Studies on amoebae and cysts associated with the isolation of Spongospora subterranea f.sp. subterranea in vitro

New evidence is presented to support the contention that the amoeba/cyst colonies isolated from surface-sterilized Spongospora subterranea f.sp. subterranea -infected potato tubers and spore balls have a saprophytic phase but are contaminants and not S. subterranea. Amoebae isolated from infected tissues and spore balls formed colonies associated with bacteria on 1% water agar at 18°C and encysted after 5–7 days. These cysts were morphologically distinct from the resting spores of S. subterranea and were formed singly or in a layer, unlike the spore ball (cystosorus) of S. subterranea . Amoebae, cysts and mixtures of amoebae and cysts in primary, secondary and tertiary subcultures failed to infect tomato roots. PCR amplification of DNA from amoebae, cysts and spore balls using the S. subterranea -specific primer pair SsF/R generated a 434-bp product from S. subterranea spore balls only and not from amoebae or cysts. When an amoeba/cyst-specific primer pair AmF/R was designed and used for PCR amplification, a single 411-bp product was generated from DNA of amoebae and cysts, but not from DNA of S. subterranea spore balls. These results are discussed in relation to earlier reports claiming the successful isolation of S. subterranea and other plasmodiophorids in vitro .     

连作土壤中大豆胞囊线虫种群数量减少的原因探讨

 通过对双河、宝泉岭和白城3个大豆胞囊线虫病抑制性土壤样品土质、定殖于大豆胞囊线虫上真菌以及土壤特性(土壤抑制性、传导性、杀真菌剂处理)的研究,表明多年连作会引起大豆胞囊线虫种群数量的减少。在此类土壤中胞囊数量一般小于100个/100g土,且空胞囊率达80%以上。胞囊上真菌分离频率高,种类较单纯,其中宝泉岭土样中定殖真菌有明显的优势种-淡紫拟青霉(Paecilomyces lilacinus)。大豆胞囊线虫衰退土壤中具有抑制因子,经物理灭菌和杀真菌剂多菌灵处理后,原有抑制特性丧失,3个土样大豆根部胞囊数量分别增加140.0%、165.5%、68.2%和141.9%、215.2%、139.3%。大豆胞囊线虫抑制性土壤具有传导特性,当抑制土与无菌土按1:9混合后胞囊数量较灭菌土分别减少70.0%、26.5%和51.8%。本文证明了连作土壤中引起大豆胞囊线虫种群数量减少的主要原因是生物因子,并初步确定为食线虫真菌的作用,同时证明土壤状况与大豆胞囊线虫的衰退也有一定关系。     

The Effect of Environment on Survival and Hatching of Heterodera avenae1

Eggs within cysts of Heterodera avenae are able to survive long periods of desiccation and can be held for several years at 5oC when stored at low relative humidity. While this provides an excellent means of storing the nematode for use in laboratory experiments, field experiments show that desiccated eggs are readily dispersed by wind. Tolerance to desiccation is similar in free and encysted eggs, which hatch at the same rate. When eggs are desiccated, water is withdrawn from the larva within the egg shell. If cysts are exposed to conditions favouring eclosion, subsequent desiccation reduces egg viability and larval emergence. The importance of defining pre-test conditions in survival studies is emphasized, and differences in the physiological state of eggs may explain different observations in Australian and European studies.     

山东泰安地区小麦禾谷孢囊线虫幼虫孵化特性初步研究

为大量获得研究所需的小麦禾谷孢囊线虫2龄幼虫,在室内离体条件下研究了预处理条件、孵化温度、低温预处理时间以及田间采样时期对山东泰安地区小麦禾谷孢囊线虫幼虫孵出量的影响.结果表明:5℃低温预处理条件下,孢囊浸水处理有利于幼虫孵出;低温预处理后,孵化温度为15℃处理的2龄幼虫孵出率高于20℃处理的孵出率;低温预处理时间为8周的2龄幼虫孵出率最高;6月和8月田间采集的孢囊幼虫孵出率显著高于7月和9月,其中8月采集的孢囊幼虫孵出率最高,为最佳采集时间.     

