First report of Hymenoscyphus fraxineus on Fraxinus excelsior in Montenegro

During the monitoring of the mycological complex on different forest tree species in the Biogradska Gora National Park in north‐east Montenegro, symptoms indicative of ash dieback caused by Hymenoscyphus fraxineus were observed on young Fraxinus excelsior trees in the protected virgin forest, including dieback of plants and branches, wilting of leaves and shoots leading to a “flag‐like” habitus, premature shedding of leaves and longitudinal bark necroses. Using standard isolation methods, slow‐growing cultures with numerous phialides, typical of the asexual phase of the ash dieback fungus, were obtained. In addition, petioles with numerous characteristic apothecia were also recorded. This is the first report of H. fraxineus on common ash in Montenegro. Possible pathways of introduction and implications of the findings are discussed.     

Fraxinus excelsior seed is not a probable introduction pathway for Hymenoscyphus fraxineus

We investigated the transmission of Hymenoscyphus fraxineus from infested seed to germinating seedlings of common ash (Fraxinus excelsior) in order to determine the potential risk associated with intra‐ and intercontinental movement of seed. Neither fungal isolations from necrotic or healthy embryos nor PCR testing with H. fraxineus‐specific primers detected the pathogen. Similarly, H. fraxineus was not detected in axenically grown seedlings generated from infested seed lots. The results help clear up prior confusion of the pathogen being seed‐borne. Any remaining surface contamination by pathogen spores could be washed off seeds as a quarantine measure.     

First definite report of natural infection of Fraxinus ornus by Hymenoscyphus fraxineus

Hymenoscyphus fraxineus was isolated from four leaf rachises with necrotic lesions of flowering ash (Fraxinus ornus) seedlings, which had been planted on a forest site in Austria where they were exposed to a massive natural infection pressure. This represents the first definite report of natural infection of this ash species by the ash dieback pathogen.     

Hymenoscyphus fraxineus can directly infect intact current‐year shoots of Fraxinus excelsior and artificially exposed leaf scars

Hymenoscyphus fraxineus, the causal agent of ash dieback, was inoculated onto intact, unwounded current‐year shoots and leaf scars of 4‐year‐old, potted Fraxinus excelsior seedlings. Pieces of ash wood colonized by the fungus were used as inoculum. Three of 25 (12%) of the inoculated intact shoots and nine of 25 (36%) of the inoculated leaf scars were infected by H. fraxineus and developed typical symptoms of ash dieback, including necrotic lesions on the shoot surface and wood discoloration as well as shoot and leaf wilting distal to the inoculation site. No symptoms occurred on control seedlings, which had been inoculated in the same way but with sterile wood pieces. Visible necrotic lesions on shoots and wood discoloration were statistically significantly longer in proximal than in distal direction from the inoculation site, a pattern which resembles symptoms after natural infection. The ash dieback pathogen was re‐isolated from nine of 12 (75%) of the symptomatic seedlings. These results provide indirect supportive evidence that the fungus infects shoots via leaves and shows that it is able, under experimental conditions using a massive mycelial inoculum, to directly infect intact, unwounded current‐year shoots of its main host in Europe.     

Ascocarp formation of Hymenoscyphus fraxineus on several‐year‐old pseudosclerotial leaf rachises of Fraxinus excelsior

The ash dieback pathogen Hymenosycphus fraxineus can form ascocarps on pseudosclerotial leaf rachises of Fraxinus excelsior not only in the year after leaf fall but also on older rachises, at least up to five growing seasons after the leaves have been shed. The significance of this finding for the epidemiology of ash dieback is discussed.     

