Pathogenicity of four Phytophthora Species on Wild Cherry and Italian Alder Seedlings

Inoculation tests were carried out in the greenhouse on wild cherry (Prunus avium) and Italian alder (Alnus cordata) seedlings, to determine their susceptibility to certain Phytophthora species (P. citrophthora, P. alni, P. megasperma and P. cinnamomi) that are commonly present in the soil. Host susceptibility was evaluated in accordance with a disease index, with the lesion length after stem inoculation, and with a root system disease index. Wild cherry was found to be highly susceptible to P. citrophthora, and was also found to be susceptible to P. alni, although to a lesser extent. Italian alder was very susceptible to P. alni, but had only low susceptibility to P. citrophthora. The other Phytophthora species caused only modest symptoms. The danger to alder and wild cherry is all the greater because these trees not only share the same pathogens, but also commonly planted together in mixed stands. The results will now have to be confirmed by using a more natural inoculation method.     

Characterization of Alder Phytophthora Isolates from Wallonia and Development of SCAR Primers for their Specific Detection

Isolates of alder Phytophthora were collected in the southern part of Belgium on riverbanks planted with Alnus glutinosa and A. incana. They were compared with strains isolated in other European countries in terms of maximum temperature for growth, oogonia shape, pathogenicity on Alnus seedlings and genetic traits. Using both molecular techniques [random amplified polymorphic DNA (RAPD) and random amplified microsatellite (RAMS)], two groups of isolates were identified, the first group being further divided into two subgroups, Ia and Ib, using RAPD. Most of the Walloon alder Phytophthora isolates as well as the standard type from UK (formally designated P. alni subsp. alni) fell into group Ia. One isolate was classified in group Ib with the German and Dutch variants (P. alni subsp. multiformis), while three isolates were placed with the Swedish variant (P. alni subsp. uniformis) in group II. In terms of morphological properties, isolates from groups Ia and Ib developed colonies with a felt‐like appearance and usually produced numerous oogonia, varying from wavy to warty after 1 week (group Ia) or 2–3 weeks (Ib) in darkness. In contrast, colonies from group II isolates were generally irregular, and smooth oogonia were produced in low quantities after approximately 1 month in culture. A polymerase chain reaction (PCR) using sequence‐characterized amplification region (SCAR) primers derived from a polymorphic amplification product generated with a RAPD primer was developed for the specific detection of alder Phytophthora. The specificity and sensitivity of this test are discussed here.     

PCR-based DNA Markers for Identifying Hybrids within Phytophthora alni

Two pairs of oligonucleotide primers were designed for the polymerase chain reaction (PCR)‐based detection and differential identification of naturally occurring interspecific hybrid types (subspecies) of Phytophthora alni, all of which cause collar rot of alder trees. Primer pairs were derived from randomly amplified polymorphic DNA (RAPD) fragments that were unique to various subspecies of this alder pathogen. The primer pair set, SAP1/SAP2 (SAP), was derived from a 0.93‐kb RAPD fragment amplified from P. alni ssp. alni. The primer pair set, SWAP1/SWAP2 (SWAP), was derived from a 1.13‐kb fragment amplified from P. alni ssp. uniformis. Patterns of SAP and SWAP amplification enabled distinction among the three subspecies. No PCR products were amplified from isolates of 31 other Phytophthora spp. examined, including P. cambivora and P. fragariae, the suspected progenitors of P. alni. The SAP and SWAP primer sets were able to detect a minimum of 10 pg of DNA from pure cultures or DNA extracted from as few as 10 zoospores. Pathogen DNA could also be amplified directly from bark lesions of artificially inoculated and naturally infected common alders and from lesions developed on common cherry‐laurel leaves used in baiting the pathogen from infested soil. Direct detection of pathogen DNA from alder tissue using SAP and SWAP primer sets should prove useful in developing measures for effective quarantine and management of P. alni.     

