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.     

栎树猝死病菌国外研究进展

栎树猝死病菌Phytophthora ramorum(Werres,De Cock&Man in’t Veld)是一种危害性极大的病原菌,主要分布于欧美。文中阐述了该病原生物学性状、传播方式、防治措施、对生态环境的影响以及分子生物学和免疫学技术的应用等方面的国外研究进展。     

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.     

栎树猝死病菌对城市绿地的风险性分析

该文利用城市绿地有害生物风险分析体系,从病原菌传入、种群定殖、潜在的危害严重度等方面分析了栎树猝死病菌的风险性。结果表明栎树猝死病菌传入我国及其在我国定殖的风险性(R)为0.87,属于极高风险;其传入风险性(P1)、定殖风险性(P2)、潜在的危害严重度(S)值分别为0.783,0.891,0.873。     

Phytophthora ramorum is a generalist plant pathogen with differences in virulence between isolates from infectious and dead‐end hosts

Variation in virulence was examined among isolates of Phytophthora ramorum from epidemiologically important or infectious (non‐oak) and transmissive dead‐end (oak) hosts from North America. Twelve isolates representative of the genetic, geographic and host range of P. ramorum in the western United States were inoculated on leaves of Umbellularia californica (bay laurel or bay) and stems of Quercus agrifolia (coast live oak). In spite of extreme genetic similarity among the isolates employed, and even within the same genotype, significant differences in lesion size were measured, suggesting virulence in this pathogen is also controlled by epigenetic factors. A strong positive correlation between lesion size on bay laurel and coast live oak provides experimental evidence P. ramorum is a generalist pathogen that lacks host specificity. Isolates from non‐transmissive oaks were significantly less pathogenic both on oaks and bays than isolates from infectious hosts. These results are essential to further our understanding of the epidemiology and evolutionary potential of this pathogen. A quantitative differential in virulence of isolates from hosts with different epidemiological roles has been described for many animal diseases, but is a novel report for a plant disease.     

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.     

Specific hybridization real‐time PCR probes for Phytophthora ramorum detection and diagnosis

Sudden Oak Death, caused by Phytophthora ramorum, poses a serious threat to native American oaks, and is also present in Europe where it has been isolated from numerous European ornamental plant nurseries. Its proven aggressiveness against plants in the Fagaceae and Ericaceae and the damage it has caused in North America have lead to it being assigned quarantine status. The timely and accurate detection of P. ramorum is a critical aid in the study of the epidemiology and biology of this pathogen. As a regulated organism, the availability of a sensitive and reliable assay is essential when attempting to achieve early detection of the pathogen. In this work, new specific hybridization probes for a real‐time PCR amplification method were found to be rapid, robust and labour‐saving, and proved suitable for routine use in a molecular diagnostic laboratory.     

栎树猝死病原在中国的适生区预测

利用CLIMEX生态气候模型和GIS系统,对栎树猝死病原在我国的适生区进行预测.模型分析表明:温度和湿度是栎树猝死病分布的主要气候限制因子,新疆、甘肃、内蒙古、宁夏和青海地区主要受干胁迫影响,广西、广东、江西和海南的大部分地区主要受热胁迫影响,这些地区均不适宜栎树猝死病菌生存.栎树猝死病菌适合于阴冷潮湿环境下生长,长江流域附近被划为边缘适宜区域,主要包括山东、河南、陕西、江苏和湖南的部分地区,其中还包括黑龙江、吉林和辽宁的东部地区和台湾省南部地区.适宜区域和最适宜区域主要集中在中国西南地区的四川、贵州、云南、重庆和中部的湖北,以及东南部的浙江、江苏、安徽、福建和台湾.这些区域正是我国杜鹃花属、栎属和石栎属等重要寄主的主要分布地区,提前预防该病害入侵是非常必要的.     

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.     

Potential susceptibility of Australian flora to a NA2 isolate of Phytophthora ramorum and pathogen sporulation potential

Phytophthora ramorum is an invasive plant pathogen and the cause of considerable and widespread damage in nurseries, gardens and natural woodland ecosystems of the USA and Europe. It is considered to be a significant plant disease as it could cause biodiversity loss and severe economic losses in plant industries in areas where it is not yet known to exist, such as Australasia. Foliar susceptibility and sporulation potential were tested using detached‐leaf assays for 70 Australian native plant species sourced from established gardens and arboreta in California using a NA2 isolate of P. ramorum. Correa‘Sister Dawn’, Eucalyptus regnans, Isopogon cuneatus, I. formosus, Leptospermum scoparium, L. lanigerum and Melaleuca squamea were identified as potentially highly susceptible host species. Hedycarya angustifolia, Olearia argophylla, Phyllocladus aspleniifolius, Pittosporum undulatum and Podocarpus lawrencei were identified as potentially resistant. All 70 species were able to be infected with P. ramorum, as confirmed by reisolation. Putative sporulating hosts include five members of the Myrtaceae, Agonis flexuosa, Corymbia ficifolia, Eucalyptus haemastoma, E. delegatensis and E. viminalis. As a part of a precautionary strategy, the potentially highly susceptible species found in this study are suitable candidates for targeted surveillance programmes in high‐risk incursion areas of Australia and within the global horticultural trade.     