Hymenoscyphus pseudoalbidus and Hymenoscyphus albidus: viridiol concentration and virulence do not correlate

Hymenoscyphus pseudoalbidus is the causal agent of ash dieback, a disease that is presently endangering Fraxinus spp. throughout most of Europe. The phytotoxin, viridiol, was previously isolated from culture extracts of H. pseudoalbidus and found to be toxic to leaves of F. excelsior. Thus, we were interested in learning to what extent viridiol is responsible for pathogenicity of H. pseudoalbidus and investigated this using twelve isolates of H. pseudoalbidus. We also included five isolates of the closely related avirulent species, Hymenoscyphus albidus, in our studies. Some, but not all, isolates of H. pseudoalbidus and H. albidus produced measurable quantities of viridiol in culture. Three tests were used to determine to what extent viridiol concentration correlates with virulence: culture extracts were tested for activity in leaf segment tests and for inhibition of germination of seedlings of Fraxinus excelsior; virulence of the isolates was tested following infection of axenically cultured ash seedlings. Activity of the culture extracts varied, as did virulence of the isolates following inoculation into seedlings. No correlations were found between viridiol concentration and activities of culture extracts in leaf segment tests or in the germination test, nor between viridiol concentration and disease symptoms when inoculated into seedlings. However, activities of culture extracts in leaf segment and in the germination test correlated, as did the results of each of these tests with virulence in the infection experiment. Apparently, as yet unidentified factors other than the concentration of viridiol play important roles in the virulence of H. pseudoalbidus.     

Tissue‐specific DNA levels and hyphal growth patterns of Hymenoscyphus fraxineus in stems of naturally infected Fraxinus excelsior saplings

Ten saplings of European ash (Fraxinus excelsior L.) naturally infected by the invasive ash dieback pathogen Hymenoscyphus fraxineus were collected in Ukraine and Norway and examined for bark necrosis and extension of discoloration in sapwood and pith in a stem region. Tissue‐specific colonization profiles were determined by spatial analyses of symptomatic and visually healthy stem tissues using a H. fraxineus‐specific qPCR assay and light microscopy. Our data suggest that hyphal growth in the starch‐rich perimedullary pith is of particular importance for both axial and radial spread of H. fraxineus, but that most of its biomass accumulates in sapwood parenchyma. The study confirms the results from earlier work and presents new information that refines the current stem invasion model.     

A versatile method for assessing pathogenicity of Hymenoscyphus fraxineus to ash foliage

We describe a method for inoculating rachises of Fraxinus excelsior (European or common ash) with Hymenoscyphus fraxineus, which is faster than previous methods and allows associated foliar symptoms to be assessed on replicate leaves. A total of ten ash seedlings were inoculated with five isolates of H. fraxineus and lesion development assessed over four weeks. A five‐point disease progress scale of symptom development was developed from no lesion (0), lesion on rachis (1), “pre‐top dead,” with curling of distal leaflets and bending of the rachis (2), top dead, with wilting and death of distal leaflets (3) to leaf abscission (4). The method revealed variation in aggressiveness of H. fraxinus isolates and may be suitable for assessing the resistance of F. excelsior and other Fraxinus species to dieback. The in vitro growth rate of H. fraxineus isolates was highly correlated with both disease progress and the length of rachis lesions on susceptible plants, indicating that it can be used as a preliminary step in selecting isolates with high aggressiveness for use in resistance screening.     

水曲柳人工林培育技术

水曲柳为东北地区珍贵乡土阔叶树种,由于其种子具有深休眠性致使繁育困难。通过20多年的系统研究,解决了制约水曲柳生产培育的技术难题。文章从种子处理、育苗、造林及抚育方面系统总结了水曲柳培育的关键技术,以期对林业生产具有指导作用。     

水曲柳优良种源选择及造林技术

揭示了水曲柳地理变异规律为生长性状变异呈经、纬向渐变为主。采用选择指数法 ,确定可在吉林省中东部生态区推广的全适性优良种源为辉南种源、大海林种源。采用环境指数法进行种源稳定性评价 ,确定出辉南、大海林种源为高产型种源。选择优良种源造林 ,树高可获得 1 8 0 0 %～ 35 .0 7%的遗传增益 ,胸径可获得 1 6 .4 6 %～4 6 .6 2 %的遗传增益 ;采用主成分分析方法 ,综合水曲柳生长性状、形态特征 ,地理气候因子验证相结合的方法 ,将水曲柳划分成 3个种源区和 2个种源亚区。提供了优良种源造林技术     

