紫花苜蓿丛枝病植原体的分子检测及鉴定

 利用植原体16S rRNA基因通用引物对云南昆明发生的苜蓿丛枝病感病植株总DNA进行巢式PCR扩增,得到1.2kb的特异片段,从分子水平证实了苜蓿丛枝病的病原是植原体。从PCR产物的RFLP酶切图谱可看出,该植原体株系的酶切图谱与马里兰翠菊黄化植原体(AY1)相同。对扩增片段进行克隆及序列测定后,利用最小进化法做Bootstrap验证的系统进化树,表明苜蓿丛枝病植原体为Candidatus Phytoplasma asteris成员之一,与植原体16SrI-B亚组成员关系密切。     

深绿木霉T2菌株对百合疫霉拮抗作用及机制

本文采用对峙培养、抗生物质测定、对扣培养、圆盘滤膜法、酶活性测定等方法,研究了深绿木霉对百合疫霉病菌的拮抗作用及机制。结果表明,深绿木霉T2菌株具有较强的营养竞争与重寄生作用;并发现其有抗生物质和溶菌酶类产生,对峙培养60 h时,深绿木霉生长速率是百合疫霉的3.68倍,能够与其竞争营养,抑制了百合疫霉的生长与扩展,深绿木霉寄生在百合疫霉菌丝上生长,导致百合疫霉菌丝降解;其代谢产物能够抑制百合疫霉的生长,48 h时难挥发性和易挥发性代谢产物对百合疫霉的抑菌率分别为85.07%和79.10%;深绿木霉T2菌株的发酵液有较高的β 1,3 葡聚糖酶活性,并在第5天达到峰值,为18.54U;深绿木霉发酵液对百合疫霉菌丝有降解作用。     

拮抗细菌B8对烟草黑胫病菌的抑制作用及其菌株鉴定

从烟草根际土壤中分离到对烟草黑胫病菌及其它植物病原菌具有抑制作用的菌株B8,平板对峙培养抑菌带内菌丝畸形、原生质凝集或外渗。其发酵原液和发酵液的无菌滤液均能抑制烟草黑胫病菌和蜡状芽孢杆菌;离体叶片接种法测定其发酵液对烟草黑胫病的防效,结果表明预防作用达100%。室内盆栽试验表明B8菌株发酵液对烟草黑胫病的预防作用可达78.1%,优于治疗作用。该菌菌体杆状,芽孢侧生、椭圆形,经形态学、生理生化性状和16S rDNA序列测定,将其鉴定为侧孢短芽孢杆菌。     

Characterization of Phytophthora spp. Causing Outbreaks of Citrus Brown Rot in Florida

ABSTRACT Epidemics of citrus brown rot from 1994 to 1997 in the south-central and east-coast citrus areas of Florida were characterized and the causal Phytophthora spp. identified. Two species of Phytophthora, P. palmivora and P. nicotianae, were consistently associated with brown rot. Epidemics caused by P. palmivora appeared to be initiated on immature fruit dropped on the orchard floor. The soilborne fungus infected and sporulated on these fruit and was then disseminated to fruit above 1 m in the canopy. In contrast, infection by P. nicotianae, the common cause of root rot, was confined to the lowest 1 m of the canopy. Fruit infected by P. palmivora produced large amounts of ellipsoidal sporangia available for splash dispersal, whereas those infected by P. nicotianae produced far fewer spherical sporangia. Isolates from brown rot epidemics were compared with P. nicotianae from citrus in Florida and Texas, P. citrophthora in California, P. palmivora, and selected Phytophthora spp. from other hosts. Brown rot symptoms produced by the different pathogenic citrus isolates on inoculated fruit were indistinguishable. Morphology, mating behavior, and isozyme patterns of brown rot isolates from 1988 to 1997 matched P. palmivora from citrus roots, other host plants, and other locations, but were different from characterized isolates of P. citrophthora in California and P. nicotianae in Florida and Texas. Cellulose acetate electrophoresis of the isozyme glucose-6-phosphate isomerase rapidly identified the causal citrus pathogen from infected fruit and soil isolation plates. Although P. palmivora is an aggressive pathogen of citrus roots, bark, and fruit, populations in orchard soils were low compared with P. nicotianae.     

