Diplanetism and microcyclic sporulation in Phytophthora ramorum

The zoosporic phase of the pathogen Phytophthora ramorum plays a crucial role in the process of plant infection, yet little is known about the fate of zoospores failing to target their hosts. Here, we describe new stages in the life cycle of P. ramorum concerning the in vitro development of monomorphic diplanetism and microcyclic sporulation in free water. Papillate cysts were formed after zoospore suspensions of isolates of the EU1 and NA1 clonal lineages were vortexed. Cysts usually germinated directly forming an emerging tube, or indirectly by releasing a secondary zoospore, which leaves behind an empty cyst with a short evacuation tube. Germinate cysts frequently developed either an appressorium or a microsporangium both terminally. We also observed microcyclic sporulation, i.e. sporangia indirectly germinated by forming a microsporangium, as in microcyclic conidiation of true fungi. Temporal progress of encysted zoospores in solution showed that percentage of germination varied significantly among and within isolates as well as between experiments, suggesting that germination is partly ruled by internal mechanisms. Diplanetism and microcyclic sporulation in P. ramorum may provide a second opportunity for host infection and may increase the chance of long dispersal in moving water.     

Quantitative comparisons of antitermite extractives in heartwood from the same clones of <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> planted at two different sites

The quantities of some extractives in the heartwood of 25 plus tree clones of Japanese cedar (Cryptomeria japonica) from two different sites were investigated to clarify the differences in antitermite properties in relation to clones and environmental factors. The measured compounds were cubebol, epicubebol, sandaracopimarinol, and ferruginol. The total amounts of extractives were calculated from gas chromatogram peak areas. The heartwoods from Tano (Tano Forest Research Station, Miyazaki University; 31° N, 131° E, 130 m asl) tended to contain more of the individual extractives and total extractives than those from Komenono (Komenono Forest Research Station, Ehime University; 33° N, 132° E, 700 m asl). There was a significant difference (calculated by analysis of variance) at the 1% level among clones at both sites in quantities of total and individual extractives. This result suggests that the qualities and quantities of heartwood extractives are largely affected by genetic factors. Spearman’s rank correlation with the average of the extractive quantities at the sites was investigated. The correlation coefficients of total extractives, cubebol, epicubebol, sandaracopimarinol, and ferruginol were 0.61, 0.85, 0.76, 0.67, and 0.74, respectively. This result means that the order of the amounts of these compounds among the cultivars could be maintained at different sites. It is apparent that both environmental and genetic factors affected the quantities of these extractives in the heartwoods of Japanese cedar.     

Acetaldehyde emission from wood induced by the addition of ethanol

A mechanism of acetaldehyde emission from wood induced by the addition of ethanol was proposed. It is known that acetaldehyde generation is due to the oxidation of ethanol via a metabolic process involving alcohol dehydrogenase (ADH) in living bodies. However, it remains unclear whether the enzymatic alcohol oxidation is applicable to wood. We investigated possible factors of wood parts, conditioning, storage sites, and heating and sterilization treatments on acetaldehyde emission using the syringe method and HPLC analysis. We reconfirmed that acetaldehyde emission was observed only when ethanol was added to wood. Greater acetaldehyde emissions were obtained in heartwood compared to sapwood in both Japanese cedar (Cryptomeria japonica D. Don) and Japanese cypress (Chamaecyparis obtusa Endl.) specimens. In addition, an acetaldehyde conversion rate of 1–2 mol% was determined in green cedar heartwood samples, while, conversely, air-dried cedar heartwood samples showed 4–5 mol%. Ethylene oxide gas sterilization had the effect of decreasing acetaldehyde emission on air-dried wood, but not on green wood. Autoclave sterilization could completely prevent acetaldehyde emissions from both green and air-dried wood. These results suggested that an original ADH in wood and an attached ADH from the outside via microorganisms onto wood were assumed to be the primary causes of acetaldehyde emissions from wood induced by the addition of ethanol.     

