新疆野苹果和秦冠的抗黑星病特性

 透射电镜观察表明, 苹果叶片上表皮角质层厚度在品种间存在显著差异, 新疆野苹果和秦冠叶片的角质层厚度显著高于富士和嘎啦的;同一品种不同龄期叶片的角质层厚度随着叶龄增长而增厚, 且黑星病严重度与叶片上表皮角质层厚度间存在显著的负相关关系。苹果品种抗病性组分分析结果表明, 新疆野苹果的病害严重度最低, 约为嘎啦的1/22, 潜育期最长, 为嘎啦的2.0倍, 无(或少有)病斑出现, 不产孢。秦冠的严重度约为嘎啦的1/14, 潜育期约为嘎啦的1.5倍, 产孢量约为嘎啦的1/26。黑星病菌在新疆野苹果和秦冠叶片上的侵染概率及病斑扩展速率均显著低于富士和嘎啦。因此, 新疆野苹果和秦冠对黑星病的抗病性表现在抗侵入和抗扩展两个方面。     

Histological and ultrastructural characterization of the leaf infection events of Colletotrichum fructicola on Malus domestica ‘Gala’

Glomerella leaf spot (GLS), characterized by black necrotic spots and severe defoliation, is a destructive foliar disease of apple. Widely grown cultivars such as Gala and Golden Delicious are highly susceptible to GLS. Currently, the infection biology of the causal pathogen, Colletotrichum fructicola, on apple leaves is unclear. In the present study, the penetration and colonization processes of C. fructicola were characterized on apple (cv. Gala) leaves using light and transmission electron microscopy. C. fructicola conidia produced germ tubes 4 hours post-inoculation (hpi) and appressoria at 8 hpi. In melanized appressoria, funnel-shaped appressorial cones formed around the penetration pore. At 12 hpi, C. fructicola produced secondary conidia. After penetration, C. fructicola began to develop infection vesicles at 36 hpi. At 48 hpi, the primary hyphae of C. fructicola were produced from infection vesicles within host epidermal cells; the host epidermal cell plasma membrane remained intact, indicating a biotrophic association. Subsequently, secondary hyphae penetrated epidermal cells and destroyed cell components, initiating necrotrophic colonization. C. fructicola also produced biotrophic subcuticular infection vesicles and hyphae. Together, these results demonstrate that C. fructicola forms special infection structures and colonizes apple leaves in a hemibiotrophic manner, involving intracellular as well as subcuticular colonization strategies. Detailed characterization of the infection process of C. fructicola on apple leaves will assist in the development of disease management strategies and provide a foundation for studies of the molecular mechanism of the C. fructicola–apple leaf interaction.     

苹果炭疽叶枯病病原Glomerella cingulata及其侵染过程

为明确苹果炭疽叶枯病病原菌围小丛壳Glomerella cingulata的侵染致病特征,在分离获得该病原菌的基础上,采用形态学观察、ITS序列分析和致病性测定对其进行了鉴定,并利用光学和扫描电子显微镜对病原菌在嘎啦苹果叶片上的侵染过程进行了研究.结果表明,在陕西咸阳地区分离获得的9株病原菌均为围小丛壳G.cingulata.25 ℃下接种9 h后,分生孢子中间产生隔膜,双胞化,并萌发产生芽管和附着胞;24 h后分生孢子的2个细胞均可萌发并形成芽管及附着胞,部分芽管顶端可产生次级分生孢子;48 h后次级分生孢子萌发形成附着胞;72 h后,附着胞下形成的侵染钉可直接入侵寄主,在表皮细胞内形成初生菌丝和次生菌丝,此时叶片表面已出现褐色斑点.接种7 d后叶片病斑处出现分生孢子盘和子囊壳.表明陕西省近年出现的苹果炭疽叶枯病病原菌为围小丛壳G.cingulata,该病菌在嘎啦叶片上的一些特殊侵染行为可能是导致该病害易在短时间内暴发的重要原因.     