Population dynamics of Rumex obtusifolius under contrasting lucerne cropping systems

The population dynamics of Rumex obtusifolius L. was analysed in a lucerne:winter cereal crop rotation by means of a matrix population model that takes into consideration two crop rotation periods: the lucerne (Medicago sativa L.) cropping period and the cereal cropping period. Several transition matrices based on life-cycle stages were calculated for each cropping period using experimental data and were used in the construction of a model that analyses the population dynamics of R. obtusifolius under different harvest dates and lengths of lucerne cropping periods. Model projections showed that populations of R. obtusifolius increased during the lucerne cropping period regardless of harvest date and decreased during the cereal cropping period. Under a late harvest date, populations decreased at each crop rotation when lucerne was grown for 3 years, remained close to the equilibrium when lucerne was left to grow for 5 years, and increased for longer lucerne cropping periods. In contrast, populations of R. obtusifolius decreased even with a lucerne cropping period of 9 years under an early harvest date. The significance of these results in relation to the biology and the non-chemical control of the species is discussed.     

Thermal Inactivation of Phytophthora nicotianae

ABSTRACT Phytophthora nicotianae was added to pasteurized soil at the rate of 500 laboratory-produced chlamydospores per gram of soil and exposed to temperatures ranging from 35 to 53 degrees C for 20 days. The time required to reduce soil populations to residual levels (0.2 propagule per gram of soil or less) decreased with increasing temperatures. Addition of cabbage residue to the soil reduced the time required to inactivate chlamydospores. Temperature regimes were established to simulate daily temperature changes observed in the field, with a high temperature of 47 degrees C for 3 h/day, and were good estimators of the efficacy of soil solarization for the control of P. nicotianae in soil. Cabbage amendment reduced the time required to inactivate chlamydospores of P. nicotianae and its effect was more pronounced at lower temperature regimes.     

烟草疫霉菌的培养及大量产生游动孢子囊和游动孢子方法的研究

烟草疫霉菌(Phytophthora nicothianae)在燕麦培养基上生长良好 ,菌丝在滴加了土壤浸出液的皮氏培养液中 ,可在 3d内产生大量的游动孢子囊 ,游动孢子囊经低温处理后再培养 2 4h可产生大量游动孢子     

Detection and identification of a phytoplasma from lucerne with Australian lucerne yellows disease

Foliar and root symptoms are described for Australian lucerne yellows (ALuY), a disease common in Australian lucerne seed crops. A phytoplasma was detected in plants exhibiting symptoms, but not in symptomless lucerne plants. Oligonucleotide primers specific to the phytoplasma 16S-23S rRNA intergenic spacer region (SR) were used in polymerase chain reaction (PCR) assays on DNA extracted from lucerne plants with and without symptoms. Identical restriction fragment length polymorphism (RFLP) enzyme profiles were obtained for PCR products amplified from 10 yellows-affected lucerne samples. RFLP profiles obtained for four restriction enzymes were different from those of the tomato big bud (TBB) phytoplasma. ALuY phytoplasma PCR products were sequenced to determine phylogeny and were found to fall within the faba bean phyllody phytoplasma group, or phytoplasma group 16srII. Transmission electron microscopy revealed phytoplasmas in the phloem of yellows-affected plant samples, but not in symptomless plant samples. Fungal, bacterial and viral agents in the aetiology of Australian lucerne yellows were ruled out.     

甲霜灵对烟草黑胫病菌细胞膜透性的影响

为阐明烟草黑胫病菌抗甲霜灵的生理机制,研究了不同浓度甲霜灵处理540 min内敏感、低抗和高抗菌株相对渗率的变化情况,结果表明:甲霜灵能损害烟草黑胫病菌的细胞膜,使膜透性不断增大;其对敏感菌株ZC002和低抗菌株10M-006细胞膜透性的影响速度和程度比较接近,都随处理浓度(0.1~100.0 μg/mL)的提高而不断增加;对高抗菌株10M-004细胞膜透性的影响速度最慢,程度最低,且随处理浓度(50.0~100.0 μg/mL)的提高而增加。高抗菌株适应甲霜灵的能力明显强于敏感菌株和低抗菌株,该适应能力随甲霜灵浓度的提高而减弱。     

