Histology of Diplodia pinea in diseased and latently infected Pinus nigra shoots

Diplodia tip blight is a serious disease of >30 conifer species worldwide. Symptoms are particularly severe on non‐native, two‐needled Pinus species, and typically include stunted, necrotic needles and shoots and a general decline of the tree. Latent Diplodia pinea infections occur in current‐year shoots of some symptomless pines, and in some apparently healthy current‐year shoots of diseased pines. Latent infections also occur in symptomless terminal buds. A histological approach was used to investigate the nature of latent infections in shoot stems and terminal buds of landscape Pinus nigra. Fungal colonization was compared in healthy, diseased and latently infected tissues. A nested‐PCR technique that is specific for D. pinea was used to differentiate latently infected tissues from uninfected ones. Latent D. pinea infections were localized in the outer stem cortex, usually in the vicinity of needle scales at leaf axes. In contrast, pathogenic D. pinea infections were characterized by fungal colonization throughout the shoot stem tissues, even very early in symptom development. The presence of necrophylatic periderms in two of the latently infected samples suggests that host defences play a role in the production and maintenance of latent stem infections. Latent infections of terminal buds appeared to originate from the distal bud scales of axillary buds in the terminal bud cluster, and not from the subtending shoot. Fungal tissues were never observed inside asymptomatic, PCR‐negative shoots.     

Accumulation of constitutive diterpenoids in the rhytidome and secondary phloem of the branch bark of Larix gmelinii var. japonica

The quantitative compositions of the major constitutive diterpenoids in the rhytidome and secondary phloem of the branch bark of Kuril larch (Larix gmelinii var. japonica) were investigated. The eight major diterpenoids were isolated from a diethyl ether extract of the branch bark of L. gmelinii var. japonica and identified as 13-epimanool (1), larixol (2), larixyl acetate (3), 13-epitorulosyl acetate (4), abietic acid (5), neoabietic acid (6), dehydroabietic acid (7), and isopimaric acid (8). The amount of each diterpenoid was subsequently quantified in both the rhytidome and secondary phloem. All of the diterpenoids were present in both bark tissues, but the amounts were significantly higher in the rhytidome than in the secondary phloem. Developed fusiform resin cavities containing oleoresins were commonly observed in dead secondary phloem captured into the rhytidome of a bark transverse section. The accumulation and distribution of these constitutive diterpenoids in the bark tissues can probably be attributed to terpenoid biosynthesis in the living secondary phloem and the rhytidome formation process. From the viewpoint of constitutive chemical defense in conifers, it is suggested that the tree body may be more effectively defended against natural enemies by the higher amount of diterpenoid amount in the outermost and dead bark tissue, the rhytidome, where the potentially poisonous and easily oxidizable diterpenoids can be safely and stably maintained.     

Detection of MLOs in declining alder trees in Southern Italy and their characterization by RFLP analysis

Mycoplasma-like organisms (MLOs) were detected and characterized by RFLP analysis in declining trees of Alnus glutinosa and A. cordata in Southern Italy for the first time. The infected trees showed symptoms of yellowing, sparse foliage, premature autumn colouration, sprouting, deliquescent branching, phloem necrosis, dieback and witches’ brooms.     

