4种短体线虫形态及rDNA-ITS的PCR-RFLP鉴定

 短体线虫隶属于垫刃目(Tylenchida Thorne, 1949),垫刃亚目(Tylenchina Chitwood, 1950),短体科(Pratylenchidae Thorne, 1949),短体亚科(Pratylenchinae Thorne, 1949),短体线虫属(Pratylenchus Filipjev, 1936),是一类重要的迁移性内寄生植物病原线虫 ^[1]。国内外已报道该属线虫百余种。其中我国已报道种类有P.alleni、 P.artemisiae、 P.coffeae、 P.crenatu、 P.cruciferus、 P.dioscoreae、 P.hexincisus、 P.loosi、 P.penetrans、 P.pratensi、 P.neglectus、 P.penetrans、 P.scribneri、 P.thornei、 P.varicaudatus、 P.vulnus及P.zeae 17种。近年来,随着世界各地贸易往来日益频繁,植物线虫的传播机会大大增加,我国口岸也先后从进境植物根围截获P.brachyurus、 P.neglectus、 P.zeae、 P.coffeae、 P.penetrans、 P.pinguicaudatus等。基于本类群线虫的重要性和国内以往的鉴定都主要单纯依据形态学特征,本研究结合形态学和rDNA的PCR-RFLP及序列分析技术对浙江和海南不同寄主上植物线虫调查时发现的4个短体线虫群体进行了鉴定。     

Ultrastructure, Morphology, and Sporogenesis of Pasteuria penetrans

ABSTRACT Pasteuria penetrans is a bacterial parasite of root-knot nematodes that shows great potential as a biocontrol agent. Scanning and transmission electron microscopy were used to study the ultrastructure, morphology, and sporogenesis of four isolates of P. penetrans. The effects of different Meloidogyne spp. and tobacco cultivars on sporangium size and morphology of endospores attached to the cuticle of second-stage juveniles (J2) of root-knot nematodes also were investigated. The P. penetrans isolates and their origins were P-20 from M. arenaria race 1 in Levy County, FL; P-100 from Meloidogyne sp. in Pasco County, FL; B-4 from Pratylenchus scribneri in Seminole County, FL; and P-120 from Meloidogyne spp. in Alachua County, FL. Sporangia of the four isolates were identical morphologically and similar in their dimensions, ranging from 2.39 to 3.42 mum in diameter and from 1.38 to 2.38 mum in height. Different Meloidogyne spp. and tobacco cultivars had no effect on sporangium size. Endospores attached to J2 were visualized in three forms: endospores retaining the sporangium wall, endospores covered with a thin exosporium, and endospores without covering. Sporogenesis of P. penetrans was similar to that of other gram-positive bacteria and generally matched the seven-stage scheme reported for Bacillus thuringiensis.     

Effects of fosthiazate and aldicarb on populations of plant-growth-promoting bacteria, root-lesion nematodes and bacteria-feeding nematodes in the root zone of potatoes

A 2-year study investigated the relationship between the nematicides aldicarb and fosthiazate and populations of plant growth-promoting (PGP) and plant-growth-inhibiting (PGI) bacteria, and root-lesion nematodes and bacteria-feeding nematodes in the root-zone soils of potatoes. Fewer ( P  < 0.05) bacterial genera and species were recovered from aldicarb-treated soils than from the fosthiazate and untreated soils but bacterial population densities were greatest in the aldicarb-treated soils. In potato plantlet bioassays using root-zone bacteria from the three soil sources, bacteria from the aldicarb-treated soils reduced ( P  < 0.01) the mean wet and dry weight of shoots and roots compared with those from the fosthiazate and untreated soils. In the field, fosthiazate (but not aldicarb) significantly increased tuber yields and reduced nematode population densities of Pratylenchus penetrans in roots and soil. Population densities of bacteria-feeding nematodes (primarily Diplogaster lheritieri ) were not affected by either nematicide. Aldicarb appeared to suppress the populations of plant-growth-promoting bacteria that contribute to enhanced growth in potatoes. This is attributed to the long-term use of aldicarb at the trial site, which may have encouraged the selection, or adaptation, of soil microorganisms that degrade carbamate pesticides but do not enhance potato growth.     