The sporicidal activity of yellow‐cedar heartwood,essential oil and wood constituents towards Phytophthora ramorum in culture

In this paper, we demonstrate that 140 mg/kg of essential oil from the wood of yellow‐cedar, incense cedar, Port‐Orford‐cedar or western juniper strongly inhibits zoospore germination and hyphal growth of Phytophthora ramorum in culture. Four individual compounds in yellow‐cedar heartwood were also tested. Zoospore germination was reduced to 0% with 10, 100 and 1000 mg/kg of nootkatin, carvacrol and valencene, respectively. Nootkatone was the least active compound, with 3.5% zoospore germination at 1000 mg/kg. Sporangia germination was 0% with 500 mg/kg of nootkatin or carvacrol. The disruption of the zoospore outer membrane and the loss of sporangial contents were often observed and indicative of sporicidal activity. Hyphal growth was inhibited by 99.9% with 50 mg/kg of nootkatin or 500 mg/kg of carvacrol, but growth resumed upon removing the inhibitors. The zoosporicidal activity of yellow‐cedar heartwood shavings stored dry for approximately 10 years was consistent with the quantity of extractable compounds they contained. Thus, spreading fresh shavings or chips of yellow‐cedar heartwood with appreciable higher concentrations of the active compounds, over areas in infection zones where spores might be difficult to control such as trails and parking lots used by hikers and bicyclists, might be useful as part of an integrated program to minimize P. ramorum spore distribution.     

Variation in pathogenicity among the three subspecies of Phytophthora alni on detached leaves,twigs and branches of Alnus glutinosa

Pathogenicity tests were carried out on leaves, twigs and branches of Alnus glutinosa using several isolates of Phytophthora alni ssp. alni, P. alni ssp. multiformis and P. alni ssp. uniformis in vitro. Healthy fresh leaves were collected from disease‐free areas and inoculated with mycelium on agar discs or by dipping in zoospore suspensions. In addition, twigs and branches were collected from both disease‐free and disease‐affected areas, inoculated with mycelium on agar discs and incubated at four temperatures (15, 20, 25, 30°C). All subspecies tested were pathogenic but with varied level of virulence. In inoculation tests on foliage, wounding was a key factor in causing infections: lesions on inoculated wounded leaves were larger than on non‐wounded leaves. In the twig and branch inoculation tests, no differences in virulence were observed among the P. alni subspecies in terms of sampling locations, but lesions differed in size according to incubation temperature, with the largest lesions occurring on tissues incubated at 25°C. The work is the first to report foliar necrosis caused by P. alni on A. glutinosa. P. alni ssp. uniformis was the least virulent of the subspecies in branch inoculations. These findings demonstrate that various tissues of A. glutinosa could act as sources of pathogen inoculum and may disseminate alder Phytophthora in natural ecosystems.     

Field survival and growth of clonal,micropropagated Eucalyptus marginata selected for resistance to Phytophthora cinnamomi

Clones of jarrah (Eucalyptus marginata), micropropagated from glasshouse-grown seedlings selected for resistance or susceptibility to Phytophthora cinnamomi, were planted in a former bauxite mine-site in the jarrah forest and inoculated with P. cinnamomi. Mortality after 13 years in resistant clones was 0–30%, while that of susceptible clones was 40–100%. Mean heights of resistant clones after 13 years were 7.8–13.6 m, while heights of surviving susceptible clones were 0.9–6.7 m. The resistance character of the seedling ortets was transmitted consistently to the clones. The field mortality of clones of some rare, apparently resistant seedlings selected from susceptible half-sib families was low after 1 year, but approached that of the susceptible clones after 2 years. The results show that Phytophthora-resistant jarrah ortets can be selected using stem-inoculation of glasshouse-grown seedlings; the resistance of the resulting clones has been validated in the field in an inoculation trial.     

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.     