枣疯病和泡桐丛枝病原植原体分离物的组织培养保藏和嫁接传染研究

分别从河北唐县赞皇大枣、辽宁凌源梨枣和河南濮阳扁核酸3个品种的枣疯病和来自山东、江西和北京的不同无性系的泡桐丛枝病树上采集丛枝枝条作为组织培养材料,获得的枣疯病和泡桐丛枝病组培苗皆表现典型的丛枝症状。其中感染植原体的赞皇大枣组培苗(Ft)和扁核酸组培苗(HPD)在未加任何激素的MS培养基和其它培养基交替继代培养1a以上仍能维持丛枝苗生长;而发病梨枣(LD)除产生丛枝外,还出现叶片黄化和植株矮化、枯梢等衰退症状。泡桐丛枝病植原体可在不同无性系组培苗上通过各种培养基交替和单纯的MS培养基继代培养,并已在实验室内连续保藏达10a,其引致丛枝症状的能力无明显的改变。用枣树Ft染病材料作接穗,以健康冬枣(DJ)和抗病婆枣(W14)砧木进行组培苗间嫁接传病试验,可使部分DJ和W14发病;而嫁接未发病的砧木多数像健苗一样正常生长。用感染山东泡桐丛枝病植原体ZD株系丛枝组培苗为接穗,嫁接健康泡桐无性系组培苗致使无性系MB33、TY2T和C125发病。用植原体16SrDNA通用引物进行PCR,确证了泡桐和枣树发病苗和嫁接发病组培苗体内存在植原体。用DAPI荧光显微镜技术比较组培苗韧皮部筛管中的植原体浓度,结果显示,Ft和嫁接发病冬枣(DJ-Ft)筛管中植原体浓度相对较高,但仍低于各泡桐无性系染病丛枝组培苗;HPD和LD浓度中等,而发病的W14砧木含有植原体的筛管数量较少、且浓度很低。在嫁接不成功或未发病的DJ和W14砧木组织及健康对照组织中皆检测不到植原体荧光。     

Rapid Micropropagation of Edible Bamboo Dendrocalamus asper

Abstract

Micropropagation protocols for Dendrocalamus asper using nodal shoots and seeds culture are described. Multiple shoots were induced through forced axillary branching. Ninety-five percent of the nodal shoot explants taken from juvenile primary and lateral branches, produced multiple shoots through axillary buds activation within 2 to 8 weeks on Murashige & Skoog's (MS) medium supplemented with 0.1-15 mg/l benzyladenine (BA). The cultured seeds also produced multiple shoots (1-20) within 6 weeks on this medium. The multiple shoot differentiation was influenced by the concentration of BA in the medium. The in vitro generated shoots were excised and subculture on MS + 3.0 mg/l BAP for further shoot multiplication. Fifteen to 20 fold rate of shoot multiplication was achieved by regular subculturing. These shoots were multiplied for more than 3 years without loss of vigor. Ninety-five percent of the shoots were rooted, when propagules (each consisting of cluster of 3 shoots) were transferred on to MS medium with 3.0 mg/l NAA or 10 mg/l indole-3-butyric acid (IBA).

To date, 18,000 plants (through axillary bud initiated from nodal ex-plants) and 6,000 plants from seed culture have been hardened and acclimatized. 12,000 plants have been field transferred.     

Host responses to natural infection by Cytonaema sp. in the aerial bark of Eucalyptus globulus

The chemical and anatomical host responses to natural fungal infection by Cytonaema sp. in the aerial bark of 3‐year‐old Eucalyptus globulus plantation trees were examined. The lesion margin (LM) of the canker‐infected bark was characterized by the formation of a layer of dark extractives visible to the naked eye. Chemical analysis of the LM by gas chromatography‐mass spectrometry (GC‐MS), high‐performance liquid chromatography (HPLC) with ultraviolet detection and HPLC‐MS using negative ion electrospray ionisation indicated the presence of a range of compounds including hydrolysable tannins, polymeric proanthocyanidins, flavonoid glycosides, formlyated phloroglucinol compounds and volatile terpenes. These compounds were either undetectable in healthy tissue or present at significantly lower concentrations than in the LM. The LM of the canker‐infected bark was morphologically distinct from healthy phloem, its characteristics varying depending on severity of canker infection. In superficial infections in which only the phloem was affected, the following LMs were observed: (i) a continuous wound periderm of multiple layers, or (ii) an incompletely differentiated and discontinuous wound periderm. In cases of severe canker infections in which the vascular cambium had been killed, the new phloem formed subsequently contained traumatic oil glands in addition to the responses observed for superficial canker infections. All LMs were characterized by the formation of new parenchyma cells that stained positive for the presence of polyphenols. The significance of the chemical and structural responses as defence mechanisms against fungi causing stem canker is discussed.     

