each amplified LChV-2 genome fragment were sequenced with an automated nucleotide sequencer at the Institute of Biochemistry and Biophysics in Warsaw. RT-PCR results showed that 6 of 27 trees were infected, with LChV-1 detected in five sweet cherry trees and LChV-2 singly infecting one sweet cherry tree cv Elton (isolate C4/14). The nucleotide sequence of the 438-bp methyltransferase gene fragment of isolate C4/14 showed 86, 85, and 84% identity to GenBank Accession Nos. AF333237, AF531505, and AJ430056, respectively, all previously reported LChV-2 sequences from cherry trees. Sequence analysis of the 1,088-bp coat protein gene showed 89 to 91% and 92 to 93% nucleotide and amino acid identity, respectively, with the aforementioned three LChV-2 isolates. The tree infected with LChV-2 was indexed by graft transmission to the woody indicator, Prunus avium cv. Canindex, which showed reddening of the leaves characteristic of LChD 3 months after inoculation. Since cherry production in Poland is 230,000 t per year, the disease may have a significant economic impact because the affected fruits are unsuitable either for consumption or sale. To our knowledge, this is the first report of LChV-2 in Poland.

Enhanced detection and isolation of the walnut pathogen Brenneria rubrifaciens, causal agent of deep bark canker

Deep bark canker (DBC) of walnut is caused by the bacterium Brenneria rubrifaciens which produces the red pigment rubrifacine. This disease of English walnut trees, is characterized by deep vertical cankers which exude sap laden with B. rubrifaciens. Although DBC is not observed on young trees, it is hypothesized that B. rubrifaciens is present in host tissue years before symptom development. Therefore, a sensitive technique would be useful in detecting B. rubrifaciens in asymptomatic trees. Tn5 mutants deficient in rubrifacine production (pig−) were generated and DNA sequences from pig− mutants were used to design two primer sets; GSP1F–GSP1R and GSP2F–GSP2R. A third primer pair, BR1–BR3 was designed from the 16S rRNA gene. The three primer pairs did not amplify the diagnostic bands from members of the following bacterial genera: Agrobacterium, Erwinia, Pseudomonas, Ralstonia, and Rhizobium. In addition, no amplification was observed using DNA from the following Brenneria species, alni, nigrifluens, quercina, or salicis. All three DNA primer sets detected B. rubrifaciens in spiked greenhouse soil and infiltrated walnut leaf tissue. PCR detection limits for BR, GSP1, and GSP2 primer pairs were 254, 254, and 2.54 × 104 colony forming units (CFU) respectively. Real-time PCR detection limit for BR primers was 8 CFU. The differential medium, yeast extract dextrose calcium carbonate agar (YDCA) was amended with novobiocin, and bacitracin, to enhance isolation from environmental samples. The improved detection and isolation methods described here will facilitate examination of B. rubrifaciens ecology under both nursery and orchard conditions.

Protection of ash (Fraxinus excelsior) trees from ozone injury by ethylenediurea (EDU): Roles of biochemical changes and decreased stomatal conductance in enhancement of growth

Treatments with ethylenediurea (EDU) protect plants from ozone foliar injury, but the processes underlying this protection are poorly understood. Adult ash trees (Fraxinus excelsior), with or without foliar ozone symptoms in previous years, were treated with EDU at 450 ppm by gravitational trunk infusion in May-September 2005 (32.5 ppm h AOT40). At 30-day intervals, shoot growth, gas exchange, chlorophyll a fluorescence, and water potential were determined. In September, several biochemical parameters were measured. The protective influence of EDU was supported by enhancement in the number of leaflets. EDU did not contribute its nitrogen to leaf tissue as a fertiliser, as determined from lack of difference in foliar N between treatments. Both biochemical (increase in ascorbate-peroxidase and ascorbic acid, and decrease in apoplastic hydrogen peroxide) and biophysical (decrease in stomatal conductance) processes regulated EDU action. As total ascorbic acid increased only in the asymptomatic trees, its role in alleviating O(3) effects on leaf growth and visible injury is controversial.