渭北富士苹果偏斜果的影响因素

为了明确引起渭北富士苹果果形偏斜的原因,于2009~2010年在西北农林科技大学白水苹果试验站,以16~17年生的乔化富士苹果为主要试材,从授粉受精和果实类型两方面对其果形进行研究。结果表明:除粉红女士授粉外,秦冠、嘎拉、新红星3个品种授粉都均降低富士苹果的果形偏斜指数,改善果形;柱头数对富士果实形状有明显影响,人为去除3~4个柱头会明显减少果实种子数,导致果实发育畸形。种子数和种子在心室中的分布决定了果实的形状,端正果每个心室几乎均有种子分布,偏斜果至少有1个心室种子败育,畸形果至少有2个心室中的种子败育。比较长、中、短果枝果形,长果枝果实的果形指数明显大于中、短果枝,而果形偏斜指数小于中、短果枝,但差异不显著。下垂果多为果形高桩的端正果,畸形果少,果形偏斜指数明显小于侧生果。     

苹果叶片应答斑点落叶病菌胁迫的蛋白质组学分析

In order to elucidate key proteins related to disease-resistance in apple leaves,the total proteins were extracted from control and infected apple leaves, and separated by two-dimensional electrophoresis. The differential expressed proteins were then identified by MALDI-TOF-TOF/MS.In total, 25 differential expressed proteins were detected by Image Master Software. After tryptic digestion, MALDI-TOF-TOF/MS analysis and database searching, 20 protein spots were finally identified, including 11 functional proteins related to photosynthesis, energy metabolism, stress and defense responses. The pathogenesis-related proteins involved in defense responses such as APX, GPX and Mal d1 were differentially expressed in apple leaves. It indicates that these proteins may play a key role in the resistance to A. alternata apple pathotype.     

基于iTRAQ定量蛋白质组技术筛选‘华月’苹果斑点落叶病抗性相关蛋白

 为了探讨苹果叶片抗病相关蛋白的表达特性,进一步解析苹果叶片自身防御反应分子机制,本文以抗病苹果品种‘华月’为试材,采用iTRAQ定量蛋白质组技术分析苹果链格孢菌处理前后,苹果叶片总蛋白差异表达情况。结果表明,与无菌水处理的叶片相比,病原菌接种后的叶片共筛选到433个差异表达蛋白,聚类分析结果较好,其中257个蛋白显著上调,176个蛋白显著下调。进一步开展差异蛋白筛选及功能分析,筛选后的53个蛋白依据功能可划分为7类,分别为代谢过程相关,防御或逆境应答反应相关,蛋白质代谢过程相关,细胞分裂相关,细胞壁修饰相关,细胞过程相关及其他功能未知蛋白。代谢通路分析表明,KEGG共富集到7个结果,64个蛋白在7条通路中得到注释,且这些蛋白对这7条信号通路均具有显著影响(P<0.05)。亚细胞定位分析结果表明,共49个蛋白得到成功定位,其中35个蛋白定位于叶绿体,表明叶片细胞中大量的叶绿体蛋白参与了病原菌胁迫应答反应。其他蛋白中,7个定位于细胞质,3个定位于细胞质膜,其余4个分别定位于细胞壁、胞外、过氧化物酶体及内质网。除了防御或逆境反应相关蛋白,光合作用及其他代谢相关蛋白也参与了苹果叶片细胞的防御反应。病程相关的PR类蛋白中包括3个过氧化物酶类蛋白(PR-9)、1个类甜蛋白(PR-5)及1个MLP样蛋白(PR-10),过敏反应相关的MLP样蛋白在果树应答逆境胁迫的研究中的报道较少,MLP蛋白的差异表达可能也与苹果叶片的防御反应相关。本研究进一步证实了苹果叶片应答链格孢菌胁迫的抗性相关蛋白中,PR类蛋白的差异表达可能是影响苹果叶片细胞抗病反应的关键因子,研究结果为进一步解析果树抗病分子机制提供了参考。     

云南葡萄产区葡萄炭疽病病原鉴定及致病力分析

为了明确引起云南葡萄产区炭疽病的病原种类,利用形态鉴定和特异性引物分子检测相结合的方法对从云南省主要葡萄产区采集的60株炭疽病菌菌株进行了鉴定。葡萄炭疽病菌菌株的菌落形态和生长速率与对照菌株尖孢炭疽菌Colletotrichum acutatum差异不明显,但其分生孢子大小显著小于尖孢炭疽菌,附着胞深褐色,球形或不规则形。胶孢炭疽菌Colletotrichum gloeosporioides特异性引物CgInt/ITS4从供试葡萄炭疽病菌菌株基因组DNA中扩增出1条约500 bp的特异性条带,而尖孢炭疽菌特异性引物CaInt2/ITS4对葡萄炭疽病菌无扩增条带。研究表明,引起云南葡萄主产区炭疽病的病原为胶孢炭疽菌;供试胶孢炭疽菌对红提葡萄均有致病力,但菌株致病力差异较大,对番茄和草莓存在交叉侵染的能力,且对多菌灵的敏感性较尖孢炭疽菌高。     