Canopy openings may prevent fir broom rust (<Emphasis Type="Italic">Melampsorella caryophyllacearum</Emphasis>) infections

Melampsorella caryophyllacearum is a heteroecious fungus that produces disease on silver fir resulting in galls that depreciate the value of trees. Currently, little is known about its epidemiology. The purpose of this study was to identify those site or management factors associated with fir broom rust infection. A 29-plot systematic sampling was carried out throughout all silver fir forests in the Spanish Pyrenees. In every sampling plot, we examined each tree for the presence of M. caryophyllacearum galls or witch’s brooms. We differentiated their position as either on the main stem or on the branches of trees. We identified the most susceptible stands by relating a compendium of tree and stand variables with the presence and severity of symptoms. Disease symptoms were observed in 11.8% (95% CI: 5.8–17.9%) of trees. Fir broom rust-infected trees tended to be those of larger diameters. The rust infection was observed in 55.2% (95% CI: 35.7–73.6%) of the stands. We identified a wide range of severities with 5 of 29 stands having more than 30% of trees infected. Fir broom rust appeared to be a widespread disease, although we observed important differences in terms of severity. The presence and severity of the rust in stands was associated with understories composed of more shade tolerant plant species and with longer periods without thinnings. Canopy opening should be experimentally tested as a possible control method.     

Weevil feeding on Scots pine affects germination of Endocronartium pini

The aim of this study was to assess the effect and importance of the feeding of the pine top weevil (Pissodes piniphilus) on the germination of Endocronartium pini (syn. Peridermium pini) in Scots pine (Pinus sylvestris), and thus to establish the possibility of the E. pini infection via insect woundings. Germination tests were therefore carried out on current and previous year needle and phloem extracts. Elucidation of the importance of pathogen infection for the insect's feeding preference was also required; for this purpose feeding preference tests were carried out with healthy and infected pine branches using the pine top weevil as a test insect. Weevil feeding increased the germination of E. pini aeciospores on pine extracts. Germination on previous year annual-shoot extracts was lower than that on current year annual-shoot extracts. The advance of the growing season increased this trend, but weevil feeding increased germination on extracts from older annual shoots to the levels found on extracts from current annual shoots. Spores germinated equally well on needle extracts and on phloem extracts. The weevils ate more often on infected branches than on healthy branches. E. pini infections may occur via woundings on branches and weevil feeding may facilitate this.     

Broom rusts of balsam fir and black spruce in Newfoundland

The broom rusts of balsam fir and black spruce occur sporadically throughout the island of Newfoundland, but they are not a serious threat to the forests. The incidence and intensity of the rusts vary, but no tree mortality can be attributed to the diseases. The average number of brooms per tree was higher in black spruce than in balsam fir. Also, more brooms occurred on brandies than on tree trunks. Height and diameter growth was less in infected trees than in uninfected trees of both the species.     

Structural responses in bark to mechanical wounding and Armillaria ostoyae infection in seedlings of Pinus sylvestris

The sequence of events leading to the restoration of an intact periderm surface in the stem bark of Pinus sylvestris following wounding and challenge with Armillaria ostoyae was determined. A sub-erized impervious tissue (SIT) was produced outside the necrophylactic periderm (NP). Al-though A. ostoyae initially infected wounded bark with intracelfularly growing hyphae, these died before penetrating deeply into the bark. Later, intercellularly growing rhizomorphs, which had also developed from the inoculum, penetrated the NP and infected the functional phloem/ cambial zone. Compared to the reactions in uninfected wounded bark, the A. ostoyae infection delayed the formation of NP and reduced the frequency of cells involved in lignification and in SIT, but increased the number of lignified cell layers. The rhizomorph infection reached deeper and lignification was more pronounced in defoliated seedlings than in non-defoliated ones. The importance of structural responses in defence against A. ostoyae is discussed.     