甘蓝黑胫病菌有致病力与无致病力菌株的区分

 自然界分离到的黑胫茎点霉(Phoma lingan)常可发现有致病力和无致病力两类菌株,这两类菌株不但在生长速度和色素产生上有所不同,在几种同功酶图上也有不同。用淀粉胶法测定有致病力菌株和无致病力菌株的同功酶系统发现它们在7种酶系统中酶带位置和数目都表现不同.在MDH、IDH、AAT、GPT、PGM和EST的酶图中无致病力菌株移动较快,而在6PG的酶图中有致病力菌株移动较快,而在6PG的酶图中有致病力菌株移动较快.此外,无致病力菌株的MDHPGM、和AAT均有两条带,而有致病力菌株上只有一条带.     

山西省窖藏薯类块茎中2种短体线虫的种类鉴定

本文采用传统形态学与分子生物学相结合的方法,对采自山西省窖藏马铃薯和薯蓣块茎中的短体线虫进行了种类鉴定.结果 表明,从马铃薯块茎中分离出的短体线虫形态学特征与斯克里布纳短体线虫Pratylenchus scrib-neri一致,其SSU序列与P.scribneri美国群体相似性达99.8％.从薯蓣块茎中分离出的短体线虫...     

Isozyme and RAPD variation among Phytophthora colocasiae isolates from South-east Asia and the Pacific

Isozyme variation was studied in 94 isolates of Phytophthora colocasiae originating from Indonesia, Papua New Guinea, the Philippines, Thailand and Vietnam. Eight polymorphic enzyme systems (HK, PGM, PGI, Gluco, MDH, ICD, 6PDH, ME) revealed 52 isozyme patterns (zymotypes), each uniquely characterized by the presence or absence of 60 electromorphs. A core sample of 20 isolates was subsequently analysed with RAPD markers. Seven primers were used successfully and all profiles were reproducible. Clear bands were revealed and, in some cases, allowed differentiation between isolates exhibiting identical zymotypes. Results indicate that throughout this vast geographic region, taro leaf blight is caused by numerous and distinct strains that are genetically variable. Variation occurs within and between countries. The geographical distribution of zymotypes shows that none is common to two different countries. Although the differences in pathogenicity are not yet established, different P. colocasiae genotypes are likely to recombine and evolve rapidly as this species is heterothallic. From these results, a long-term breeding strategy is recommended for taro ( Colocasia esculenta ) based on recurrent selection using a wide genetic base composed of carefully selected parents from diverse geographic origins to maximize multigenic resistance in progenies.     

Influence of aldicarb on root lesion nematodes, leaf disease and root rot in wheat and barley

The effects of aldicarb on populations of root lesion nematodes (primarily Pratylenchus penetrans ) and on grain yields of spring barley and wheat were examined in the field over 3 years, 1981*83. The incidence of barley net blotch ( Pyrenophora teres ), wheat leaf blotch ( Leptosphaeria nodorum ), and common root rot ( Cochliobolus sativus ) was also recorded in 1982 and 1983. Aldicarb treatments reduced the size of root lesion nematode populations in soil and roots in all years, except in the mid-season soil sample in 1983. The severity of leaf disease was decreased only in 1982, but the incidence of root rot was not significantly affected by the nematticide. Although aldicarb increased cereal grain yields by approximately 15% there was no significant relationship between numbers of root lesion nematodes in roots and soil and fungal disease symptoms on barley and wheat.     

甘肃省白银市保护地蔬菜根结线虫病病原鉴定

2012年5月-7月在甘肃省白银市采集了番茄、黄瓜、豇豆等几种保护地蔬菜的25个根结线虫群体.直接解剖获得雌虫,通过雌虫会阴花纹观察,结合酯酶和苹果酸脱氢酶(MDH-EST)测定,鉴定出所收集的25个群体中有16个为南方根结线虫[Meloidogyne incognita (Kofoid&White)],9个为爪哇根结线虫[M.javanica (Treub)].南方根结线虫为优势种群,本次试验也是首次在甘肃省发现爪哇根结线虫.     