水曲柳的芽接繁殖技术研究

通过用美国大叶白蜡作砧木进行芽接试验,结果表明:美国大叶白蜡和水曲柳的优良性状得到了互补,嫁接亲和力强,提高了抗逆性和生长量。在各嫁接方法中,T形芽接效果较好,平均成活率为95.5%;在4个时间段内,5月31日采用与T形芽接组合成活率最高,达到98.6%;在考虑当年高生长量时,同样是5月31日采用T形芽接高生长量最高,达到43.60cm;在3种砧木高度的处理中,以嫁接高度为10cm的处理,平均成活率最高,达到89.5%。     

Hymenoscyphus fraxineus mycelial growth on media containing leaf extracts of different Oleaceae

We assessed the mycelial growth rate of Hymenoscyphus fraxineus, the causal agent of ash dieback, on agar media containing leaf extracts of seven common Mediterranean species of the Oleaceae (Fraxinus excelsior, F. angustifolia, F. ornus, Ligustrum vulgare, Olea europaea, Phyllirea latifolia and Syringa vulgaris). The pathogen grew on all media, but growth rates showed significant differences among media and H. fraxineus isolates. Growth rates were highest on media containing F. excelsior and F. angustifolia, intermediate on media containing O. europaea and P. latifolia and lowest on those containing F. ornus and L. vulgare.     

水曲柳种子真菌群落研究

落地的水曲柳种子自然染菌率较高,未染菌的种子占6.7%;经分离鉴定,从水曲柳种子上共分离出22种真菌,其中接合菌2种、子囊菌1种、半知菌18种、待鉴定1种,交链孢菌是优势种群。在种仁、种皮、种翅3个部位上分离到的真菌种类、数量差别较小。     

水曲柳早期选择研究

以吉林省中东部水曲柳天然林和人工林平均优势木为材料,通过树干解析研究生长性状早晚期相关性,结果表明:不同林分类别个体生长变异随树龄增大而减小,幼-成龄相关系数随树龄增大而增大,其中天然林变异系数递减率在25 a生后趋于平稳,各生长性状幼-成龄相关性在25 a生时达到显著或极显著水平;人工林变异系数递减率在9~12 a生以后趋于平稳,各生长性状幼-成龄相关性在9 a生时达到显著或极显著水平。天然林除树高外,其他各生长性状早期选择效率均在25 a生时达到最大值,而后逐渐减小;人工林除材积外,9~12 a生时各性状早期选择效率达到最大值。综合分析不同性状生长变异规律、早晚期相关及选择效率,初步确定天然林和人工林最佳早期选择树龄分别为25 a生和9 a生。     

水曲柳枝芽特性的研究

本研究以水曲柳枝芽为试材,采用石蜡切片与外部形态观测相结合的方法,目的在于明确水曲柳枝芽生长特性和发育进程,以期完善该树种的发育生物学的基础理论研究。     

水曲柳的栽培技术初探

简要介绍了水曲柳的生物学特性及经济价值,并对该树种育苗地的选择、种子处理、播种等方面做出说明。     

The introduction of Hymenoscyphus fraxineus to Northern Ireland and the subsequent development of ash dieback

Ash dieback caused by Hymenoscyphus fraxineus was first recorded in Northern Ireland (NI) in November 2012. The disease was observed only on recently (<6 years) planted trees. An in-depth case study in 2015 of an ash plantation with severe symptoms indicated that many of the trees were infected at the time of planting. Apothecia were observed developing from pseudosclerotia beneath the epidermis of dead branches still attached to the tree, suggesting a possible mechanism whereby H. fraxineus could be disseminated without leaf or rachises infection. Apothecia also formed on roots, indicating that infections may also occur in the soil. Often young trees were killed by the formation of large basal lesions which did not arise from stem infections higher up. On first detecting the disease on the island of Ireland the Governments of NI and the Republic of Ireland published an “All-Ireland Chalara Control Strategy.” Part of that strategy was a ban on the importation of ash plants from regions where the disease was known to be present, to prevent the introduction of further inoculum, and the implementation of an ‘eradication and containment’ policy with the aim of preventing the establishment and spread of the disease. While these measures may have slowed disease establishment, they were ultimately unsuccessful and by 2018 ash dieback was widespread and established throughout the whole of NI in plantations and in the wider environment.     