Anatomical and cellular responses of Pinus monticola stem tissues to invasion by Cronartium ribicola

White pine blister rust (Cronartium ribicola) causes extensive damage to white pines and their associated ecosystems across North America. The anatomical and cellular characteristics of C. ribicola colonization in Pinus monticola branch and stem tissues were studied as a basis for understanding host tree reactions that may be related to resistance. Samples examined showed typical fusiform swelling and some had produced aecia. The reaction of phloem and xylem tissues was compared with non-infected tissue using light and electron microscopy. Cortical parenchyma and phloem polyphenolic parenchyma cells underwent mitotic division, cell swelling, and ca sixfold greater accumulation of phenolic compounds in colonized vs. control stems. In the cortex and secondary phloem, haustoria were common in parenchymatous cells, and hyphae were abundant in the intercellular spaces, but cell death was rare, unless aecia had ruptured the stem cortex. Hyphae were also common in xylem rays, tracheids and between tracheids. Disease-induced changes in the cambial zone included development of cambium-derived xylem traumatic resin ducts. Results demonstrate that diverse host defence responses were activated in the bark of apparently susceptible trees, but lack of mechanical damage by C. ribicola to the phenol-containing host cells and the resin duct system allowed extensive colonization and development of aecia despite elicitation of these stem defences. Interactions between P. monticola and C. ribicola are discussed and compared with other conifer–fungus pathosystems.     

Penetration of Picea sitchensis root bark by Armillaria mellea,Armillaria ostoyae and Heterobasidion annosum

Penetration of root bark tissues of Picea sitchensis by Armillaria ostoyae, Armillaria mellea and Heterobasidion annosum was examined in the absence of wounds, in superficial wounds (rhytidome tissues removed to expose the secondary phloem) and in wounds to the depth of the vascular cambium (deep wounding). Both species of Armillaria penetrated bark without prior wounding, but neither species formed rhizomorphs in this treatment. Armillaria ostoyae penetrated to 39 cell layers in depth by 48 days after inoculation of unwounded bark, whereas A. mellea penetrated 25 cell layers in the same time. Armillaria mellea penetrated superficial wounds significantly more rapidly than did A. ostoyae. Both species produced rhizomorphs within wounded host tissues. Inoculation of deep wounds with Armillaria resulted in a greater depth of bark necrosis with A. mellea than with A. ostoyae. In the absence of wounding, H. annosum failed to penetrate root bark tissues, but in both superficial and deep wounds hyphae penetrated beyond the ligno–suberized boundary zone (LSZ) by 12 days after inoculation. Where no inoculations were made, superficial or deep wounding led within 25 days to the restoration of a structurally continuous LSZ, and by day 48 the wound periderm (WP) was fully differentiated. In inoculated wounds, however, formation of the LSZ and WP was delayed or inhibited in most trees, particularly following inoculation with A. ostoyae or A. mellea. Suberization in the LSZ and WP remained diffuse and discontinuous 48 days after inoculation. Moreover, the presence of WP did not prevent further penetration of the tissues by the pathogens. Variations between trees in the depth of pathogen penetration were noted, possibly indicating differing susceptibilities of individual host genotypes. The possible host factors involved in resistance to penetration of root bark tissues by Armillaria and Heterobasidion are discussed.     

白刺幼苗芽库及枝系构型对不同氮添加水平的响应

[目的]研究不同氮添加梯度下白刺幼苗芽库组成及空间结构的差异,探讨了幼苗的养分限制、氮素利用以及根系和插穗的品质与芽库的关联,阐明了白刺芽库响应氮素有效性所采取的适应性策略。[方法]本研究通过盆栽控制实验,对唐古特白刺幼苗的芽库特征进行了测量分析。[结果]氮添加显著增加了白刺幼苗芽和营养枝数量,显著降低了休眠芽和休眠枝数量,并对二级枝和三级枝出芽率具有明显促进作用;随着氮添加量的递增,营养枝有从基部向顶端移动趋势;氮平衡指数与白刺幼苗出芽强度、分枝强度、二级枝和三级枝出芽率均呈二次非线性正相关关系;植株氮含量、氮积累量和地下部分形态特征分别与芽和营养枝数量呈正相关关系,与休眠芽和休眠枝数量呈负相关关系。[结论]氮添加影响幼苗芽库容量。随着氮添加水平的提高,各指标多数在N3和N4处理达到最大值后逐渐降低,因此6个氮添加水平中36 mmol·L-1和48 mmol·L-1是白刺幼苗最适氮添加量。氮添加对幼苗芽库内组分的相对位置产生了不同影响趋势,所以白刺幼苗可以通过改变芽库容量和空间分布来响应养分有效性变化。     

