杨树溃疡病病原——葡萄座腔菌科真菌间关系的系统学初探

利用ITS rDNA序列对我国杨树溃疡病的葡萄座腔菌科病原真菌进行系统发育学分析,共得到4种葡萄座腔菌科病原真菌:Botryosphaeria dothidea,Neofusicoccum parvum,Diplodia seriata和D.mutila。它们与相应的无性型或有性型一一对应。其中,D.seriata在我国是第一次报道为杨树的病原。研究了试验菌株与国内外已报道菌株间的系统发育学关系,分析了它们与环境间的联系。结果表明B.dothidea具有高度的种内遗传多样性。这些结果对葡萄座腔菌科真菌在全球的分布作了必要的补充,也为杨树溃疡病的研究和防治提供进一步的理论基础。     

Lasiodiplodia species associated with dying Euphorbia ingens in South Africa

Various species of Euphorbia occur in South Africa, including herbaceous, succulent and woody types. The largest of the succulent Euphorbia spp. in South Africa is Euphorbia ingens. These trees have been dying at an alarming rate in the Limpopo province during the course of the last 15 years. Investigations into the possible causes of the death have included the possible role of fungal pathogens. Amongst the fungi isolated from diseased trees were species in the Botryosphaeriaceae. The aim of this study was to identify these fungi using morphology and DNA sequence data of two gene regions (TEF 1-α and ITS). Results showed that Lasiodiplodia theobromae and L. mahajangana were present, representing the first report of Lasiodiplodia species on a succulent Euphorbia species. Pathogenicity studies showed that these Lasiodiplodia species can cause infections on healthy E. ingens trees, implicating them as contributors to the decline of E. ingens.     

Impact of Botryosphaeria,Diplodia and Neofusicoccum species on two Eucalyptus species and a hybrid: From pathogenicity to physiological performance

Species of Botryosphaeriaceae are associated with canker and dieback of Eucalyptus spp. worldwide, but little is known about their effect on the host physiology. The aim of this study was to evaluate the impact of Botryosphaeriaceae isolates from nine species in three genera (Botryosphaeria, Diplodia and Neofusicoccum), previously isolated from eucalypts, on three different Eucalyptus hosts (seedlings of E. nitens, cuttings of E. globulusand of E. globulus× E. cypellocarpa). An approach combining standard pathogenicity trials with evaluation of plant morpho‐physiological parameters was used. The size of the lesions produced revealed differences in fungal aggressiveness and host susceptibility. Isolates of Neofusicoccum kwambonambienseand Diplodia corticolawere the most aggressive, while Botryosphaeria dothidea and Diplodia seriataisolates were the least aggressive. In general, hybrid E. globulus× E. cypellocarpa plants developed smaller lesions, followed by E. nitens and E. globulus. Eucalyptus nitensplants showed minimal modifications on the morpho‐physiological profile when infected, although more severe symptoms and mortality were observed. This is probably due to a more variable genetic background of the plants. However, in general, no direct association between putative fungal aggressiveness and plant physiological disorders could be found. Results suggested that under optimal growth conditions plants manage to cope with pathogen attack and maintain their physiological performance.     

可可毛色二孢菌侵染后的烟草酵母双杂交cDNA文库构建

正葡萄是世界上最重要的经济果树之一,据联合国粮食及农业组织统计,截止到2013年,中国葡萄栽培面积已达715.5万hm2,葡萄总产量超2 300万kg。长期以来,葡萄病害是影响葡萄产业健康发展的重要因素。近年来研究发现由葡萄座腔菌科真菌(Botryosphaeriaceae)引起的葡萄溃疡病(Botryosphaeria dieback)是葡萄上的主要枝干病     

Development of isolate‐specific markers for Neofusicoccum parvum and N. luteum and their use to study rainwater splash dispersal in the vineyard