黄花石斛组织培养繁殖技术研究

进行黄花石斛种子在不同培养基及添加物中的萌发率试验研究。结果表明,以1/2MS+6-BA0.5 mg/L+NAA0.1 mg/L+10%椰汁+1 g/L活性C的萌发率为最佳;带腋芽的茎段和顶芽以1/2KC+6—BA1 mg/L+NAA0.1 mg/L+10%椰汁+1 g/L活性C诱导原球茎增殖较佳;生根培养以1/2MS++NAA0.1 mg/L生根较多。炼苗基质以泥碳、蕨根、栎树屑混合基质栽培成活率最高。     

Feeding responses of the western dry-wood termite <Emphasis Type="Italic">Incisitermes minor</Emphasis> (Hagen) (Isoptera: Kalotermitidae) against ten commercial timbers

Five Japanese timbers, four timbers from the USA, and one Malaysian timber were evaluated for their resistance to the invasive dry-wood termite Incisitermes minor (Hagen) using laboratory choice and no-choice feeding tests with holed specimens. The highest survival rates of I. minor in both the heartwood and sapwood no-choice feeding tests were more than 70% after 3 months. When offered sapwood and heartwood choice feeding tests and the combined choice feeding tests, the highest survival rates of I. minor were more than 75% after 3 months. With regards to the percentage of wood mass losses in the no-choice and choice feeding tests, karamatsu (Larix leptolepsis), buna (Fagus crenata), and Douglas fir (Pseudotsuga menziesii) were classified as “resistant” species among the ten sapwood specimens. In the heartwood no-choice and choice feeding tests, the resistant species were buna, karamatsu, Douglas fir, sugi (Cryptomeria japonica), akamatsu (Pinus densiflora), and western red cedar (Thuja plicata). The ranking of the resistance of the ten commercial timbers against I. minor was buna > karamatsu > sugi > western red cedar > Douglas fir > rubber > western hemlock > hinoki > spruce.     

Antioxidant activity of extracts from the wood and bark of Port Orford cedar

Heartwood, sapwood, and inner and outer bark of Port Orford cedar were extracted with methanol, and the extracts evaluated for antioxidant activity. The total phenol content (TPC) of the extracts was determined by the Folin-Ciocalteu method and expressed as gallic acid equivalent (GAE). Butylated hydroxytoluene was used as a positive control in the free-radical-scavenging activity tests and ethylenediaminetetraacetic acid dihydrate disodium salt served as a positive control in the metal-chelating activity assay. All wood extracts showed significant freeradical-scavenging activity. In the radical-scavenging assay of 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (the ABTS assay), the inner bark extracts exhibited the strongest free-radical-scavenging activity. The 50% inhibitory concentrations (IC₅₀) in the radical-scavenging assay against 1,1-diphenyl-2-picrylhydrazyl hydrate radical (DPPH) of the heartwood, sapwood, and inner and outer bark extracts were 64.77, 29.03, 10.31, 19.87 μg·ml⁻¹, respectively. In the metal-chelating activity system, the sapwood extract demonstrated significant activity. The greatest TPC, 537.5 mg GAE/g dry extract, was detected in the inner bark. The lowest TPC of 136.9 mg GAE/g dry extract was observed in the heartwood dry extract. The results indicate that the antioxidant activities of the extracts are in accordance with the amounts of phenolics present; the inner and outer barks of Port Orford cedar are rich in phenolics and may provide good sources of antioxidants.     

Application of activable tracers to investigate radial movement of minerals in the stem of Japanese cedar (<Emphasis Type="Italic">Cryptomeria japonica</Emphasis>)

Two activable tracers, Rb and Eu, were injected into the sapwood of Japanese cedars (Cryptomeria japonica D. Don) to investigate the radial movement of minerals in their stems in the resting period. Eight trees of four cultivars, two of which genetically form wet heartwood, were treated near the end of the growing period. At 40 days after the treatment, Rb was detected in the outer heartwood, whereas Eu was not. Radial movement of Rb was more rapid in trees with wet heartwood than in those with normal heartwood. At 204 days after the treatment, more Rb was detected in the heartwood than was found on the first sampling, whereas no Eu was detected in the heartwood. The difference in radial movement between Rb and Eu was considered mainly to be the result of selective transport of beneficial minerals by Japanese cedar. The difference in the rate of radial movement of Rb between wet and normal heartwood became more conspicuous at 204 days after treatment. We concluded that the movement of Rb from the sapwood to the outer heartwood was by active transport through the rays, whereas that in the heartwood was by diffusion due to the gradient of Rb concentration.     