荧光假单胞菌拮抗菌株对烟草疫霉的抑菌机制及控病效果

为探讨烟草根际生防细菌对烟草疫霉Phytophthora nicotianae的抑菌机制,从重庆地区连作烟田健康烟株根际土壤分离获得5株荧光假单胞菌Pseudomonas fluorescens拮抗菌株。通过平板对峙及代谢产物抑菌试验筛选对烟草疫霉具有高效拮抗作用的菌株,其中,P-72-10菌株抑菌效果最强,抑菌带半径达13.0 mm,相对抑制率为68.57%,且该菌株代谢产物对烟草疫霉菌丝生长有明显的抑制作用,相对抑制率达25.39%～46.03%;显微观察发现该菌株可引起烟草疫霉菌丝的分支增多,菌丝顶端膨大呈畸形,多数菌丝中间或顶端细胞的细胞壁加厚、原生质浓缩和产生类似厚壁孢子的细胞。在温室盆栽条件下P-72-10菌株对烟草黑胫病也表现出良好的控病效果,对抗病和感病品种的相对防效分别为53.57%和66.37%。     

Specific detection of Phytophthora nicotianae using the polymerase chain reaction and primers based on the DNA sequence of its elictin gene ParA1

Six primers based on the sequence of the flanking and coding regions of the elicitin gene ParA1 of Phytophthora nicotianae were tested for specific detection of the fungus by the polymerase chain reaction (PCR). One combination, IL7/IL8, with IL7 in a flanking region and IL8 in a coding region of the gene, gave an intense 378 bp signal with a diverse collection of isolates of P. nicotianae, that included some from black shank disease of tobacco and others from a variety of hosts. The sequence of the amplification product obtained with an isolate that produces elicitin and one that does not, was homologous with the known sequence of the ParA1 gene. The same primer combination gave no signal with sixteen other Phytophthora species tested except for two isolates P. palmivora with which it gave a weak 800 bp signal. It gave no signal with DNA from healthy tobacco and tomato plants but P. nicotianae was detected in inoculated tobacco and tomato plants. Small numbers of zoospores (>100) trapped onto a nitrocellulose membrane after filtration from suspension were also detected after two successive rounds of PCR.     

Differential Sensitivity of Phytophthora parasitica var. nicotianae and Thielaviopsis basicola to Monomeric Aluminum Species

ABSTRACT Aluminum (Al) is toxic to many plant pathogens, including Thielaviopsis basicola and Phytophthora parasitica var. nicotianae. Because fungi-toxicity of Al has been described in soils over a wide pH range, multiple species of Al may be responsible for pathogen suppression. The goals of this work were to determine the sensitivity of T. basicola and P. para-sitica var. nicotianae to Al over a range of pH values, quantify the toxicity of monomeric Al species to production of sporangia of P. parasitica var. nicotianae and chlamydospores of T. basicola, and detect the accumulation of Al in pathogen structures. A complete factorial treatment design was used with Al levels ranging from 0 to 100 muM and pH levels ranging from 4 to 6 in a minimal salts medium. The chemistry of test solutions was modeled using GEOCHEM-PC. Colonies were grown in 5% carrot broth, and after 1 or 2 days, the nutrient solution was removed, colonies were rinsed with water, and Al test solutions were added to each of four replicate plates. After 2 days, propagules were counted and colonies were stained with the Al-specific, fluorescent stain lumogallion. The oomycete P. parasitica var. nicotianae was sensitive to multiple monomeric Al species, whereas sensitivity of T. basicola to Al was pH-dependent, suggesting that only Al(3+) is responsible for suppression of this fungal pathogen. Chlamydospore production by T. basicola was inhibited at pH values <5.0 and Al levels >20 muM, whereas sporangia production by P. parasitica was inhibited at Al levels as low as 2 muM across all pH values tested. The lumogallion stain was an effective technique for detection of Al in fungal tissues. Aluminum accumulated in sporangia and zoospores of P. parasitica var. nicotianae and in nonmelanized chlamy-dospores of T. basicola, but not in cell walls of either organism. The differential sensitivity of the two organisms may indicate that true fungi respond differently to Al than members of the oomycota, which are more closely related to plants.     

Root rot of lavender caused by Phytophthora nicotianae

Lavender (Lavandula angustifolia) plants with rotted roots and discoloured vascular systems consistently yielded cultures of fungi that were identified as Phytophthora nicotianae van Breda de Haan (= P. parasitica). Inoculation experiments using either zoospore suspensions or mycelial fragments were successful in reproducing symptoms originally observed on wilting and dying plants. Lavender is a new host for P. nicotianae.     