Spatial pattern of paulownia witches’ broom and its effects on the growth of Paulownia elongata in Shaanxi Province, China

The spatial pattern of paulownia witches’ broom was investigated using an Iwao’s (m*&#8722;m) model in Shaanxi Province, China. Iwao’s (m*&#8722;m) model revealed that spatial pattern of paulownia witches’ broom exhibited a uniform distribution in sample plots. In this model, characteristics of contagiousness coefficient (α) justified the probability of infection by virus-transmission vectors. For tree age less than six, susceptible individuals were aggregated as clusters in the plots and then mutually repelled over age. A preliminary survey of stand volume show that from age five to six, stand volumes of disease grade II showed excessive vegetative growth and returned to normal level between ages ten and 11. Loss rate of volume increment with disease grade II decreased, and then returned to raising trend both at grades III and IV. This phenomenon is possibly caused by an over-compensation effect and by environmental factors. The redundancy of growth hormones in branches, shoots, leaves, buds and roots increased the stem volume of entire trees.     

PCR primers for identification of Sirococcus conigenus and S. tsugae,and detection of S. conigenus from symptomatic and asymptomatic red pine shoots

Regions of diversity in the internal transcribed spacer (ITS) sequences of Sirococcus species were exploited to design primer pairs used in a PCR-based method for the identification of the conifer shoot blight pathogen Sirococcus conigenus and the closely related fungus Sirococcus tsugae. The specificity of each primer pair for the respective fungus, detection limits and utility for detection from host material were confirmed. The S. conigenus primers were then used to detect this pathogen in tissues of symptomatic or apparently healthy red pine shoots collected at six locations in Wisconsin and Michigan and results compared with those obtained using a cultural assay. For needles, bark and wood of symptomatic shoots, the mean frequencies of detection of S. conigenus using the PCR-based methods were consistent (≥7.5 out of 10) and always greater than for the cultural assay. Detection from symptomatic shoots using the cultural assay was more frequent from needles than from bark or wood. Both the PCR-based method and the cultural assay detected S. conigenus in similar frequencies from asymptomatic shoots, although less frequently than from symptomatic shoots. The efficiency of the PCR-based method and its utility for direct testing of host material should make it particularly useful in areas where multiple shoot blight pathogens are found.     

Improving plantlet yield in Pinus pinaster somatic embryogenesis

Abstract

Somatic embryogenesis is expected to play a significant role in the future forest tree improvement programmes. The main bottleneck of this technique is still the progression from immature embryogenic cultures to mature cotyledonary embryos able to develop properly into well-growing plants. In this work, we present an improved protocol focused on increasing the maturation and conversion rate of Pinus pinaster Ait. embryogenic cultures. Results showed that the optimisation of the nutrient composition in the maturation medium increased the number of mature embryos by 25% (187.8 embryos per gram of fresh mass in average compared to 144.5 embryos in regular medium). It was also shown that 12-month cryostorage did not reduce viability or embryogenic ability of maritime pine cultures. A further increase in the yield of the protocol could be obtained by using benzyladenine in the conversion medium, promoting the bud-break of axillary buds that yielded 5.7 shoots in average per somatic embryo. Rooting of axillary shoots reached 84.3%. This methodology offers an alternative to overcome some problems associated with low somatic embryo production since the plantlet yield could be increased fivefolds.     

MICROPROPAGATION IN TISSUE CULTURE OFDAHURIAN LARCH

Dormant buds of Larix gmelinii(4-30years old)were cultured in vitro.Axillary budsgrew on the explants,and60％-65％ of the explants’axillary buds,a differentiation rate of 60％ wasobtained on the explants collected from the 30-year-old trees.The maximum number of axillarybuds was 26 in one induction per initial explant.Bud clusters were separated into individual budsand most of them elongated into shoots.A few roots grew on the shoots.The MS(Murashige andskoog)and SH(Schenk and Hildebrandt)were more efficient media than the WPM.(Woody PlantMedium).The best hormone Combinations for the axillary bud inductions were BA1+NAA0.01 andBA2+NAA0.2(mg/L).The procedure was as follows:(1)Apical buds were explanted on theno—hormone basal agar medium and grown for 1 or 2 weeks;(2)Explants were transferred onto thebud—inducing medium and grown for 2 weeks and then(3)Cultured on the basal medium withouthormones for axillary bud elongation;(4)Bud clusters were separated and cultured continuously toa minimum height of1     