旱稻孢囊线虫（Heterodera elachista）孵化特性研究

为了解旱稻孢囊线虫的孵化特性,从水稻根系及根系附近土壤分离孢囊,在室内离体条件下研究不同温度及水稻根系分泌物、土壤浸液、水稻根汁等因子对2龄幼虫孵化的影响,并观察不同温度下2龄幼虫的存活能力.结果表明:旱稻孢囊线虫孵化的适宜温度范围为28～32℃,且在该温度范围初孵2龄幼虫的存活率高、存活时间相对较长,35℃下孵化率及初孵2龄幼虫的存活率、存活时间均明显下降,40℃下无线虫孵化,20℃下孢囊线虫孵化率仅为0.9％,4℃下可延长2龄幼虫的存活时间.水稻分泌物、水稻土壤浸液和水稻20倍根汁对旱稻孢囊线虫孵化具有刺激作用,5倍根汁和4 mmol/L氯化锌溶液对线虫的孵化有抑制作用.上述结果可为制定旱稻孢囊线虫防治措施提供依据.     

贵州省大豆孢囊线虫的发生与分布

大豆孢囊线虫病是威胁大豆生产的重大病害之一。2015-2016年,采用随机取样的方法对贵州省大豆孢囊线虫的发生和分布情况进行了调查。调查范围包括贵阳、六盘水、安顺、毕节、黔西南和遵义等6个地区下辖的14个县(区),共采集土样287份。调查结果表明,贵州省大豆孢囊线虫的总检出率为28.2%,其中毕节地区的大方县和赫章县孢囊检出率最高,分别达到46.7%和45.0%,而在遵义市、贵阳市和黔西南州孢囊检出率为0。六盘水地区的水城县孢囊发生量最大,平均孢囊数为3.4个/200g土,最大孢囊数达到17个/200g土;安顺地区的镇宁和关岭县大豆孢囊线虫发生程度最轻,每200g土样平均孢囊数分别为0.3和0.2个,最大孢囊数仅为2个/200g土。分离的孢囊主要形态特征与大豆孢囊线虫一致,并且ITS序列与甘肃大豆孢囊线虫种群序列一致性达100%。根据形态学观察和ITS序列分析比对,将贵州省采集到的孢囊线虫鉴定为大豆孢囊线虫。本研究结果可为有效防控贵州省大豆孢囊线虫病提供依据。     

Testing potato clones for resistance to Globodera rostochiensis (Ro1) and Globodera pallida (Pa2 and Pa3) by root-ball screening1

Clones from the international test assortment were tested for resistance to Globodera pallida (Pa2 and Pa3) and estimated by root-ball counting and by extraction of the total number of cysts to show the practicability of the root-ball test. New clones in a breeding programme were tested by root-ball counting for resistance to Globodera rostochiensis (Ro1) and G. pallida (Pa2 and Pa3), and the statistical aspects are discussed. The conclusion is that testing new clones early in a breeding programme by root-ball counting of cysts with four to five replicates is a sufficient method to separate the susceptible clones from resistant and moderately resistant ones.     

Mouse (Mus musculus) as intermediate host of Sarcocystis sp. from the goshawk (Accipiter gentilis)

Sporocysts from the goshawk (Accipiter gentilis) were experimentally transferred to the mouse (Mus musculus). It was found that the goshawk is the host of one of the sarcosporidians inducing muscle sarcocystosis in mice. Thin-walled, sporulated oocysts expelled by the goshawk measured 16.5-19.0 X 12.0-13.0 micron. Those which measured 12.0-13.5 X 8.2-9.0 micron were widely elliptical, with rounded poles. No asexual reproduction of parasites was detected in the viscera of mice. The cysts started to appear in skeletal muscles on day 20 after oral infection with 10,000 or 100,000 sporocysts per mouse. The cysts measuring 15-630 X 18-65 micron contained widely oval metrocytes (2.8-4.3 X 1.5-2.8 micron) or banana-shaped cystozoites (6.0-8.0 X 2.0-3.8 micron). Three months after infection the cysts were found also in the tongue of mice. No morphological differences were observed between the oocysts-sporocysts from owls (Tyto alba and Asio otus) and goshawk, not even between the muscle cysts of these sarcosporidians in mice. The possibility of passaging the species Sarcocystis dispersa from the long-eared owl through the digestive tract of goshawk is discussed.     