A Survey of Phytophthora Species on Hainan Island of South China

During the period 1997–2007, a comprehensive study of the occurrence and distribution of Phytophthora species was conducted on Hainan Island of South China. To date, 14 species of Phytophthora have been recovered and their distribution determined. Phytophthora nicotianae ( =P. parasitica ) is the most important species attacking a wide variety of crops, followed by Phytophthora capsici and Phytophthora citrophthora . In contrast to Phytophthora colocasiae attacking taro leaves throughout the entire island, Phytophthora cyperi was found only once on Digitaria ciliaris in Danzhou. It is of interest to note that Phytophthora heveae, Phytophthora katsurae and Phytophthora insolita are commonly found in forest soil/water of protected mountains without causing any plant diseases. Although Phytophthora species are usually terrestrial or found in fresh water, one isolate of Phytophthora resembling closely the asexual isolates of P. insolita in Hainan was obtained from decaying Rhizophora leaves submerged in seawater. An unidentified Phytophthora species producing non-papillate; internally proliferating sporangia was isolated from the soil in which Ceriops tagel and Bruguiera serangula were growing in a salt water shrimp farm.     

Restriction pattern analysis of deoxyribonucleic acid isolated from callus and cell suspension of actinorhizal and non-actinorhizal Betulaceae

Using cell suspensions, a method was elaborated to isolate high-molecular-weight genomic deoxyribonucleic acid (DNA; 65 MDa or more) from members of the Betulaceae: Alnus incana (L.) Moench, Alnus glutinosa (L.) Gaertn. and Betula papyrifera Marsh. The method was also effective for isolation of DNA from callus cells. Based on the chemical lysis of protoplasts, this procedure yielded 130 μg (callus) to 250 μg (cell suspension) of DNA (g fresh cells)⁻¹, with a ratio A₂₀₀/A₂₈ of 1.7–2.0. The purified DNA obtained, formed distinct bands when restricted fragments were electrophoresed. Among the 10 endonucleases used for restriction analysis of Alnus glutinosa, Alnus incana and Betula papyrifera genomes, PvuI1 (EC 3.1.23.33) was unique in giving identical patterns for the two Ainus species. An unusual pattern occurred when Al-2 DNA was restricted with Ava II (EC 3.1.23.4). It formed a ladder with a repeating fragment unit of 181 base pairs long. With the enzymes tested, no differences in restriction patterns were observed among clones of Alnus incana (AI-2 vs AI-2), Betula papyrifera (BP-4 vs BP-8) and subclones of Ainus glutinosa AG-1 (PLFJ709 vs LF1709), suggesting genetic stability of the Betulaceae cultures.     

Host range differentiation of spore-positive and spore-negative strain types of Frankia in stands of Alnus glutinosa and Alnus incana in Finland

Host compatibility of different spore-positive (Sp⁺)and spore-negative (Sp^?) strain types of Frankia from alder stands in Finland was studied in Modulation tests with hydrocultures of Alnus glutinosa (L.) Gaertner, A. incana (L.) Moench and A. nitida Endl. Root nodules and soil samples from stands of A. incana (Lammi forest and Hämeenlinna forest) were dominated by Sp ⁺ types of Frankia (coded AiSp⁺ and AiSp⁺ H. respectively), which caused effective root nodules in test plants of A. incana, but failed to induce nodules in A. nitida. In A. glutinosa Frankia strain types AiSp ⁺ and AiSp ⁺ H caused small, ineffective root nodules with sporangia (coded Ineff ^?), which were recognized by the absence or near absence of vesicles in the nodule tissue. Ineffective nodules without sporangia (coded Ineff ^?) were induced on A. glutinosa with soil samples collected at Lammi swamp. The spore-negative strain type of Frankia was common in root nodules of A. glutinosa in Finland (Lammi swamp) and caused effective Sp^? type root nodules (coded AgSp ^?) in hydrocultures of A. incana, A. glutinosa and A. nitida. A different Sp ⁺ strain type of Frankia. coded AgSp⁺ Finland, was occasionally found in stands of A. glutinosa. It was clearly distinguished from strain type AiSp ⁺ by the ability to produce effective nodules on both A. glutinosa and A. incana. The nodulation capacities of soil and nodule samples were calculated from the nodulation response in hydrocutlure and served as a measure for the population density of infective Frankia particles. Sp ⁺ nodules from both strain types had equal and high nodulation capacities with compatible host species. The nodulation capacities of Sp type root nodules from A. glutinosa were consistently low. High frequencies of Frankia AiSp⁺ and AiSp⁺ H were found in the soil environment of dominant AiSp ⁺ nodule populations on A. incana. The numbers of infective particles of this strain type were insignificant in the soil environment of nearby Sp ^? nodule populations on A. glutinosa and in the former field at Hämeen-linna near the Sp⁺ nodule area in Hämeenlinna forest. Strain type AgSp^? had low undulation capacity in the soil environment of both A. incana and A. glutinosa stands, Explanations for the strong associations between Frankia strain types AiSp⁺ and AiSp ^? H and A. incana and between strain type AgSp^? and A. glutinosa are discussed in the light of host specificity and of some characteristics of population dynamics of both strain types. The possible need to adapt the concept of Frankia strain types Sp ⁺ and Sp ^? to strains with some variation in spore development was stressed by the low potentials of strain type AiSp ⁺ H to develop spores in symbioses with hydrocultures of A. incnna.     