Aqueous leaf extract of Ligustrum vulgare inhibits ascospore germination and mycelial growth of Hymenoscyphus fraxineus

Hymenoscyphus fraxineus, the causal agent of common ash dieback, possesses a low level of genetic diversity in Europe. The introduction of novel strains of this fungus must be prevented, due to the possible emergence of new virulence alleles, which could result in the infestation of the small proportion of hitherto resistant or tolerant ash trees. More comprehensive knowledge of the host spectrum of H. fraxineus is necessary for preventing further introductions. It is possible that H. fraxineus manifests itself in hosts beyond the genus Fraxinus, though this proposition has received little attention thus far. Two in vitro experiments were set up to investigate whether privet (Ligustrum vulgare) could serve as a host: germination rate of fresh H. fraxineus ascospores and colony growth of H. fraxineus were tested on agar media containing leaf extracts of privet, common ash (Fraxinus excelsior) and manna ash (Fraxinus ornus). Two different media with leaf extracts were tested, one with high (60%) and one with low (12%) extract content. Barely any significant differences were recorded in the case of the media with low extract content. Significant effects occurred only at the higher extract content level: germination was completely inhibited on the privet medium. Mycelial growth on the privet medium was slower than on both the common ash and manna ash media and, in addition, one of the three H. fraxineus strains was completely inhibited. These observations indicate the presence of inhibitors in privet. It is therefore unlikely to be a suitable host for H. fraxineus.     

水曲柳珍稀树种扦插繁殖技术的研究

在全光下应用双长臂旋转扫描喷雾装置进行水曲柳(Fraxinusmandshurica)扦插试验,通过对插穗的母树年龄、伐根萌条的枝条部位、切口形状、ABT生根粉配置浓度等指标的研究,结果显示,从10、15年生树冠上剪取1、2、3年生枝条,40年生树冠上剪取2、3年生枝条,40年生伐根上剪取1年生枝条做插穗,对生根率的影响差异达到了极显著水平,按生根率高低排序:伐根当年萌生枝条84.76%>10年树的1年枝条82.5%>15年生树的1年枝条79.68%>10年生树的2年枝条56.21%>15年生树的2年枝条55.63%>10年生树3年枝条52.46%>15年生树3年枝条50.62%>40年生树冠的2年生枝条48.36%>40年生树冠的3年生枝条45.59%,切口形状、ABT生根粉浓度和浸泡插穗基部时间对生根率的影响差异极显著,插穗以斜切口,ABT生根粉浓度用质量分数为2.0×10-4的溶液,浸泡插穗基部根时间以4 h为最佳组合,平均生根率为84.76%,最高为93.14%,伐根萌生枝条部位对生根率的影响差异不显著。     

Control of Hymenoscyphus fraxineus,the causal agent of ash dieback,using composting

Viability of Hymenoscyphus fraxineus inocula following temperature treatments for different exposure times was examined in vitro and in aerated flask‐ and large‐scale composting tests using green waste. After an exposure for up to 10 days at 20°C, 97.3% of H. fraxineus mycelium and pseudosclerotia plate cultures remained viable. No viability was detected following a 3‐day exposure to 40°C or a 1‐day exposure to 45°C although pseudosclerotia were more tolerant than mycelium to an exposure to 35°C. Primordial apothecia of H. fraxineus emerged from 62%–100% of infected ash rachises collected from two infected sites and stored at 4°C for 0–5 months; exposure to compost for up to 10 days at 20°C did not affect this emergence. No emergence of H. fraxineus apothecia was observed from ash rachises that were exposed to compost at 45°C for 1 day or at 35°C or 40°C for 3 days in flasks or at 40°C for 1 day or at 30°C for 5 days in a large‐scale composting system. Based on a fitted model, estimates of the survival of H. fraxineus inoculum in infected ash rachises exposed to compost at 50°C for 1 day were 0.081% of that in the untreated H. fraxineus ash rachis inoculum. Increasing loss in viability of H. fraxineus inoculum in infected ash rachises during longer and warmer exposures to compost at 35°C–45°C corresponded with a reduced concentration of pathogen DNA detected in the rachises using real‐time PCR. However, exposure of rachises to compost at >53°C resulted in a smaller reduction in pathogen DNA detected than exposure to compost at lower temperatures, possibly due to the inhibition of enzymatic degradation of DNA at elevated temperatures.