Radial movement of sapwood-injected rubidium into heartwood of Japanese cedar (<Emphasis Type="Italic">Cryptomeria</Emphasis><Emphasis Type="Italic">japonica</Emphasis>) in the growing period

Rubidium solution was injected in the sapwood of a Japanese cedar cultivar in the growing period, and its radial movement in stem was traced to investigate the accumulation of alkali metals in the heartwood. Sapwood-injected Rb was detected in outer heartwood at 10 days after the treatment, and continued increasing at 20 days after. Radial movement of Rb toward heartwood was considered to occur soon after the treatment, and to decline at a certain point of the time after Rb injection ceased. However, Rb continued moving in heartwood probably by diffusion even after the cease of Rb injection. In a series of injection experiment, radial movement of injected Rb is not corresponding to the seasonality of both cambial activity and cytological changes of ray parenchyma accompanied with heartwood formation. From the results on Rb’s behavior, we conclude that accumulation of K and other alkali metals in heartwood of Japanese cedar has two steps, active transport from sapwood to outer heartwood via ray, and diffusion in heartwood, and that these processes proceed independently from both cambial activity and cytological changes of ray parenchyma.     

Assessment of decay risk of airborne wood-decay fungi

The decay risk of airborne wood-decay fungi was investigated by using an air sampler. Japanese cedar disks 7.8 cm in diameter and about 3 mm in thickness with moisture content of about 100% were placed in a “BIOSAMP” air sampler and exposed to 1000 l air. Air sampling was carried out from June to September at the same sampling site in Tsukuba, Japan. The exposed disks were then incubated for 16 weeks in a damp container kept at 26° ± 2°C. During the incubation period, wood mass loss ranged from −15 to 807 mg with a mean mass loss of 244 mg. Factors affecting mass loss were explored. Wood moisture content and ratio of heartwood area proved to be significant factors. In addition, six weather factors were found to influence mass loss. Disks that were sampled on a cloudy day showed significantly higher mean mass loss compared to those sampled on a sunny day. Subculturing of filamentous fungi from 16-week incubated disks suggested one-third of the isolated fungi produced ligninolytic enzymes.     

PCR‐RFLP markers identify three lineages of the North American and European populations of Phytophthora ramorum

Phytophthora ramorum, the cause of sudden oak death and ramorum blight, has three major clonal lineages and two mating types. Molecular tests currently available for detecting P. ramorum do not distinguish between clonal lineages and mating type is determined by cultural methods on a limited number of samples. In some molecular diagnostic tests, cross‐reaction with other closely related species such as P. hibernalis, P. foliorum or P. lateralis can occur. Regions in the mitochondrial gene Cox1 are different among P. ramorum lineages and mitochondrial genotyping of the North American and European populations seems to be sufficient to differentiate between mating types, because the EU1 lineage is mostly A1 and both NA1 and NA2 lineages are A2. In our study, we were able to identify P. ramorum isolates according to lineage using polymerase chain reaction‐restriction fragment‐length polymorphism (PCR‐RFLP) of the Cox1 gene, first by using ApoI to separate P. ramorum from other species and EU1 from North American populations, and then AvaI to distinguish between NA1 and NA2 genotypes. However, P. foliorum had the same restriction profile as P. ramorum NA1 isolates.     