Molecular Characterization of Natural Hybrids of Phytophthora nicotianae and P. cactorum

ABSTRACT Hybrid isolates of Phytophthora nicotianae x P. cactorum from five different hosts (Cyclamen, Lavandula, Lewisia, Primula, and Spathiphyllum spp.) were identified by their atypical morphology and their well-defined heterozygous isozyme patterns. The hybrid nature of these isolates was tested by restriction fragment length polymorphism analysis of the internal transcribed spacer (ITS) region of rDNA, generating fragments typical for both P. nicotianae and P. cactorum. In hybrid isolates, polymerase chain reactions (PCR) with primers derived from unique parts of the ITS region (ITS-PCR) of both species yielded a combination of unique amplicons typical of both parental species. Eleven hybrid isolates, three isolates of each parental species and two atypical isolates from Rhododendron and Idesia spp. close to P. cactorum, were analyzed for amplified fragment length polymorphisms (AFLP). Consistent differences in AFLP patterns existed among the hybrid isolates, strongly indicating that these hybrids have arisen from independent hybridization events between P. nicotianae and P. cactorum. The two atypical isolates morphologically resembling P. cactorum were identical to the latter species in ITS-restriction fragment length polymorphism and response to the specific PCR primers but were intermediate between P. nicotianae x P. cactorum and P. cactorum in isozyme profiles and AFLP patterns. Since the introduction of hydroponic systems in greenhouses in the Netherlands, outbreaks of Phytophthora diseases are occurring in previously unaffected host species. This may be due to interspecific hybridization events resulting in novel pathogenic behavior.     

烟草黑胫病菌对两种保护性杀菌剂的敏感性测定

采用菌落生长速率法测定了2005年从云南、贵州、山东烟区病株上分离到的38个烟草黑胫病菌Phytophthora nicotianae Breda de haan var.nicotianae Waterhouse 菌株对两种保护性杀菌剂的敏感性。结果表明:百菌清(B)对各参试菌株的EC50值在2.33~49.47 μg/mL之间,平均值为9.35(±2.77) μg/mL;代森锰锌(Z)对各参试菌株的EC50值在3.51~82.05 μg/mL之间,平均值为10.59(±4.06) μg/mL。B及Z对除XR001外的37个菌株的EC50值分别呈连续的双峰(主峰明显)和单峰频次分布;B对构成敏感性正态频次分布主峰的30个菌株的EC50均值5.99(±0.78) μg/mL 和Z对组成连续敏感性正态频次分布的37个菌株的EC50均值8.66(±1.09) μg/mL 可分别作为烟草黑胫病菌对B及Z的敏感性基线。     

Protection of Citrus Rootstocks Against Phytophthora spp. with a Hypovirulent Isolate of Phytophthora nicotianae

ABSTRACT Phytophthora root rot of citrus in Florida is caused by Phytophthora nicotianae and P. palmivora. A naturally occurring isolate of P. nicotianae (Pn117) was characterized as hypovirulent on citrus roots. Pn117 infected and colonized fibrous roots, but caused significantly less disease than the virulent isolates P. nicotianae Pn198 and P. palmivora Pp99. Coincident inoculation of rootstock seedlings of Cleopatra mandarin (Citrus reticulata) or Swingle citrumelo (C. paradisi x Poncirus trifoliata) with the hypovirulent Pn117 and the virulent isolates Pn198 and Pp99 did not reduce the severity of disease caused by the virulent Phytophthora spp. When either rootstock was inoculated with the hypovirulent Pn117 for 3 days prior to inoculation with virulent isolates, preinoculated seedlings had significantly less disease and greater root weight compared with seedlings inoculated with the virulent isolates alone. Recovery of the different colony types of Phytophthora spp. from roots of sweet orange (C. sinensis) or Swingle citrumelo was evaluated on semiselective medium after sequential inoculations with the hypovirulent Pn117 and virulent Pp99. Pn117 was isolated from roots at the same level as the Pp99 at 3 days post inoculation. Preinoculation of Pn117 for 3 days followed by inoculation with Pp99 resulted in greater recovery of the hypovirulent isolate and lower recovery of the virulent compared with coincident inoculation.     