Epiphytic Occurrence of Pseudomonas syringae pv. papulans (Rose) in France, Where Blister Spot has Never Been Seen

Pseudomonas syringae pv. papulans (PSP) the causal agent of blister spot, on the apple cultivar Mutsu in the USA, Canada and Italy, has not been described in France. A study on epiphytic populations of P. syringae isolated from French apple orchards revealed two isolates called KA54 and E121, whose biochemical characterisation showed high similarities with PSP strains. Identical symptoms were obtained with KA54, E121 and PSP strains, after vacuum inoculation of detached immature fruits of the cultivar Fuji, and young leaves of the cultivars Fuji, Mutsu, Gala and Golden Delicious. Koch's postulate was verified. These results indicate the presence of PSP in France. Differential characterisation criteria including serological, molecular and pathogenicity tests are proposed.     

Modelling the effects of wetness duration and fruit maturity on infection of apple fruits of Cox's Orange Pippin and two clones of Gala by Venturia inaequalis

The effects were investigated of fruit maturity and duration of wetness on infection of apple fruits by Venturia inaequalis , and subsequent scab development. Incubation rate (inverse of median incubation period) increased linearly with increasing temperature (5–20°C) on detached 5-week-old fruits of cv. Royal Gala. Fruits were highly susceptible in the early stages of development, but became increasingly resistant as they matured. Inoculation of attached 12-week-old and detached near-mature fruits did not result in any lesions, while inoculation of attached 4-, 5-, 7- and 9-week-old fruits resulted in various levels of infection. Fruits of cv. Mondial Gala were more susceptible than those of cv. Cox's Orange Pippin. On cv. Mondial Gala, a wet period of 9 h resulted in ≈ 90% infection of 4-week-old fruits, but only 9% infection of 9-week-old fruits. Numbers of scab lesions on an apple generally followed a Neyman type A rather than a Poisson distribution, indicating a certain degree of aggregation of lesions on a fruit. A two-parameter generalization of the Poisson model described the observed incidence–density relationship well. A longer duration of wetness was required to result in a similar level of scab infection on old fruits to that on young fruits. On cv. Mondial Gala, wet periods of 9 and 32 h were required for ≈ 90% incidence of fruit scab on 4- and 7-week-old fruits, respectively. A mathematical model was developed to relate the incidence of fruit scab to duration of wetness and fruit maturity. The potential use of these results in practical disease management is discussed.     

Aggressiveness of eight Venturia inaequalis isolates virulent or avirulent to the major resistance gene Rvi6 on a non‐Rvi6 apple cultivar

For sustainable management of scab‐resistant apple cultivars, it is necessary to understand the role of aggressiveness in the adaptation of Venturia inaequalis populations and particularly the costs to the organism of acquiring additional virulence. The aims of the present study were (i) to identify the quantitative variables that are most important in determining the differences in aggressiveness among groups of V. inaequalis isolates, and (ii) to ascertain whether virulent and avirulent isolates of V. inaequalis differ significantly in aggressiveness. The aggressiveness of eight isolates that differed in their virulence to the major resistance gene Rvi6 was compared on the non‐Rvi6 apple cv. Gala. Three components of aggressiveness, namely lesion density, the number of spores per square centimetre of leaf area, and the number of spores per lesion, were evaluated 21 days after inoculation, and the kinetics of lesion density over time were analysed in terms of maximum lesion density, length of latent period and rate of lesion appearance. On the second youngest but fully developed leaf at the time of inoculation, maximum lesion density in the virulent group was 20% lower and the latent period 7% longer, than in the avirulent group. However, the alternative hypothesis, namely that isolates had adapted to quantitative resistance present in cv. Gala depending on their cultivar of origin, could not be rejected. The analysis of the kinetics of lesion density by a non‐linear mixed‐effect model proved useful in the assessment of aggressiveness.     