Migratory passerine birds in Britain carry Phytophthora ramorum inoculum on their feathers and “feet” at low frequency

In this study, we investigated whether birds could be vectors facilitating long‐distance spread of Phytophthora ramorum in Britain. Migratory bird species associated with the main sporangium‐producing host plants and most likely to pick up P. ramorum spores were considered. Swabs were taken from the flank and “feet” of 1,014 birds over a 12‐month period (April 2011–March 2012) in the west of Britain and subsequently analyzed for the presence of P. ramorum using nested PCR. Ten positive samples from 10 birds were identified: three in Cornwall, one in Devon, three in Gloucestershire, two in north Wales and one in Merseyside. Phytophthora ramorum was detected on samples from four species of thrushes (Redwing Turdus iliacus, Fieldfare T. pilaris, Blackbird T. merula and Song Thrush T. philomelos) and one species of warbler (Chiffchaff Phylloscopus collybita). All birds that tested positive were sampled in late autumn and winter (October–February), when long‐distance movements (over 100 km) would have stopped. The low incidence of P. ramorum found using PCR suggests that the incidence of inoculum, whether viable or not, on birds was low. The apparently low incidence of inoculum on birds suggests migratory passerine birds can carry P. ramorum inoculum on their feathers and “feet,” albeit at low frequency. The dates of positive samples indicate that birds would not have been moving long distances at the time but further work is needed to estimate the extent of their contribution to the spread of P. ramorum in Britain.     

Antinociceptive activity of Eupatorium buniifolium aqueous extract

The antinociceptive properties of the aqueous extract of Eupatorium buniifolium aerial parts were investigated in chemical and thermal models of nociception in mice. Oral doses of 250 and 500 mg/kg produced an inhibition of 42.3% and 73.9%, respectively, of the abdominal writhes induced by acetic acid in mice. This antinociceptive effect was not reversed by pretreatment with naloxone. In the formalin test, the administration of 500 and 1000 mg/kg p.o. had no effects in the first phase (0-5 min) but produced a dose-dependent analgesic effect on the second phase (15-30 min) with inhibitions of the licking time of 38.9% and 57.3%, respectively. In the hot plate test, no effect was seen at the dose of 500 mg/kg p.o.     

Cesium absorption through bark of Japanese cedar (<Emphasis Type="Italic">Cryptomeria japonica</Emphasis>)

Absorption of radiocesium (¹³⁷Cs and ¹³⁴Cs) through bark, and its subsequent translocation into wood and needles, has been suggested as a potential source of tree contamination, but the process is not well understood. Field experiments were conducted to confirm whether Cs could enter a Japanese cedar tree through the bark and how Cs moves within a tree. Stable Cs (¹³³Cs) was applied to the bark at 1.2-m height on 10- and 26-year-old Japanese cedars. The ¹³³Cs concentrations were determined in the bark, sapwood, and heartwood (for 26-year-old cedar only) of stem disks from several heights, as well as in current-year needles from the canopy. The ¹³³Cs concentrations were considerably higher in the sapwood and heartwood of stem disks from 1.2-m height in treated trees than in untreated trees, suggesting that ¹³³Cs penetrated the bark to enter the wood. The average ¹³³Cs concentrations were higher in the heartwood than the sapwood, indicating ¹³³Cs accumulation in the heartwood. High ¹³³Cs concentrations in the needles of treated trees implied acropetal movement of ¹³³Cs to actively growing organs. Our results demonstrate that Cs can enter Japanese cedar trees through the bark and that Cs is transported radially to the heartwood and vertically to the apex.     