Isoenzyme diversity in Xanthomonas campestris pv. mangiferaeindicae

A collection of 26 strains of Xanthomonas campestris pv. mangiferaeindicae isolated from three different host species in eight countries was investigated for variation in isozyme patterns. Three enzyme systems were analysed: esterase (EST), phosphoglucomutase (PGM) and superoxide dismutase (SOD). Four groups of strains were identified: nonpigmented strains isolated from mango and pepper-tree in Australia, Comores, India, Reunion Island, South Africa, and Taiwan; nonpigmented Brazilian strains from mango; nonpigmented strains from ambarella isolated in the French West Indies; heterogeneous yellow pigmented strains from mango (Brazil and Reunion Island). The value of isozyme profiling as markers of the pathogenicity groups in X. c . pv. mangiferaeindicae is discussed.     

不同杀线剂对穿刺短体线虫的杀灭效果研究

为寻找杀灭短体属线虫有害生物的有效药剂,以穿刺短体线虫为供试线虫,测定了5种不同杀线剂对该线虫的杀灭效果。结果表明,5种供试杀线剂分别处理该线虫0.5h、1h和24h后,对该线虫均有一定的杀灭效果,顺序依次为:阿维菌素﹥噻唑磷﹥灭线磷﹥丁硫克百威﹥棉隆,其中阿维菌素和噻唑磷对穿刺短体线虫的杀灭效果较好,可作为备选药剂进一步研究。     

安徽省盆景基地盆景根际线虫种类鉴定

报道了１９９４￣１９９６年安徽主要盆景基地盆景根际线虫的分离鉴定结果。鉴定线虫１０个属１６个种,重要种类有斯克里布纳根腐线虫和食真菌线虫。     

Differential effect of nematicide treatments on tuber yields in early- and late-maturing potato cultivars

A 3-year field study examined the effects of 1,3-dichloropropene or oxamyl on tuber yields in four early- and five late-maturing potato cultivars. The nematicide treatments increased total tuber yield by ≈ 12% for early-maturing cultivars, but by less than 2% for the late-maturing cultivars. The treatments reduced the numbers of root lesion nematodes (primarily Pratylenchus penetrans ), which were the dominant plant parasitic nematodes at the site. Covariance analysis indicated that treatment effects on total tuber yield were no longer significant after adjusting for root lesion nematodes in soil at harvest, confirming that these nematodes had a direct effect on the host. As expected, there were significant inverse relationships ( P  = 0·05) between total tuber yields and numbers of root lesion nematodes in soil and in roots. However, the application of nematicides in late-maturing cultivars was found to be of no practical value. Crop production systems designed to reduce or optimize pesticide applications can form one criterion in the development of more environmentally sensitive management strategies. The merit of utilizing chemicals for root lesion nematode control are considered with regard to cultivar maturity.     

Vertical Distribution of the Plant-Parasitic Nematode, Pratylenchus penetrans, Under Four Field Crops

ABSTRACT The vertical distribution of Pratylenchus penetrans was monitored in four fields cropped with maize, black salsify, carrot, or potato. Soil samples were collected at 21-day intervals from May 2002 until April 2003 from five plots (2 x 5 m(2)) per field. Per plot, 15 cores were taken to a depth of 70 cm and split into seven segments of 10 cm each. Within the plots, segments from corresponding depths were pooled. After mixing, 200-g subsamples were taken and nematodes were extracted by zonal centrifugation from the root fraction and the mineral soil fraction separately. In most crops, the root fraction contained more than 50% of the total number of P. penetrans. Because the ratio between the numbers of nematodes in the root fraction and mineral soil fraction changes during the growing season, numbers of P. penetrans found in the mineral soil fraction cannot be used to estimate the total number in the soil. Therefore, both fractions have to be processed to obtain a reliable estimate of the density. No nematodes were recovered below 50 cm soil depth, except in the maize field where nematodes were found at 70 cm. The optimum sampling depth for maize, black salsify, carrot, and potato was 45, 25, 25, and 35 cm, respectively. The percentage of nematodes per soil layer was independent of the sampling date, indicating that a defined optimum sampling depth will be applicable throughout all seasons. The cumulative vertical distribution, modeled with a logistic equation, can be used to estimate the sampling error when samples are collected at different depths.     