Seasonal patterns in water uptake for Medicago sativa grown along an elevation gradient with shallow groundwater table in Yanchi county of Ningxia,Northwest China

Lucerne(Medicago sativa L.) is a deep-rooted perennial leguminous forage with high evapo-transpiration rate exceeding the annual precipitation in semi-arid areas of Northwest China.Groundwater might be the potential water sources of lucerne in the area with shallow groundwater table.In this study,stable isotopic compositions of oxygen and hydrogen(δ18O and δD) of different water sources and xylem sap were analyzed to determine the seasonal(April,June,July and August) and topographic(three slope positions) variations in water sources for lucerne growing in Ningxia eastern semi-arid area adjacent to Mu Us Desert characterized by shallow groundwater table.Iso Source software was used to calculate the probable contribution of different water sources to the total plant water uptake.Stomatal conductance,stem water potential,carbon isotope discrimination(?~(13)C) of whole plant were also determined for evaluating the water status of lucerne growing at different slope positions.The results showed that soil water content increased as the elevation decreased.Oxygen compositions of soil water in the 0–40 cm profile fluctuated considerably.Soil water δ~(18)O values in deep profile(3.5 m) were similar to those in groundwater,implying the recharge of groundwater to this soil layer.Highest water utilization rate from deep soil profile(below 350 cm) was recorded for lucerne grown at the slope position 1(groundwater table depth of 3.5–3.9 m) in April,June and July.The lucernes at slope position 2(groundwater table depth of 5.8–6.4 m) and slope position 3(groundwater table depth 7.1–8.3 m) mostly used water from deep soil layers(below 350 cm) during dry period,and turned to use water from superficial soil layer in wet period.Higher yield,?~(13)C value of whole plant and stomatal conductance were observed for lucerne grown at the slope position 1 than those at other slope positions.These results indicated that groundwater is a significant water source for transpiration of lucerne grown in Ningxia semi-arid area with shallow groundwater table where lucerne grassland is suggested to be established so as to obtain better yield performance.     

几丁寡糖对烟草黑胫病的控制效应及其机制

在室内条件下,采用菌丝生长速率法测定了几丁寡糖对烟草黑胫病菌的抑制作用,继而在温室盆栽和人工接种条件下,分别测定几丁寡糖、木霉、几丁质、几丁寡糖+木霉、几丁寡糖+木霉+几丁质等5种处理对烟草黑胫病的防治效果。结果表明,几丁寡糖在离体条件下对烟草黑胫病菌菌丝生长无抑制作用,但在盆栽试验中对烟草黑胫病具有显著防治效果。5种处理中,几丁寡糖单独处理对烟草黑胫病的防治效果最高,为57.81%,其次为几丁寡糖+木霉+几丁质和几丁寡糖+木霉处理,分别为53.49%和52.49%。在温室条件下对烟草植株施用几丁寡糖及人工接种黑胫病菌后10天内,几丁寡糖能显著提高烟草体内的SOD、POD、PPO和几丁质酶活性,且活性峰值分别比对照提高96.30%、136.36%、10.34%和9.10%。     

新疆主要农作物疫霉菌种类鉴定

 1993~1995年,对侵染新疆主要农作物的疫霉菌进行了较全面的调查研究,共从9个地区的28种作物上采集表现根腐、根颈腐、果腐症状(包括病土)的病样1531个,结果从其中17种作物上分离到452个疫霉菌株。根据形态特征、生理生化特性、致病性和菌体可溶性蛋白电泳测定,鉴定为7个种:辣椒疫霉(Phytophthora capsici Leon.),恶疫霉[P.cactorum(Leb.et Cohn) Schroeter],掘氏疫霉(P.drechsleri Tucker),菸草疫霉(P.nicotianae van Breda de Haan),苎麻疫霉(P.boehm eriaeSaw.),柑桔褐腐疫霉[P.citrophthora (Sm.et Sm.) Leonian],隐地疫霉(P.cryptogea Pethyb.etLaff.)。这些疫菌是造成新疆茄果类、瓜类、棉花、苹果、梨、枸杞、草霉、红花、白术等幼苗及成株大量死亡及烂果的主要病原,在农业生产中具有十分重要的意义。