台湾桤木和四川桤木种源苗木对水分胁迫的生理响应

采用PEG6000,研究了4种台湾桤木种源和2种四川桤木种源苗木对水分胁迫的生理响应。结果表明：（1）随着水分胁迫强度增加,叶片质膜相对透性增大,四川桤木种源质膜透性比台湾桤木种源增加幅度大。（2）在轻度的水分胁迫下,6个种源SOD活性均呈上升趋势,POD活性的增加只发生在PEG10％的水分胁迫强度以内,轻度水分胁迫下,MDA含量上升幅度小于重度胁迫强度;在PEG30％胁迫处理时,台湾桤木叶片MDA含量平均上升30．22％,而四川桤木上升了40．44％,表明台湾桤木种源膜脂过氧化程度较四川桤木种源轻,受害程度相对要小一些。（3）水分胁迫下,叶片脯氨酸含垃均有不同程度的增加．在PEG20％水分胁迫处理下,各种源脯氨酸积量增加的幅度为：台湾桤木苗栗种源（AF２）（68．9％）〉台湾桤木福建南平种源（AF４）（60．2％）〉台湾桤木台东种源（AF２）（54．7％）〉台湾桤木台中种源（AF1（47．6％）〉四川桤木广元种源（AC2）（39.7%）＞金堂种源（AC１）（39.1%）。水分胁迫条件下,叶片可溶性糖含量呈下降趋势。６种种源苗木在水分胁迫条件下,由于脯氨酸的积累,有一定的渗透调节能力,但这种渗透调节能力又是十分有限的。（４）采用隶属函数法,对各种源的抗旱生理进行综合评价,其耐旱牛理强弱的顺序是：AF2、AF1、AF3、AF4、AC2、AC1,台湾桤木比四川桤木具有较强的耐旱生理适应性。从耐旱生理角度讲,凡是适合四川桤木生长的区域,基本上亦适合台湾桤木,在引种载培中,应选择抗旱性稍强一些的台湾桤木苗栗种源和台中种源。     

5种桤木属植物的核型分析

利用改良去壁低渗法对桦木科（Betulaceae）桤木属（Ainus Mill.）东亚地区5种植物进行染色体数目与核型研究。结果显示：所试验物种中染色体形态比较一致,多是由中部（m）及近中部（sm）着丝点染色体组成。其中A.nitida染色体数为2n=28,核型公式为K（2n）=28=22m 4sm(2SAT) 2st(2SAT);A.hirsuta染色体数为2n=42,核型公式为K(2n)=42=36m 6sm; A.cremastogyne,A.formosana 染色体数均为2n=56,核型公式分别为K(2n)=56=2M 40m(1SAT) 14sm(1SAT)和K(2n)=56=46m 8sm 2st;A.firma染色体数为2n=112,核型公式为K(2n)=112=80m 28sm 4st。     