Unique bands were identified in single isolates of Neofusicoccum parvum and Neofusicoccum luteum using universally primed polymerase chain reaction (UP‐PCR) analysis of isolates obtained from grapevines and non‐grapevine hosts in New Zealand, Australia, South Africa and the USA. Primers were designed to amplify a 1550 bp portion of the 1573 bp marker band from N. parvum isolate B2141 and a 510 bp portion of the 524 bp marker band from N. luteum isolate G51a2. A PCR‐RFLP assay was developed to distinguish the N. parvum isolate B2141 from other N. parvum isolates, based on a polymorphism found in the marker band using the TaqI restriction endonuclease. For N. luteum isolate G51a2, the designed primers were specific at an annealing temperature of 63°C in the PCR. The sensitivity threshold of the N. parvum and N. luteum isolate‐specific markers was 50 pg and 5 pg, respectively, when used in standard PCR with purified genomic DNA. The sensitivity of the N. parvum isolate‐specific marker was increased to 0·5 pg by nested PCR. The specificity test of both isolate‐specific markers with six other Botryosphaeriaceae spp. showed that they were specific to their respective species and isolates. Both markers were able to detect the conidia of N. parvum and N. luteum marker isolates in rainwater samples collected at different distances from an inoculation point in the vineyard. The results showed that rain splash could disperse the conidia of both of these species up to 2 m from the inoculum point in a single rainfall event.     

芒果蒂腐病病原葡萄座腔菌科真菌种类鉴定

In order to identify the pathogen of Botryosphaeriaceae causing mango stem-end rot, tissue isolation method was used to process mango samples collected from Hainan, Guangxi, Yunnan, and Sichuan Provinces in China, and 58 fungal isolates were obtained. Among them, 39 isolates were belonged to Botryosphaeriaceae, accounting for 67.24%. These isolates were identified based on morphological characteristics and phylogenetic analysis with internal transcribed spacer (ITS) and elongation factor 1 alpha (EF-1α). The results showed that there were four main species of Botryosphaeriaceae, including Neofusicoccum parvum (33.33%), Lasiodiplodia theobromae (30.77%), Botryosphaeria dothidea (28.21%), and L. pseudotheobromae (7.69%). The mango stem-end rot caused by L. pseudotheobroma was firstly reported in China. The virulence of the four species was tested by in vitro inoculation and the results showed that all isolates were pathogenic, among which L. theobromae, L. pseudotheobroma, and N. parvum were the “strong” grade, and B. dothidea was the “middle” grade.     

山核桃干腐病病原菌的鉴定

葡萄座腔菌科Botryosphaeriaceae真菌是很多木本植物溃疡病和枯梢病的重要病原菌。已有报道认为:山核桃干腐病病原菌为茶藨子葡萄座腔菌Botryosphaeria dothidea,但对于葡萄座腔菌科其他真菌与山核桃干腐病的关系还没有深入研究。分离得到了山核桃干腐病相关病原菌,通过比较其形态学特征和核糖体DNA内转录间隔区(rDNA ITS)序列特征,将分离得到的供试菌株分为3组。根据形态学特征和基于ITS序列的系统发育分析,3组菌株分别鉴定为Botryosphaeria dothidea,B.fabicercianum和B.obtusa。其中B.dothidea为优势菌株,分离频率为71.42%,B.fabicercianum和B.obtusa分离频率分别为14.28%。经致病性测定,上述分离菌株均能引起健康山核桃Carya cathayensis枝条发病,但致病性存在差异。在山核桃上发现的B.obtusa和B.fabicercianum为首次报道。     

Detection of botryosphaeriaceous species in environmental samples using a multi‐species primer pair