Gene × environment tests discriminate the new EU2 evolutionary lineage of Phytophthora ramorum and indicate that it is adaptively different

A new evolutionary lineage of the destructive introduced tree pathogen Phytophthora ramorum, EU2 lineage, was recently discovered attacking larch and other hosts in Northern Ireland and south west Scotland, UK. Sixteen ‘medium × agar concentration × incubation temperature’ stress environments were tested to find a rapid and repeatable method to discriminate the known EU2 lineage from the EU1, NA1 and NA2 lineages in culture, in particular from the EU1 already prevalent across the UK; and to investigate whether EU2 might be adaptively different. At 25°C ¹ on both Carrot agar and V8 juice agar, the mean radial growth rates of all four lineages were significantly different, with NA2 > EU2 > EU1 > NA1. At this temperature, EU2 colonies were not only phenotypically distinct from EU1 and all other lineages but on average grew three times as fast as EU1. This indicates that EU2 is adaptively different from EU1. Twelve days growth in the environment ‘V8A/2% agar/25°C ¹ gave excellent discrimination of all four lineages in three repeat experiments, including clear discrimination of EU2 from EU1. Each lineage exhibited a distinctive colony pattern. The utility of this test environment was examined further by screening fresh UK isolates of unknown lineage from new larch outbreak sites alongside standard isolates. The lineage assignments predicted were corroborated by gene sequencing and RFLP profiling. These results also revealed that the EU2 lineage was present at several new larch sites in south west Scotland, whereas isolates from geographically adjacent areas such as the Isle of Mull, north west Scotland, the Isle of Man and north west England were all of EU1 lineage.     

Individual tree growth differences in a clonal eucalypt blanking trial at four years

Achieving a well-stocked stand at time of planting is important to compartment productivity and yield in clonal eucalypt stands for pulpwood production. The window period in which individuals that replace those that died after initial planting (blanks) still contribute meaningfully to final yield is set by the competition dynamics between individuals within the stand. The longer the period between planting and blanking, the greater the advantage achieved by original plantings in dominating site resources (light, water and nutrients) and suppressing blanks. A blanking trial was established to determine growth differences of blanked and original individuals and productivity per unit area. Factorial combinations of blanking period after planting, mortality pattern and extent of mortality were tested. Analysis of measurements at four years showed that the period before blanking is the only factor that significantly accounts for the variation in plot volume between treatments, although the percent mortality and pattern of mortality by percent mortality together also have influence on plot volume. Late blanking results in a higher proportion of suppressed trees that contribute little to total volume. Asymmetric competition is more pronounced where late blanking is carried out and where mortality is spread, rather than clumped.     

番荔枝不同家系果实表型性状的差异

为了筛选出适宜广西石漠化地区栽培的番荔枝（Annona squamosa）新品种,2012年从越南岩溶地区引进了番荔枝15个家系的果实,通过对其果实表型性状差异性分析,结果表明：各家系之间果实纵径、单果质量、糖度以及种子的长度、种宽、种厚、千粒重均有显著差异,而各家系之间果实横径差异不显著,果实纵径最大是27号,果实横径最大是11号,单果质量最重是23号,糖度最高是20号。     

印楝属植物表型变异分析

[目的]结合分子数据确定印楝属内3个种及云南省引种栽培过程中发现的变异类型,特别是变异类型的遗传背景及它们之间的遗传关系.[方法]采用方差分析、聚类、主成分分析等方法,对云南省引种栽培的印楝、泰楝、菲楝以及引种栽培过程中发现的一个变异类型的20个表型性状进行分析.[结果]印楝属植物的形态学特征存在广泛变异,其中,小叶面积变异幅度最大,变异范围为6.09~21.50 cm²,变异系数为51.70%,其次为种子宽与小叶对数,二者变异幅度分别为0.47~0.95 cm和7.00~12.33,变异系数分别为30.34%和30.31%;主成分分析显示,印楝属植物的形态变异主要来源于小叶基部偏斜程度、小叶叶形、果实长、小叶面积、复叶长度、小叶对数.对印楝属植物和外类群近缘属植物川楝进行UPGMA聚类分析,结果表明:印楝属植物可以归为2个大类群,第一类群包括印楝、泰楝、变异类型,第二类群只包含菲楝;第一类群又可分为2个小分支,印楝和变异类型聚为1个小分支,而泰楝单独聚为1个小分支.[结论]印楝属植物叶表型性状存在广泛变异,变异类型与印楝遗传相似度最大,但是否为印楝的变种或印楝和泰楝的杂交个体有待进一步确定.     

Within-stem variation in wood properties of triploid hybrids of Populus tomentosa at three clonal trials

To obtain a better understanding of variability in wood properties along the stem in triploid hybrids of Populus tomentosa L., basic wood density (BWD) and fiber traits were measured at four sample heights for each tree sampled from three clonal trials. The BWD showed a similar trend at each site in that density was initially decreased then increased with increasing height position. For fiber traits, the fiber length (FL) decreased linearly with the height of the trunk, whereas fiber width and coarseness changed relatively slightly with increasing height position. In addition, the vertical wood properties distribution patterns were different from clones so that some clones showed more markedly change in height positions than others. The joint analysis indicated significant height position effect for BWD and fiber traits, in accordance with site and clonal effect. Significant site × height interaction was found on wood properties, whereas the significant clone × height interaction was only obtained at fiber width. Moreover, variation in the relationships between FL and BWD or other fiber traits showed no consistent pattern with increasing height position.