Features of the intrageneric Alnus-Frankia specificity

Host specificity between local Frankia strains and native alders [Alnus incana (L.) Moench and A. glutinosa (L.) Gaertn.] was evaluated in inoculation experiments. Pure cultures of Frankia , whether originating from A. incana or A. glutinosa , were infective and effective on both host species. These pure cultures were isolated from spore-negative (Sp⁻) nodules. From spore-positive (Sp⁺) nodules no Frankia isolates were obtained. This strain type resisted all our isolation attempts and therefore crushed nodules had to be used for Sp⁺ type inoculations.
The Sp⁺ type Frankia populations differed in their host specificity. Sp⁺ nodules from A. glutinosa were effective on both alder species, but Sp⁺ nodules from A. incana induced effective nodules only on the original host; on A. glutinosa only small (1-3mm) prenodule-like structures were found. Such A. glutinosa plants died on N-free medium, thus showing that these nodules were ineffective. In the effective nodules the middle cortex was dominated by infected cells filled with vesicle clusters. In the ineffective nodules only a few cortical cells were infected and sporangia predominated in these cells. Surprisingly enough they also contained vesicle-like structures as demonstrated in electron micrographs.     

Characterization and pathogenicity of three Phytophthora spp. recovered from rivers in Bulgaria

Many Phytophthora species are pathogens on fruit trees and may cause destructive diseases. In the current study, we examined six Phytophthora isolates recovered from rivers in Bulgaria, representatives of the following three species: Phytophthora chlamydospora, P. pseudocryptogea and P. syringae. Morphological traits, cardinal temperatures and growth rates of the isolates were described. We found considerable variation in the size of sporangia and significant difference in the mycelial growth rates of the two P. pseudocryptogea isolates, along with multiple polymorphic sites in the ITS region of one of them. In the cases of the other two Phytophthora species, no such differences were found between the isolates. Both P. chlamydospora isolates had a lower optimum growth temperature compared with the reported in the literature for this species. In pathogenicity tests against leaves and fruits of apple, pear, cherry, apricot and plum, the isolates proved to be capable of causing infections with varying severity. P. chlamydospora showed to be the most aggressive towards the leaves, while P. pseudocryptogea isolates induced the highest percentage of decay on the fruits of all tested tree species, which may suggest partial organ or tissue specificity. The demonstrated infection capacity of the described isolates points out the investigated Phytophthora species as a potential threat for the orchards in Bulgaria, if favourable conditions are available.     

欧洲赤杨根瘤及其内生菌的形态学研究

对在云南生长的欧洲赤杨自然发生的根瘤及其内生菌──Ｆｒａｎｋｉａ进行了光学显微镜和扫描电镜的观察,结果表明,这种根瘤与原产地生长的欧洲赤杨所结根瘤十分相似;与原产地在云南的蒙自桤木的根瘤结构也很相近。从这种根瘤中分离到的内生菌的纯培养具有典型的Ｆｒａｎｋｉａ的特征。     

三种猕猴桃根腐病致病菌多重实时定量PCR检测技术的建立及应用

[背景]近年来,随着猕猴桃种植面积的不断扩大,病害的频繁发生已逐渐影响猕猴桃的产量和品质。恶疫霉(Phytophthora cactorum)、樟疫霉(P.cinnamomi)和雪松疫霉(P.lateralis)是一类可以引起猕猴桃根腐病的致病疫霉菌。[目的]建立并优化可以同时检测3种致病疫霉的多重实时定量检测技术,并调查猕猴桃主要产区的致病菌分布情况。[方法]基于Ypt1 (ras-related protein gene)基因设计恶疫霉、樟疫霉和雪松疫霉的特异性TaqMan探针和引物,建立并优化多重实时荧光定量PCR检测体系。利用近缘种检验检测体系特异性并进行灵敏度测试,应用该检测体系分析猕猴桃主要产区根际土壤中3种致病疫霉的Yt1基因含量。[结果]供测试的11个恶疫霉近缘种、11个樟疫霉近缘种、13个雪松疫霉近缘种及非目标菌种DNA样品中均无荧光信号,反应结果为阴性,而在恶疫霉、樟疫霉和雪松疫霉DNA样品中分别检测出HEX、FAM和ROX荧光信号,反应结果为阳性。三种疫霉的检测灵敏度均达到100fg。此外,通过对猕猴桃主产区陕西省周至县和眉县果园共166份土壤样品的检测发现,恶疫霉的分布最广泛且Ypt1基因含量最高,樟疫霉和雪松疫霉则相对较少。[结论]建立的猕猴桃根腐病致病疫霉多重实时定量检测体系特异性强、灵敏度高,适合于恶疫霉、樟疫霉和雪松疫霉的检测及定量分析。该技术可为猕猴桃疫霉病害的早期诊断、监测及预防提供指导。     