Assessment of the eradication measures applied to Phytophthora ramorum in Irish Larix kaempferi forests

Phytophthora ramorum is the causal agent of the sudden larch death epidemic in Ireland and the UK. Within the EU, it is a quarantine pathogen and eradication measures are required if it is detected in horticultural or forest environments. Eradication measures in forests include the clearance of susceptible tree hosts from the infected stand along with all host known to support pathogen sporulation within a 250‐m buffer zone of the infected stand. Between 2010 and 2016, these measures have affected over 18,000 ha of Larix kaempferi forests in Ireland and the UK, but the epidemic continues to spread. An assessment of the efficacy of the eradication measures has not been published to date. Here, we provide details of the detection frequency of P. ramorum from aerial (rainwater) and terrestrial (soil, watercourses, plant material) sources in three forest locations in Ireland that had significant areas of L. kaempferi affected by P. ramorum before their removal. Monitoring of six plots with differing infection and eradication management histories was carried out from September 2013 to 2015. Presence of P. ramorum was confirmed by plating plant material onto selective media, followed by morphological identification. Phytophthora ramorum was detected in 65 of 1283 samples, in all sample types and in 17 of the 20 months sampled. Only three of the 295 soil samples were positive for P. ramorum, with all of these coming from an area under perennial standing water. The most positive samples came from a plot where symptomatic Larix trees had not been removed and the findings occurred consistently over the 2‐year study. Plots where infected Larix had been removed were rarely positive for P. ramorum across all the sample types indicating a level of success from the eradication measures in reducing pathogen levels on the sites.     

Relationship between field resistance to Phytophthora ramorum and constitutive phenolic chemistry of coast live oak

Sudden oak death, caused by Phytophthora ramorum, has resulted in high levels of coast live oak (CLO) mortality. However, some CLO survive in areas with high disease pressure and may thus be resistant. We tested the hypothesis that such field‐resistant trees contain constitutively higher levels of phenolics than susceptible trees. Phloem was sampled from the trunks of two groups of trees (one previously inoculated, one naturally infected with P. ramorum) categorized over the course of several years as putatively resistant (PR, no symptoms), in remission (IR, showed symptoms but then recovered) and symptomatic (S). Individual and total soluble phenolics from these trees were quantified. There were no significant differences in individual or total soluble phenolics between groups of naturally infected trees. However, inoculated PR and IR trees were characterized by higher constitutive levels of ellagic acid, a tyrosol derivative, and an unidentified phenolic than S trees. Ellagic acid and tyrosol‐like compounds in CLO phloem are promising resistance biomarker candidates.     

Isolation of Phytophthora lateralis from Chamaecyparis foliage in Taiwan

Following the discovery in 2008 of Phytophthora lateralis in forest soil under old‐growth yellow cedar (Chamaecyparis obtusa var. formosana) in north‐east Taiwan, further sampling was undertaken in the same region. Soil, root and symptomatic foliage samples were collected from five separate sites where C. obtusa was the dominant species in cloud forests at ca. 1800–2500 m. Soil and fine root samples were baited with cedar needles; both direct isolation and cedar needle baiting were used on foliage samples. Phytophthora lateralis was obtained from soil at three of the sites, but only from three of the 27 soil samples overall. Only one of 25 root samples yielded the pathogen, and this was associated with infested soil. Three foliage samples with symptoms visible as dark brown to black frond tips also yielded P. lateralis; these came from two different sites. This is the first record of P. lateralis infecting the foliage of C. obtusa. Moreover, when some of the symptomatic Chamaecyparis foliage segments were incubated, sporangia of P. lateralis formed on the necrotic tissues, sometimes in the axils of needle segments. The study provides evidence that P. lateralis has both a soil/root infecting phase and an aerial or foliar infecting phase in Taiwan, which is consistent with its unusual combination of water‐dispersed (non‐papillate) and aerially dispersed (caducous) sporangia. It also demonstrates the importance of investigating the biology, aetiology and ecological behaviour of Phytophthoras in their native, endemic environments.     

In vitro screening of angiotensin I-converting enzyme inhibitors from Japanese cedar (Cryptomeria japonica)

Screening and isolation of angiotensin I-converting enzyme (ACE) inhibitors from Japanese cedar (Cryptomeria japonica) based on the in vitro ACE inhibitory assay were attempted. The ethanol extract from outer bark showed the highest inhibitory activity (IC₅₀ is 16g/ml) among 24 extracts prepared from roots, leaves, heartwood, sapwood, inner bark, and outer bark by successive extraction with four solvents. The fractionation of the outer bark ethanol extract followed by the bioassay resulted in the isolation of two strong ACE inhibitors, catechin and dimeric procyanidin B3. The bioassay of three flavan-3-ols including (+)-catechin and six flavones revealed that most of these compounds have high ACE inhibitory activity. The results suggest that the phenolic hydroxyl group at the C7 position and heterocyclic oxygen atom of these compounds are important for expressing the inhibitory activity.     