Rapid detection and quantification of viable potato cyst nematodes using qPCR in combination with propidium monoazide

Potato cyst nematodes (PCN), Globodera pallida and Globodera rostochiensis, are obligate parasites of solanaceous plants, causing severe losses in several potato growing areas throughout the world. To date, management of PCN is related to nematode population densities estimated as eggs per gram of soil, without considering the actual number of viable juveniles within the cysts. In classical nematology, the standard method to determine PCN viability is based on a staining assay, using Meldola's blue dye (MB) followed by microscopic visualization of MB‐treated nematodes. Although MB is considered to be reliable in staining embryonated juveniles within eggs and cysts, it is a time‐ and labour‐consuming assay. In the present work, a real‐time PCR (qPCR)‐based method combined with propidium monoazide (PMA), a photoreactive DNA‐intercalating dye, was developed for the quantification of viable PCN. This dye renders exposed DNA of dead cells unable to be amplified by PCR, and thus only DNA from viable/intact PCN juveniles is amplified and detected. The novelty of the present method lies in the simultaneous quantitative and qualitative estimation of viable PCN inocula using species‐specific primers and TaqMan probes. The PMA–qPCR viability method (v‐PCR) developed for the two Globodera species successfully discriminated dead from living specimens in heat‐treated samples and eggs in old and newly formed cysts. Interestingly, the detection of DNA from 34‐year‐old nematode cysts stored at room temperature was observed. In conclusion, the proposed v‐PCR method should prove to be very useful for the routine determination of PCN viability from field samples.     

Identification of Fungal rDNA Associated with Soil Suppressiveness Against Heterodera schachtii Using Oligonucleotide Fingerprinting

ABSTRACT To understand the nature of a soil with suppressiveness against Heterodera schachtii, an rDNA analysis was used to identify fungi associated with H. schachtii cysts obtained from soils possessing various levels of suppressiveness. Because H. schachtii cysts isolated from these suppressive soils can transfer this beneficial property to nonsuppressive soils, analysis of the microorganisms associated with the cysts should lead to the identification of the causal organisms. Five soil treatments, generated by mixing different amounts of suppressive and fumigation-induced nonsuppressive soils, were infested with second-stage juveniles of H. schachtii and cropped with mustard-greens. Fungi were identified through an rDNA analysis termed oligonucleotide fingerprinting of ribosomal RNA genes (OFRG). Cysts obtained from soil mixtures consisting of 10 and 100% suppressive soil predominantly contained fungal rDNA with high sequence identity to Dactylella oviparasitica. The dominant fungal rDNA in the cysts isolated from the soil mixtures composed of 0.1 and 1% suppressive soil had high sequence identity to Fusarium oxysporum. Polymerase chain reaction (PCR) amplifications performed with sequence-selective primers corroborated the treatment-specific distribution of rDNA clones obtained by the OFRG analysis. When these sequence-selective PCR primers were used to examine H. schachtii cysts from biocidal soil treatments that produced various levels of suppressiveness, only the D. oviparasitica-like rDNA was consistently identified in the highly suppressive soils.     

甘肃省小麦禾谷孢囊线虫的发生及分布

为了解甘肃省小麦孢囊线虫病发生程度和分布情况,2009-2012年于小麦抽穗至灌浆期,采用Z字形取样法,取根际0~20cm土壤,用漂浮法分离孢囊,统计100g土样的孢囊数量,用形态学方法鉴定线虫。从甘肃省12个地区50个县区,134个乡镇共采集到463个小麦田间土样。结果表明,孢囊平均检出率为47.9%,其中冬麦区孢囊平均检出率为61.5%,春麦区平均检出率为34.2%,冬麦和春麦混播区为52.3%,冬麦、春麦及冬春麦混播区平均孢囊数分别为3.5、6.8和3.9个/100g土样。其中平凉市灵台县和兰州市永登县孢囊线虫发生最为严重,个别地块每100g土样中孢囊数超过40个。根据孢囊形态学特征的观察,将甘肃省采集到的孢囊线虫鉴定为禾谷孢囊线虫(Heterodera avenae)。研究结果可为有效防治甘肃省麦类孢囊线虫病提供依据。     