滑坡和泥石流灾害与环境因子的关系

明确滑坡和泥石流灾害发生百分率与地学因子间的关系,对相关预报和危险度评价研究具有重要意义.基于全国18431个滑坡和泥石流灾害点的定位信息,结合1 km×1 km栅格内6个环境因子（高程、高差、坡度、坡向、植被类型、植被覆盖度）的空间分布数据,利用频率比数分析方法分析了这些环境因子与滑坡和泥石流灾害的关系.结果表明：滑坡和泥石流更多地分布于我国三大自然阶梯的第1和第2过渡带中海拔较低的地区;1 km×1 km栅格内高程差为300 m时,滑坡和泥石流灾害发生的可能性最大;当坡度为30°时,滑坡和泥石流灾害爆发的可能性最大;山区林地和坡耕地是最易发生滑坡和泥石流的两个地类;植被覆盖度在80%～90%时,滑坡和泥石流灾害的发生频率最高.     

不同光环境对桤木幼苗生长和光合特性的影响

通过设置3个光照强度(100%、56.2% 和12.5%),模拟森林幼苗生长的旷地(采伐迹地)、林窗和林下光环境,研究不同光照强度对外来种台湾桤木和乡土种四川桤木幼苗的生长、光合特性以及生物量积累与分配的影响.结果表明： 低光环境限制了两种桤木幼苗形态指标的增长,适当遮荫的林窗环境比旷地更有利于幼苗的生长.台湾桤木幼苗具有较高的比叶面积和相对生长速率,较大的单叶面积、叶长、叶宽、株高和基径,较少的叶片数和较低的叶面积比、叶柄长.低光环境下,台湾桤木幼苗的最大净光合速率、光饱和点和表观光量子效率较高,光补偿点和暗呼吸速率较低.随着光照强度的降低,台湾桤木幼苗具有更高的根生物量比和更低的叶生物量比;四川桤木幼苗则相反,加剧了动物取食和机械损伤的风险.     

中国桤木属植物的细胞学研究(Ⅰ)

对桦木科桤木属自然分布于中国西南地区的4个种进行了细胞学研究,结果分别为:川滇桤木K(2n)=56=30m+24sm+2st;桤木K(2n)=56=38m+16sm+2M;蒙自桤木K(2n)=56=28m+26sm+2st;毛桤木K(2n)=56=42m+14sm;其中川滇桤木的核型属于2A型,其余均为2B型;上述种类染色体数目均为2n=56。4种材料的核型均为首次报道。     

Correlations between the ages of Alnus host species and the genetic diversity of associated endosymbiotic Frankia strains from nodules

Nodule samples were collected from four alder species: Alnus nepalensis, A. sibirica, A. tinctoria and A. mandshurica growing in different environments on Gaoligong Mountains,Yunnan Province of Southwest China and on Changbai Mountains, Jilin Province of Northeast China. PCR-RFLP analysis of the IGS between nifD and nifK genes was directly applied to uncultured Frankia strains in the nodules. A total of 21 restriction patterns were obtained. The Frankia population in the nodules of A. nepalensis had the highest genetic diversity among all four Frankia populations; by contrast, the population in the nodules of A. mandshurica had the lowest degree of divergence; the ones in the nodules of A. sibirica and A. tinctoria were intermediate. A dendrogram, which was constructed based on the genetic distance between the restriction patterns, indicated that Frankia strains from A. sibirica and A. tinctoria had a close genetic relationship. Frankia strains from A. nepalensis might be the ancestor of Frankia strains infecting other Alnus species. From these results and the inference of the ages of Alnus host species, it is deduced that there was a co-evolution between Alnus and its microsymbiont Frankia in China.     