Morphological and molecular characterisation of Pratylenchus oleae n. sp. (Nematoda: Pratylenchidae) parasitizing wild and cultivated olives in Spain and Tunisia

A new mono-sexual root-lesion nematode species, Pratylenchus oleae n. sp., parasitizing roots of olive plants cv. Koroneiki in commercial fields at Ouled Chamekh (central Tunisia), and wild and cultivated olive (cv. Picual) plants in Agua Amarga (southern Spain) is described. The new species is characterised by the female having a lip region slightly offset and bearing three annuli, stylet 16.5 (14.5-17.0) μm long, with prominent rounded knobs, pharyngeal overlapping rather long (22–36) μm, lateral fields areolated and with four incisures and diagonal lines in middle band, spermatheca rounded but non-functional, tail short, conoid-rounded to subcylindrical, usually annulated terminus, males unknown, and a specific D2-D3, ITS1, 18S-rRNA, hsp90 and COI sequences. Morphologically this species is related to P. cruciferus, P. delattrei, and P. kumamotoensis. The results of the phylogenetic analysis based on sequences of the D2-D3 expansion regions of 28S, partial 18S and ITS rRNA genes confirmed the close relationship of P. oleae n. sp. with P. dunensis, P. penetrans, P. pinguicaudatus, from which was clearly separated. A PCR-based diagnostic assay was also developed for identification of P. oleae n. sp. using the species-specific primers Poleae_fw1_4 and Poleae_rv1 that amplify a 547-bp fragment in the internal transcribed spacer (ITS1) region of ribosomal DNA, which clearly separate from other root-lesion nematodes damaging olive such as P. penetrans and P. vulnus.     

Reproductive fitness and pathogenicity of selected Radopholus populations on two banana cultivars

Ten populations of Radopholus similis from various locations and one population of Radopholus sp. from Indonesia were tested for their reproductive fitness and specific pathogenicity on Musa AAA, Cavendish cv. Poyo under controlled experimental conditions in a constant environment chamber. In addition, five of these populations were tested on Musa AAA, Ibota cv. Yangambi. Reproductive fitness of the populations tested on the two cultivars, measured as the ratio of the final number of nematodes per root system (P_f) to the number of nematodes inoculated (P_i), differed significantly. Greatest fitness was observed among R. similis populations collected from banana in different African countries (Cameroon, Uganda and the Ivory Coast) and one population from arecanut in Sri Lanka. In contrast, a population from tea in Sri Lanka and the population of Radopholus sp. from turmeric in Indonesia were the least fit. Specific pathogenicity was estimated at 8 weeks and 12 weeks after inoculation using three plant growth parameters: fresh root weight, fresh shoot weight and plant height, compared to uninfected control plants. Reduction in plant root weight was the best indicator of pathogenicity. While the R. similis populations from Uganda and the Ivory Coast were highly pathogenic, other populations with great reproductive fitness (i.e. isolates from Cameroon and Sri Lanka) did not significantly reduce root weight. In cv. Poyo, no linear correlation was found between final numbers of nematodes per gram of root (P_f) and the decrease of root weight.     

Distribution analyses of Meloidogyne spp. and Pratylenchus coffeae sensu lato in coffee plots in Costa Rica and Guatemala

The distribution of the plant-parasitic nematodes Pratylenchus coffeae sensu lato and Meloidogyne spp. were studied in two plots, one in Guatemala ( P. coffeae and M. paranaensis ) and the other in Costa Rica ( P. coffeae and M. exigua ). The quantity of nematodes per g fresh weight root were counted for each coffee tree sampled. The distributions were aggregated, and generally fitted well to negative binomial distributions. Population aggregation was greater when a smaller number of nematodes were involved, suggesting that initial colonization develops in foci. Analyses of the relationships between population levels of the species suggested that there was competition between Pratylenchus coffeae and Meloidogyne spp. This competition was expressed differently depending on the relative population density of the different species.     