Methods for Detection of Potato Cyst Nematodes1

Methods are reviewed for inspection of growing potato crops and of soil sampling for detection and estimation of potato cyst nematodes (Heterodera rostochiensis Woll. and H. pallida Stone). Modifications of the Dutch soil-sampling method are now widely used in Europe, including U.K. The statistical principles of soil sampling for Heterodera cysts are discussed. It is concluded that the efficiency of the method for detection is best improved by increasing both the quantity of soil examined and the number of sampling points per area sampled; the quantity is the most important factor which determines the level of infestation that can be detected. The area represented by a standard sample should be the same as that for which a separate decision will be made; variations in this area are unimportant because in effect one is sampling from an infinite population. It is concluded also that effective systems of control on potato-growing land, and removal of soil from exported plants and potatoes, offer better prospects than soil sampling for preventing transport of cysts; a combination of these methods provides the best safeguard, e.g. for seed potatoes.     

河北省大豆孢囊线虫分子鉴定及其分布

2003年,在河北省邯郸、邢台、石家庄、保定、秦皇岛、唐山、张家口等地采集大豆根际土壤样品65份,应用大豆孢囊线虫特异性引物对采集的孢囊线虫进行了分子鉴定,结果表明从邯郸的永年、磁县、峰峰,邢台的南和、广宗、清河,保定的南大园、西马池、望都、定州,石家庄的栾城、鹿泉、平山及石家庄市郊,秦皇岛的抚宁、昌黎,唐山的迁西、迁安、丰润的大豆和张家口的红云豆上采集的孢囊线虫的孢囊及其二龄幼虫均为大豆孢囊线虫;卢龙和滦县的样品中没有发现大豆孢囊线虫的孢囊及其二龄幼虫,且孢囊的检出率与二龄幼虫的检出率的趋势一致。     

控制大豆孢囊线虫生物种农剂的研制及沈阳田间小区防效的初步试验

以“辽豆15”为材料,采用田间小区试验,探讨了自行研制的生物种衣剂对大豆孢囊线虫数量的影响。试验结果表明：2007年在沈阳汪家的试验中,处理4（复配菌株F1024,B482,A4+微量元素组合3+助剂）对大豆根外孢囊的抑制率达65.66%,与对照有显著差异。2008年对处理4进行了精细的试验设计,并在2个不同地点布置试验,结果处理5（复配菌株F1024,B482,A4+元素组合+助剂）在汪家和康平2个试验点的根外孢囊数量及根内线虫数量都与对照差异达到显著水平,2个试验点的根外孢囊抑制率分别为 62.13%和46.45%,根内线虫抑制率分别为62.50%和81.78%。     

Identification of the Beet Cyst Nematode Heterodera schachtii by PCR

PCR-RFLPs of ITS-rDNA and PCR with species-specific primers were developed for identification of cysts and juveniles of the beet cyst nematode Heterodera schachtii. Restrictions of PCR product by MvaI or ScrFI distinguish H. schachtii, H. betae, H. trifolii and H. medicaginis. RFLP profiles with eight restriction enzymes for these four nematode species are presented. Based on Internal Transcribed Spacer sequences of populations from several Schachtii group species, a specific primer for H. schachtii was designed, permitting amplification of the target sequence from juveniles and cysts of the beet cyst nematode. A duplex PCR protocol tested with a wide range of nematode samples is described.     

不同轮作模式下小麦禾谷孢囊线虫的发生动态和种群密度

轮作是防治小麦禾谷孢囊线虫的重要农业措施,为了明确青海省春麦区不同轮作模式对小麦禾谷孢囊线虫的控制效果,采用田间大区试验法对生产中应用的6种轮作模式进行了研究。结果表明:不同轮作模式下,小麦禾谷孢囊线虫种群密度变化差异极显著,其中小麦与马铃薯、油菜、蚕豆轮作两年或以上能有效降低小麦禾谷孢囊线虫种群密度,土壤中的孢囊量减少39.31%~84.39%,单孢虫口数量减少73.21%~95.35%,虫口密度减少83.76%~97.82%;不同作物间,小麦与马铃薯或蚕豆的轮作效果(虫口密度减少74.39%~79.37%)显著优于小麦与油菜的轮作效果(虫口密度减少67.16%)。在同一地块相同条件下,种植油菜、蚕豆、马铃薯、小麦4种作物,小麦禾谷孢囊线虫均能正常孵化,4月底土壤中的2龄幼虫(J2)量增加,5月上旬达到高峰期,5月中旬开始,土壤中的J2、孢囊量、虫口密度和单孢虫口数量均急剧下降,6月至7月份下降幅度小,趋于稳定;田间空孢囊率于5月中旬至6月中旬急剧增加,7月份趋于稳定,8月份以前,4种作物田禾谷孢囊线虫的孵化动态和种群密度变化趋势一致,8月中旬,小麦田随着新孢囊脱落到土壤中,禾谷孢囊线虫种群密度开始上升,小麦收获后土壤中的孢囊量比播种前增加28.62%,虫口密度增加41.30%;而油菜、蚕豆、马铃薯田土壤中的孢囊量比播种前减少32.27%~48.36%,虫口密度减少70.91%~81.73%,8月中旬至10月份小麦田禾谷孢囊线虫种群密度极显著高于油菜、蚕豆、马铃薯田。     