Elicitin诱导烟草微敏反应和防卫基因的表达

 研究了一种α型elicitin-parasiticein对烟草微敏反应(microscopic hypersensitive response, micro-HR)和防卫反应分子表征基因表达的诱导作用。用150 nmol/L的parasiticein喷洒烟草叶片12 h后, 经曲利本蓝(trypan blue)染色, 光镜下可观察到有细胞坏死, 说明parasiticein引起了micro-HR, 而水和低浓度的parasiticein不能引起micro-HR。用parasiticein注射叶片可引起肉眼可见的HR, 注射用parasiticein浓度远比喷洒引起micro-HR的浓度低。Parasiticein还可诱导对TMV的抗性, 浓度在30 nmol/L时比150 nmol/L的效果好。用parasiticein注射烟草30 min, HR表征基因hsr203J和hin1开始表达, 在9 h内逐渐降低, 12~16 h肉眼可观察到HR。Parasiticein喷洒烟草后, 病程相关蛋白(pathogenesis-related, PR)基因PR-1b在诱导的第1 d到第5 d都有一定量的表达。可见, parasiticein能同步诱导过敏反 应和抗病 性及其分 子表征基 因的表达 。     

Ulvan effect on conidial germination and appressoria formation of Colletotrichum gloeosporioides

Ulvan is an algal polysaccharide known for its ability to induce resistance to plant diseases such as the Glomerella leaf spot of apple caused by Colletotrichum gloeosporiodes. This study was aimed at investigating microscopically, in tests in vitro and in vivo, whether ulvan interferes in the development of pre-infective structures of C. gloeosporioides. Conidial germination and appressoria formation were monitored hourly on agar and cellophane, and at 48 h on water- and ulvan-treated susceptible as well as resistant apple leaves. Amendment of agar with ulvan (10 mg ml^?1) enhanced the germination and resulted in longer germ tubes at 7 h of incubation. On cellophane it significantly delayed appressoria formation up to 8 h, but later after 14 h increased the number of appressoria per conidium. Spraying of susceptible leaves with ulvan 6 days before inoculation decreased disease severity by 50%. This was associated with inhibition of appressoria formation and stimulus in growth of germ tubes, without interfering with conidial germination, when compared with both water-treated control and resistant plants. Appressorium formation occurred preferentially on anticlinal walls of epidermal cells and its location was not influenced by host resistance or by ulvan treatment. This study suggests a new mode of action for ulvan interfering with appressorium formation that could protect apple plants against C. gloeosporioides infection.     

葡萄胶孢炭疽菌对3种麦角甾醇脱甲基抑制剂类杀菌剂的敏感性

系统比较了从云南省主要葡萄产区采集的57株葡萄胶胞炭疽菌Colletotrichum gloeosporioides对三唑类脱甲基抑制剂烯唑醇、腈菌唑及咪唑类脱甲基抑制剂咪鲜胺的敏感性、交互敏感性及敏感性与生存适合度的相关性等指标。结果表明:供试菌株对咪鲜胺的EC50值在0.01~1.58 mg/L之间,高于其对烯唑醇(EC50值为0.05~25.45 mg/L)和腈菌唑(EC50值为0.49~192.93 mg/L)的敏感性;部分菌株对烯唑醇和腈菌唑的敏感性显著降低,而对咪鲜胺仍保持较高的敏感性;供试菌株对烯唑醇和腈菌唑的敏感性具有显著的相关性,而对咪鲜胺的敏感性与对烯唑醇和腈菌唑的敏感性之间则无显著相关性。低敏感性菌株的致病力和菌落生长速率与敏感菌株无显著差异,具有较高的生存适合度。     

Latency- and Defense-Related Ultrastructural Characteristics of Apple Fruit Tissues Infected with Botryosphaeria dothidea

ABSTRACT Apple fruit tissues infected with Botryosphaeria dothidea were examined by transmission electron microscopy using susceptible cv. Fuji and resistant cv. Jonathan. Immature (green) and mature (red) fruits of cv. Fuji with restricted or expanding lesions were also examined to reveal subcellular characteristics related with latent and restricted disease development. In infected susceptible mature fruits, cytoplasmic degeneration and organelle disruption commonly occurred, accompanying cell wall dissolution around invading hyphae. Cell wall dissolution around invading hyphae in subepidermis was rare in immature, red halo-symptomed cv. Fuji and resistant cv. Jonathan fruits. In infected immature fruits of cv. Fuji, presumably at the latent state of disease development, cellular degeneration was less severe, and invading hyphae contained prominent microbody-lipid globule complexes or the deposition of thin electron-dense outer layer around cell wall of intercellular hyphae. Both mature fruits with red halos and resistant apple fruits formed cell wall protuberances at the outside of cell walls. In addition, electron-dense extramural layers were formed in the resistant apple fruits. Aberrant hyphal structures such as intrahyphal hyphae were found only in resistant fruit tissues, indicating the physiologically altered fungal growth. These ultrastructural changes of host tissues and fungal hyphae may reflect the pathogenesis of apple white rot under varying conditions of apple fruits.     