HIGH INFECTION PRESSURE OF ESFY PHYTOPLASMA THREATENS THE CULTIVATION OF STONE FRUIT SPECIES

Stone fruit species are affected by severe disease caused by European stone fruit yellows phytoplasma (ESFY; ‘Candidatus Phytoplasma prunorum’). ESFY phytoplasma is transmitted to the host plants of Prunus spp. by the vector Cacopsylla pruni. The disease is graft-transmissible as well. The occurence of ESFY phytoplasma was monitored from 2004 to 2006 in a mother plant orchard of stone fruit species planted with virus free material in 2001 in the Primorska region of Slovenia. The total of 158 samples of mother plants were analysed in this period. The symptomatic and asymptomatic trees were analysed using molecular methods (PCR or nested-PCR). Among 15.7 % of sampled apricot trees (Prunus armeniaca) in the orchard, ESFY phytoplasma was detected in 70.0 % of samples. In the case of Japanese plum (Prunus salicina) samples were taken from one third of all Japanese plum trees and the presence of ESFY phytoplasma was confi rmed in all samples. In the European plum trees (Prunus domestica) the incidence of phytoplasma was determined in 51.0 % of sampled trees, where the plants in most cases did not show symptoms. ESFY phytoplasma was also detected in peaches and nectarines (Prunus persica) in 13.0 % of sampled trees while no detection of the phytoplasma was confi rmed in the samples of cherry trees (Prunus avium). With the survey performed in a mother plant orchard it was observed that especially young trees did not show typical symptoms and the infection was latent. In the year 2005, ESFY phytoplasma was detected in all tested samples of the vector Cacopsylla pruni captured in the vicinity of the mother plant orchard.

Phenolic Chemistry of Coast Live Oak Response to Phytophthora ramorum Infection

Since the mid 1990s, Phytophthora ramorum has been responsible for the widespread mortality of tanoaks, as well as several oak species throughout California and Oregon forests. However, not all trees die, even in areas with high disease pressure, suggesting that some trees may be resistant to the pathogen. In this study, the chemical basis of host resistance was investigated. Three field experiments were carried out in California between December 2004 and September 2005. The levels of nine phenolic compounds (gallic acid, catechin, tyrosol, a tyrosol derivative, ellagic acid, and four ellagic acid derivatives) extracted from the phloem of trees that had been either artificially inoculated with P. ramorum or trees putatively infected with P. ramorum (based on canker symptoms) were quantified by high-performance liquid chromatography (HPLC). Significant differences in phenolic profiles were found between phloem sampled from the active margins of cankers, healthy phloem from asymptomatic trees, and phloem sampled 60 cm away from canker sites, although the magnitude and direction of the responses was not consistent across all experiments. Concentrations of gallic acid, tyrosol, and ellagic acid showed the greatest differences in these different tissues, but varied considerably across treatments. Gallic acid and tyrosol were tested in in vitro bioassays and showed strong dose-dependent inhibitory effects against P. ramorum, P. cinnamomi, P. citricola, and P. citrophthora. These results suggest that phloem chemistry varies in response to pathogen infection in California coast live oak populations and that changes in phloem chemistry may be related to apparently resistant phenotypes observed in the field.

Detection of Diplodia pinea in asymptomatic pine shoots and its relation to the Normalized Insolation index

Summary Diplodia pinea (syn. Sphaeropsis sapinea), a common pathogenic fungus, causes considerable damage in Italy, particularly to pine stands in which trees are subjected to environmental stress. The occurrence of D. pinea in symptomless Pinus nigra shoots was investigated and related to the amount of radiation received by the trees growing on a site in a year, expressed as the Normalized Insolation index (NIi). Twenty‐seven pines were selected from nine locations in Trentino (northern Italy). For each pine the incidence of the fungus in apparently healthy shoots was determined by both culturing on an agar medium and application of real‐time PCR. The incidence of D. pinea determined by culturing samples taken from asymptomatic trees was 59% (16 of 27 trees), compared with 85% found using real‐time PCR (23 of 27 trees). Detection of the pathogen in healthy pine tissue was positively correlated (p < 0.05) with the NIi values, using both detection methods.

Asymptomatic carrier trees in pine stands naturally infected with Bursaphelenchus xylophilus

Abstract A rapid and accurate diagnostic method is needed to detect asymptomatic carrier trees to assist in exterminating pine wilt disease caused by the pathogenic pine wood nematode (PWN), Bursaphelenchus xylophilus. We extracted DNA by the CTAB method from pine trees located in two natural stands, a Japanese black pine stand and a Japanese red pine stand, known to be infected with PWN, and detected the PWN by the nested-PCR method. Many trees of both pine species harboured PWN, although some of them had no external and/or internal symptoms such as yellowing of needles and disrupted resin exudation. Some Japanese black pine trees remained asymptomatic during the study and survived for 1 or more years after PWN infection without displaying any wilting symptoms. The diagnostic method adopted in the present study is useful and sensitive enough for practical use to detect the asymptomatic carrier trees and may prove to be an effective method to assist in control of the pest in the field.