The influence of heartwood on the pulping properties of Acacia melanoxylon wood

The pulping wood quality of Acacia melanoxylon was evaluated in relation to the presence of heartwood. The sapwood and heartwood from 20 trees from four sites in Portugal were evaluated separately at 5% stem height level in terms of chemical composition and kraft pulping aptitude. Heartwood had more extractives than sapwood ranging from 7.4% to 9.5% and from 4.0% to 4.2%, respectively, and with a heartwood-to-sapwood ratio for extractives ranging from 1.9 to 2.3. The major component of heartwood extractives was made up of ethanol-soluble compounds (70% of total extractives). Lignin content was similar in sapwood and heartwood (21.5% and 20.7%, respectively) as well as the sugar composition. Site did not influence the chemical composition. Pulping heartwood differed from sapwood in chemical and optical terms: lower values of pulp yield (53% vs 56% respectively), higher kappa number (11 vs. 7), and lower brightness (28% vs 49%). Acacia melanoxylon wood showed an overall good pulping aptitude, but the presence of heartwood should be taken into account because it decreases the raw-material quality for pulping. Heartwood content should therefore be considered as a quality variable when using A. melanoxylon wood in pulp industries     

华中樱桃种子生物学特性研究初探

为探究华中樱桃 Cerasus conradinae种子生物学特性,以 湖北省赤壁市的野生华中樱桃的自然成熟未落地的果实为试验 材料,观察其种子形态,并测定种子长度、宽度、厚度、千粒 重、吸水特性和发芽特性。结果表明：华中樱桃种子为卵形,长 7.64±0.73mm,宽5.28±0.55mm,厚4.39±0.47mm,千粒重 62.77±2.91g,属中粒种子;种子浸种0~4 h吸水较快,后渐趋 缓,浸种24 h近饱和,吸水率为33.23%,种子饱和状态时吸水率 为33.30%;用750 mg/L 赤霉素（GA3）溶液浸种处理的种子发芽 效果较清水浸种好,但差异不明显,两者发芽率分别为65.3%和 62.7%;华中樱桃幼苗个体间差异大。     

Chemical,anatomical and technological properties of Snakewood [<Emphasis Type="Italic">Brosimum guianense</Emphasis> (Aubl.) Huber]

The very decorative heartwood of Brosimum guianense is internationally well known. Snakewood, as it is colloquially known, is represented in wood databases (e.g. the DELTA or InsideWood) as well as in lists of commercial timbers of many timber trading companies. The very decorative heartwood is hardly available and gains prices of up to 25 €/kg in form of half stems. In the present study, the chemical composition and especially the subcellular cell structure was analysed by means of UV microspectrophotometry to explain the high natural durability and some extraordinary physical properties in addition to the anatomical composition. The heartwood consists of approximately 39% lignin, 54% carbohydrates and 0.4% lipophilic compounds of unspecified origin. The fibres are very thick-walled. Numerous sclerotic tyloses and organic deposits are present in the vessel. The extractives in high content are also components of parenchyma cells as well as in tyloses, respectively. These detected phenolic extractives, partly of flavonoid character, are also part of the cell wall. Calcium oxalate crystals are deposited in the upright and square cells of rays and sporadically in axial parenchyma cells. These facts are reasons for the famous natural durability of Snakewood. The sapwood density ranges from 1.1 to 1.4 g/cm³ for heartwood (12% mc). The compression strength (119 N/mm²), the bending strength (241 N/mm²), the modulus of elasticity (23,200 N/mm²) and the hardness (196 N/mm²) indicate exceedingly high elastomechanical properties.