内蒙古中西部地区小麦禾谷孢囊线虫的发生分布

2005—2008年调查了内蒙古中西部地区7个盟市的20个春小麦种植区,共采集分离并鉴定了255份根际土壤和根系。调查结果表明,禾谷孢囊线虫（Heterodera avenae Woll.）在呼和浩特市、乌兰察布市、包头市、巴彦淖尔市、鄂尔多斯市、锡林郭勒盟等地区都有分布。其中,在乌兰察布市的察哈尔中旗、呼和浩特市的内蒙古农科院和内蒙古农业大学农场禾谷孢囊线虫虫口密度最大,每250 g土壤的孢囊平均含量分别达到了38.4、29.4个和16.4个。     

小麦孢囊线虫病传播扩散途径研究初报

为了明确小麦孢囊线虫病的扩散传播途径,调查病区农用机械、麦田翻耕后田间及其周边流水中小麦孢囊线虫的孢囊数量.结果表明,收割机、旋耕机和播种机上土样的孢囊检出率分别为27.1％、51.6％和44.0％,麦稻轮作的病田中,浪渣的孢囊检出率为58.3％,翻耕后麦田积水、沟渠和小河流中的孢囊检出率分别为33.3％、22.7％和7.7％.说明农业机械的跨区作业和水流是小麦孢囊线虫病传播、扩散的重要途径.     

Indirect haemagglutination reaction with antigen of Sarcocystis gigantea (Railliet, 1886) Ashford, 1977

The water extract from cryolyzed whole muscle cysts of Sarcocystis gigantea from sheep, in spite of the high lectin content, is a suitable antigen for the detection of specific antibodies by means of indirect haemagglutination reaction (IHA). The agglutinating effect of lectin from parasitic cysts can be eliminated with a 0.5% concentration of lactose dissolved in all solutions used for IHA. In sera of slaughterhouse sheep, positive titres ranging from 1:80 to 1:1 280 were registered. Positive reactions in lower titres were observed also with antibodies against S. dispersa, S. cuniculi and Sarcocystis sp. from pigs. Sensibilized erythrocytes can be stored in refrigerator at least for 1 week.     

Enige proeven ter bestrijding van cysten vanHeterodera rostochiensis tussen de wortels vanConvallaria-bloeikiemen

Conclusies Bij de concentratie van organisch kwik (Aaventa), waarbij de larven vanHeterodera rostochiensis niet meer uit de cysten worden gelokt en dus dood zijn, wredenConvallaria-kiemen zeer ernstig beschadigd of gedood, zodat het middel niet bruikbaar is.Het is mogelijk eieren en larven in de cysten te doden door ze in bevroren toestand gedurende lange tijd in contact te houden met lage concentraties nematicide stoffen. De nematiciden blijken evenwel bij de voor de bestrijding bruikbare, lage concentraties onder het vriespunt deConvallaria-kiemen zwaar te beschadigen.Summary A concentration of organic mercury (Aaventa) strong enough to kill the larvae in the cysts ofH. rostochiensis also damaged or killed the lily- of the-valley pips.The cysts can be killed by immersing them, together with the pips, in low concentrations of nematicides and then placing them for several months, in a freeze chamber. However, a treatment of the pips with low concentrations of nematicides followed by storage at low temperatures, useful in controlling the cysts, severely damaged the pips (Tables 2 and 3).Medewerker van de Werkgroep Onderzoek Bestrijding Aardappelcystenaaltje T.N.O.