Infection of apple fruit by Sphaeropsis pyriputrescens in the orchard in relation to Sphaeropsis rot in storage

Sphaeropsis rot, caused by Sphaeropsis pyriputrescens, is an important postharvest disease of apple in the United States. The objectives of this study were to determine the timing of apple fruit infection in the orchard in relation to development of Sphaeropsis rot in storage and to identify infection courts and mode of penetration by S. pyriputrescens on apple fruit. Fruit of apple cvs Red Delicious, Golden Delicious, and Fuji were inoculated in the orchard from 3 weeks after petal fall to 2 weeks before harvest at 5 to 6-week intervals in three consecutive seasons. All fruit were harvested and stored at 0?ºC to monitor decay development. Light and scanning electron microscopy were used to examine the infection courts and mode of penetration of the fungus on/in the host tissues. At harvest, the fungus was re-isolated from the stem (pedicel), sepal, anther, or filament of the inoculated fruit, but decay did not develop on fruit. Sphaeropsis rot developed on inoculated fruit during cold storage beginning 1–3 months after harvest. Stem-end rot was prevalent on cv. Golden Delicious, whereas calyx-end rot was prevalent on cv. Fuji. Both stem- and calyx-end rots were common on cv. Red Delicious. Infection also occurred at lenticels on fruit skin, particularly on cv. Golden Delicious, but at low incidence. Relationships between the incidence of Sphaeropsis rot in stored apple fruit and the timing of inoculation in the orchard varied with cultivar and year. On cv. Red Delicious apples, the incidence of Sphaeropsis rot generally increased as the timing of infection approached harvest. Histological studies indicated that infection took place through natural openings of plant organs such as stomata on stems and sepals and lenticels on fruit skin. Fungal penetration also was observed at micro-cracks on the stem and sepal and at trichome sockets where mechanical damage occurred in sepals. Direct penetration was observed on the stem and sepal of fruit, but most invasions were restricted between the cuticle and the epidermis. Our results indicate that wounding is not required for infection of apple fruit by S. pyriputrescens, though it may facilitate infections.     

Characterization of a new Apple dimple fruit viroid variant that causes yellow dimple fruit formation in ‘Fuji’ apple trees

A 303-nucleotide viroid was isolated from an apple tree (Malus × domestica, ‘Fuji’) cultivated in Japan. The viroid had 84.9% overall nucleotide sequence homology to Apple dimple fruit viroid (ADFVd), a member of Pospiviroidae, reported from Italy. This viroid differed from the Italian variant by 47 mutations (38 substitutions, six deletions and three insertions), and most of these mutations occurred on either side of the central conserved region. The leaves and branches of the infected trees did not have any disease symptoms, but the fruits were dimpled and yellow. The infected scions were top-grafted onto a healthy ‘Fuji’ apple tree, which tested positive for this viroid in a northern hybridization analysis, and yellow dimple fruits were produced in the second growing season. We propose that this viroid is a new variant of ADFVd and causes yellow dimple fruit formation in ‘Fuji’ apple trees.     

苹果枝条表皮应答轮纹病菌侵染的蛋白质组学分析

 蛋白质提取方法是双向电泳分析的关键。本研究中,我们通过优化提取条件,建立了适于苹果枝条表皮总蛋白提取的技术体系。在此基础上,开展了苹果枝条表皮应答轮纹病菌侵染的蛋白质组学研究,以期明确轮纹病菌胁迫下苹果枝条表皮细胞参与抗病反应的关键蛋白。具体方法是以未接菌及接菌后的苹果枝条表皮为材料,分别提取总蛋白,利用双向凝胶电泳技术结合质谱(MALDI-TOF-TOF/MS)检测分析差异表达蛋白。经质谱检测及数据库检索,共有22个蛋白点得到成功鉴定,按照功能划分为光合作用相关、糖类及能量代谢相关、防御反应相关、蛋白质合成相关及未知功能蛋白等5类。其中参与防御反应的病程相关蛋白(APX、 β-1,3-葡聚糖酶、Mal d1、PR-1)可能是苹果枝条表皮细胞应答轮纹病菌胁迫过程中参与抗病反应的关键蛋白。