大豆疫霉根腐病抗源筛选

由大豆疫霉菌引起的大豆疫霉根腐病是大豆生产的重要病害,该病已在我国大豆主要产区发生,并在局部地区造成较大产量损失。利用抗病品种是防治大豆疫霉根腐病最有效的方法。本研究目的是筛选大豆疫霉根腐病抗源,为病害防治和抗病品种的选育提供参考。用下胚轴创伤接种方法对120个栽培大豆品种（系）进行接种,鉴定其对10个具有不同毒力大豆疫霉菌菌株的抗性。有110个品种（系）分别抗1～10个大豆疫霉菌菌株,其中以河南大豆品种（系）对疫霉菌的抗性最丰富,安徽、湖北和山西大豆品种（系）也具有抗性多样性。120个大豆品种（系）对10个大豆疫霉菌菌株共产生57个反应型,有4个抗性反应型分别与单个抗病基因的反应型一致,有7个抗性反应型与2个已知基因组合的反应型相同,其他抗性反应型为新的类型。一些大豆品种（系）中可能存在有效的抗大豆疫霉根腐病新基因。     

黑龙江省主要栽培大豆品种(系)对大豆疫霉根腐病的多抗性评价

用下胚轴伤口接种方法接种鉴定黑龙江省60个栽培大豆品种和育成品系对5个具有不同毒力大豆疫霉菌菌株41-4、PMCl、USAR4、PSZJ6和USAR17的抗性.有50个品种(系)抗1个或1个以上茵株或表现中间类型,其中有5个、8个、16个和21个品种(系)分别对4个、3个、2个和1个菌株表现抗性或中间类型.60个品种(系)对5个菌株共产生12种反应模式,其中呈RRSSR反应类型的品种(系)可能含有Rpslα或Rpslc基因,品系农大3861可能含有Rps3c基因,呈SSSSS反应模式的品种(系)可能含有Rps7基因,或不含抗病基因;其它9种反应模式与含有已知单基因品种或单基因组合的反应模式不同,可能具有未知抗病基因.该研究结果表明,黑龙江省具有较丰富的抗大豆疫霉根腐病大豆品种(系),大部分品种(系)的抗性是有效的,可合理地用于大豆生产和抗疫霉根腐病育种.     

台湾桤木引种的光合生理特性研究

以台湾桤木 Alnusformosana 引种2年生苗木为研究对象,探讨了台湾桤木的光合生理生态特性.结果表明: 1 叶片净光合速率 Pn 具有明显的日变化.中部叶Pn日变化在生长中期表现为双峰曲线型,高峰分别出现在9:00和15:00左右,峰值分别为15.03μmol·m-2·s-1和12.97μmol·m-2·s-1;在生长初期和末期为单峰曲线型.不同部位叶片Pn大小为:中部叶>顶部叶>基部叶. 2 不同部位叶片Pn表现出不同的季节变化特征.中部叶和顶部叶一年有两个高峰,高峰均出现在6月和8月;基部叶只有一个高峰,出现在6月.光合作用具有较广的温度适应范围.自然条件下叶片最大净光合速率为17.67μmol·m-2·s-1;人工条件下为20.2μmol·m-2·s-1,在新的环境条件下表现出较强的光合适应能力. 3 叶片LCP为33.32～67.47μmol·m-2·s-1,LSP为1332～1656μmol·m-2·s-1,具有较低的光补偿点和较高的光饱和点,表现出较强的弱光利用能力和强光利用潜力. 4 台湾桤木CO2补偿点为54.17～74.98μmol·mol-1,CO2饱和点在800μmol·mol-1左右;羧化效率为0.0270～0.0468.表现出较大光合作用潜力和较广的生存适应能力. 5 通径系数分析表明,Cond、Ci、Vpdl和RH是影响叶片光合速率变化最主要的直接作用因子.     

色赤杨光合作用与根瘤氮代谢的关系

在放线菌结瘤植物(Actinorhizal plants)与放线菌Frankia的共生体系中,固氮酶(N_2ase)活性与所提供的光合产物的量密切相关。通过在同一天的不同对间内,对同株色赤杨光合作用和根瘤中的N_2ase比活、NH_4~ 含量、还原糖含量以及总氮量的变化所做的同步测定结果表明,N_2ase比活的最高峰迟后于光合强度的最高峰;在根瘤内部,NH_4~ 含量和还原糖含量都与N_2ase比活呈负相关,而总氮量则与N_2ase比活呈正相关。本文对这一现象进行了讨论,并且推测还原糖作为光合作用产物的衍生物,直接影响根瘤的固氮作用,它不仅为N.2ase提供固氮所需的能量,而且为固氮产物NH_4~ 提供受体。