海南省穿刺巴斯德芽茵资源调查及其系统发育分析

穿刺巴斯德芽菌Pasteuriapenetrans是根结线虫的专性寄生菌,是最有潜力的根结线虫生防因子。为了解海南省穿刺巴斯德芽菌资源,本文首次对海南省根结线虫病区进行调查,发现穿刺巴斯德芽菌的分布与土壤类型、寄主植物、气温等因素相关。在获得的27个巴斯德芽菌16SrDNA克隆中,均与穿刺巴斯德芽菌具有较高同源性。其中,菌株wf2与穿刺巴斯德芽菌P．penetrans16SrDNA同源性为95．8％,在发育树上处于独立的分支,为巴斯德芽菌属的1个新种;菌株dal、da2、qh和th与P.penetrans16SrDNA同源性为97．5％~97．8％,在发育树上也处于独立的分支,很可能是巴斯德芽菌属的新种;另22株可归属为穿刺巴斯德芽菌。调查结果反映出海南省具有丰富的巴斯德芽菌资源。     

四种短体线虫的形态和分子生物学鉴定

 采用形态学和分子生物学方法对来自荷兰的藏橐吾和铃兰、泰国的高山榕、缅甸的香蕉等种苗(球)分别鉴定出玻利维亚短体线虫Pratylenchus bolivianus、铃兰短体线虫P. convallariae、咖啡短体线虫P. coffeae和斯佩奇短体线虫 P. speijeri。对这4种线虫的形态特征进行较为详细的描述后,认为头环、口针、侧区、生殖系统和尾形等形态特征是种类鉴定的重要依据;进一步利用引物28S-D2A/28S-D3Br扩增测序得到上述4种线虫的28S rRNA基因D2/D3区序列,序列分析发现玻利维亚短体线虫从欧洲到美洲的不同种群之间的遗传距离变异较小,仅为0~0.007;铃兰短体线虫同一种群不同个体之间的遗传变异较大,为0.006~0.029;咖啡短体线虫不同种群之间的平均遗传距离为0.013;斯佩奇短体线虫不同种群之间的平均遗传距离为0.010;咖啡短体线虫P. coffeae和斯佩奇短体线虫P. speijeri亲缘关系很近,二者种间平均遗传距离仅为0.026。本研究再次证明线虫28S rRNA基因D2/D3区基因序列可作为短体线虫种间鉴定的依据。     

四种短体线虫的形态和分子生物学鉴定

采用形态学和分子生物学方法对来自荷兰的藏橐吾和铃兰、泰国的高山榕、缅甸的香蕉等种苗(球)分别鉴定出玻利维亚短体线虫Pratylenchus bolivianus、铃兰短体线虫P.convallariae、咖啡短体线虫P.coffeae和斯佩奇短体线虫P.speijeri。对这4种线虫的形态特征进行较为详细的描述后,认为头环、口针、侧区、生殖系统和尾形等形态特征是种类鉴定的重要依据;进一步利用引物28S-D2A/28S-D3Br扩增测序得到上述4种线虫的28S rRNA基因D2/D3区序列,序列分析发现玻利维亚短体线虫从欧洲到美洲的不同种群之间的遗传距离变异较小,仅为0～0.007;铃兰短体线虫同一种群不同个体之间的遗传变异较大,为0.006～0.029;咖啡短体线虫不同种群之间的平均遗传距离为0.013;斯佩奇短体线虫不同种群之间的平均遗传距离为0.010;咖啡短体线虫P.coffeae和斯佩奇短体线虫P.speijeri亲缘关系很近,二者种间平均遗传距离仅为0.026。本研究再次证明线虫28S rRNA基因D2/D3区基因序列可作为短体线虫种间鉴定的依据。     

The main parasitic nematodes on agricultural crops in Albania and their control1

From studies carried out on soil and root samples from the different agricultural crops in Albania, the following nematode genera have been detected: Meloidogyne, Ditylenchus, Tylenchulus, Globodera, Heterodera, Xiphinema, Pratylenchus, Helicotylenchus, Paratylenchus, Longidorus and Rotylenchus. The commonest species of nematodes are: M. incognita, X. pachtaicum, D. dipsaci, D. destructor, T. semipenetrans, G. rostochiensis, H. schachtii and P. penetrans. Trials on chemical control of root-knot nematodes on tomato have indicated that methyl bromide (500 kg ha^-1) and dichloropropane/dichloropropene are still the best chemicals. Certification of garlic crops, with selection of bulbs before and after sowing, has increased garlic yields to 5–10t ha^-1. Chemical methods to control D. dipsaci are not usually economical.