Bark phenolics of American beech (Fagus grandifolia) in relation to the beech bark disease1

The amount of total extractable phenols was determined for bark sections obtained from behind cankers naturally induced by Nectria coccinea var. faginata and from behind mechanically inflicted wounds on stems of American beech. Healthy bark from susceptible trees was found to contain a similar level of phenolics as bark from trees determined to be resistant to the beech bark disease. Six months after wounding, wound-altered bark from susceptible trees was found to be lower in phenols than woundaltered bark from resistant trees. Inoculation of wounds with N. coccinea var. faginata resulted in decreased phenolic levels in bark sections nearest the wound surface, and increased phenolic levels in sections nearest the vascular cambium, several mm distant. Phenolic levels in injured or infected bark appear to follow similar patterns as those resulting from injury or infection of xylem tissues.     

Factors affecting Chondrostereum purpureum infection of Salix

Factors affecting Chondrostereum purpureum infection of Salix were studied in the laboratory and field. Basidiospores passively entered all xylem tissues but not cambial, phloem or bark tissues. A small number entered stems up to 70 mm from the wound surface although most were retained within tissues 30 mm from the surface. Basidiospores required free water for germination and the optimum temperature for germtube extension was 26°C. Hyphae ramified extensively throughout xylem tissues eventually plugging vessels. Cell walls and starch granules were penetrated with enzymatic assistance. The depth of C. purpureum penetration was affected by wound diameter and inoculum concentration but not stem age. Wounds of Salix were susceptible to C. purpureum throughout the year and greater depth of penetration was obtained using mycelium as inoculum than basidiospores. The depth of C. purpureum penetration decreased with duration of wound exposure prior to inoculation. A characteristic flora of saprophytic fungi and bacteria inhabited wound tissues.     

Fungi associated with Ips typographus on Picea abies in southern Poland and their succession into the phloem and sapwood of beetle‐infested trees and logs

This study dealt with the species distribution and frequency of fungi associated with the bark beetle Ips typographus (Scolytidae) on spruce trees of various states of health in southern Poland. The spruce trees were assessed by their degree of defoliation and damage of their crowns and trunks after attack by I. typographus. The state of health of trees from which samples were obtained was related to varying stages of brood development of I. typographus. Fungi were isolated from phloem taken from and around insect galleries and the sapwood underneath brood systems. Samples were taken from ‘healthy‐looking’, weakened, wind‐fallen and wind‐broken trees as well as from trap trees. The mycobiota associated with I. typographus was quite diverse in respect of the number of detected species, 65 fungal taxa were obtained from the phloem of trees infested by I. typographus, and 36 taxa occurred in the sapwood underneath insect galleries. The spectrum of fungi mainly consisted of ascomycetes and anamorphic fungi. The ophiostomatoid fungi were represented by 14 species and were the most numerously represented group in all niches examined. The most frequent ophiostomatoid species were Ceratocystis polonica, Ophiostoma ainoae, O. bicolor, O. penicillatum, O. piceae and O. piceaperdum. The frequency of occurrence of ophiostomatoid fungi differed significantly between the phloem and sapwood as well as in relation to the varying states of health of the spruce trees. These quantitative differences in the mycobiota of I. typographus between spruce trees belonging to different health categories can be explained by successional patterns of fungal colonization of host tissues following attack by I. typographus. The pathogenic species C. polonica was the primary invader, occurring most frequently in the sapwood of ‘healthy‐looking’ trees. Ophiostoma bicolor, O. penicillatum and O. piceaperdum also occurred during the early stages of brood development of I. typographus on ‘healthy‐looking’ trees, but they mainly colonized the phloem. In contrast, O. ainoae, O. minuta and O. piceae likely follow the aforementioned species as secondary and tertiary invaders into the phloem and the sapwood of spruce trees.