Botryosphaeriaceous species are significant grapevine trunk pathogens worldwide, which can be difficult to identify to species level using conventional morphological methods. This study developed and optimized a quick, reliable molecular identification method that could facilitate investigations into the epidemiology of these diseases in vineyards. The multi‐species primers, BOT100F and BOT472R, amplified a 371–372 bp portion of the rRNA gene region from the six botryosphaeriaceous species commonly found in New Zealand vineyards. In silico analysis indicated that they would amplify DNA from six of the 12 lineages of the Botryosphaeriaceae, including all of the main species pathogenic to grapevines. A detection sensitivity of 1 and 0·1 pg DNA in standard and nested PCR, respectively, was achieved and this was calculated as equivalent to 2·5 conidia. Validation of the primers for environmental samples showed that their specificity was not compromized by the presence of competing DNA templates extracted from wood and soil. Single stranded conformational polymorphism (SSCP) analysis of the amplicons could resolve Neofusicoccum australe, N. luteum, Diplodia mutila and D. seriata, but did not differentiate between N. parvum and N. ribis. The optimized PCR‐SSCP was used to identify botryosphaeriaceous species present in rainwater traps collected over 1 year in a vineyard known to contain infected vines. It could detect multiple species in individual samples and demonstrated differences in the dispersal patterns of conidia from different species. Given the specificity and sensitivity of this method it could prove useful in epidemiology studies involving the numerous botryosphaeriaceous species that infect a wide range of host species.     

Damage to foliage and stems caused by fungal pathogens in young eucalypt plantations in Zambia

Plantations consisting of non-native tree species were established on the Copperbelt of Zambia to supplement timber supply from natural forests and to meet the increasing demand of round wood and fuel wood for the copper mines. In early 2004, a disease characterised by severe leaf defoliation and death of juvenile trees suddenly occurred in plantations of the Copperbelt Forestry Company. This study was undertaken to determine the nature of the disease and extent of damage caused with a view of providing a framework for sustainable management of the plantations. Disease incidence, severity and impact on plantations using the crown damage index were assessed for three consecutive years. Eucalyptus grandis was more affected than E. cloeziana and leaf spot incidence and severity were higher in Kalibu, Kitwe District, than in Kafubu, Kalulushi District. The incidence in Kalibu was 90.4%, causing tree crown damage of 18.6% at the end of the assessment in 2006 compared to 17.5% incidence and 1.02% crown damage recorded in Kafubu. Fungi collected from leaf tissues included species of Aulographina, Cryptosporiopsis, Cylindrocladium and fungi in the Teratosphaeriaceae. Stem pathogens were identified as fungi in the Botryosphaeriaceae and Kirramyces sp. Diseases induced by these pathogens are increasingly becoming major threats to young eucalypts, resulting in poor stem form. Factors favouring disease development are described and these need to be properly managed to minimise the impact of diseases in addition to the need to establish selection and breeding programmes to develop tolerant genotypes to ensure a sustainable future for the forestry industry in Zambia.     

Virulence affected by assay parameters during grapevine pathogenicity studies with Botryosphaeriaceae nursery isolates

This study assessed the symptoms that developed when 114 Botryosphaeriaceae isolates from grapevine nursery plant materials were used to inoculate excised green shoots and 1‐year‐old rooted canes of Sauvignon blanc. The experiments showed that all isolates and species were able to produce lesions. Overall, the Neofusicoccum species were shown to be highly pathogenic in both tissue types while the Diplodia species were highly pathogenic on canes but not on green shoots. Isolates of the most prevalent species, N. luteum and N. parvum, showed varying lesion lengths on excised green shoots and canes. An evaluation of the factors associated with lengths of lesions showed that they were significantly affected by experimental batch which reflected inherent host and environmental factors over time. Reisolation from inoculated canes also indicated that most isolates of all species except D. seriata were able to spread internally beyond the lesions. Genetic variability analysis using UP‐PCR showed that N. luteum isolates were genetically diverse but no association was observed between the phylogenetic group and degree of pathogenicity caused by the isolates. This study demonstrated that all Botryosphaeriaceae species from grapevine nurseries were pathogenic to grapevines and that the lesion lengths varied between species, among isolates within a species and among nursery sources, and was affected by the test method.