烟草根黑腐病菌的PCR分子检测

 根据烟草根黑腐病菌(Thielaviopsis basicola)与其它烟草病原真菌核糖体基因转录间隔区(internal transcribed spa-cer,ITS)序列间的差异,设计了一对特异性引物TB-5/TB-3,用于T. basicola的分子检测。利用该对引物对包括T. basicola在内的13个烟草病原菌菌株的基因组DNA进行PCR扩增,结果表明:只有T. basicola能扩增到一条400bp左右的特异性条带,其它菌株及阴性对照均无扩增产物。对烟草组织和土壤的检测结果也表明,该对引物能特异性的检测到T. basicola基因组DNA的存在。该引物对T. basicola基因组DNA检测的灵敏度为100fg/μL。     

Characterization of the Suppressiveness of Hairy Vetch-Amended Soils to Thielaviopsis basicola

ABSTRACT Factor(s) involved in soil suppressiveness to Thielaviopsis basicola when hairy vetch was used as a green manure were studied in a cotton production system. Soil suppressiveness was assessed in vitro at hairy vetch amendment levels of 0, 0.25, and 0.75% (wt/wt) by observing chlamydospores, using a nylon fabric technique. Chlamydospore germination in all soils was below 5%, and microscopic examination showed no germ tube lysis or visible propagule destruction. Viability (chlamydospore germination on T. basicola-carrot-etridiazol-nystatin [TB-CEN] medium) was reduced by 29% within 48 h after hairy vetch amendment. Viability also was reduced in atmospheres of amended soils, suggesting that the suppressiveness was due to a volatile factor. In a field study, chlamydospore viability in amended soils was reduced by 16%. T. basicola hyphal growth was more sensitive to ammonia than Rhizoctonia solani or Pythium ultimum, and chlamydospore mortality of T. basicola was 100% in petri dish atmospheres with 0.4 ppm of ammonia (50% lethal dose = 0.15 ppm). Soil atmospheric ammonia was 0.08 and 0.10 ppm for 0.25 and 0.75% amendment levels, respectively, both at 3 and 7 days after incorporation. In the field, 0.11 and 0.14 ppm of ammonia were detected in soil atmospheres 3 and 7 days after incorporation, respectively. The levels of ammonia detected were sufficient to account for the loss in T. basicola chlamydospore viability, indicating that ammonia is responsible for the suppressiveness observed.     

Survival of endoconidia of Thielaviopsis basicola in soil

Lysis and viability of endoconidia ofThielaviopsis basicola artificially introduced into clay loam and sandy soil were microscopically examined at intervals. Most of the endoconidia remained morphologically unchanged in moist clay loam for more than one year, while in moist sandy soil all had disappeared by lysis within 4 months. One percent of the endoconidia that persisted for 15 months in clay loam germinated when tested with V-8 juice. Endoconidia transformed into chlamydosporelike structures when incubated in phosphate buffer solutions supplemented with glucose and asparagine but not in those without these compounds. The significance of these phenomena for the survival of the pathogen in soil is discussed.Samenvatting Kiemkracht en lysis van endoconidia vanThielaviopsis basicola gedurende een incubatieperiode van maximaal 15 maanden in luchtdroge en vochtige klei- en zandgrond werden microscopisch onderzocht. Het merendeel der endoconidiën in luchtdroge en vochtige kleigrond en in luchtdroge zandgrond onderging geen morfologische verandering gedurende de 10 maanden van incubatie, terwijl in vochtige zandgrond alle endoconidiën reeds binnen 4 maanden door lysis waren verdwenen. Eén procent van de endoconidiën bleek na een 15 maanden durende incubatie in vochtige kleigrond nog te kunnen kiemen. Van de endoconidiën die gedurende een verblijf van tween wintermaanden in zandgrond in het veld geen morfologische verandering ondergingen was eveneens nog 1% kiemkrachtig. Deze waarnemingen doen vermoeden, dat endoconidiën mede verantwoordelijk kunnen zijn voor de overleving vanT. basicola in natuurlijke grond. Omvorming van endoconidiën in chlamydospore-achtige structuren werd waargenomen in fosfaatbuffers waaraan glucose en asparagine was toegevoegd, echter niet in fosfaatbuffers zonder deze verbindingen. De vorming van deze structuren in natuurlijke grond kon niet worden aangetoond.     

防治烟草根黑腐病拮抗芽孢菌株的筛选

由基生根串珠霉菌(Thielaviopsis basicola)引起的烟草根黑腐病是烟草生产上重要根部真菌病害,近年在我国烟草种植区有加重危害的趋势,目前防治措施主要依赖化学药剂,给卷烟卫生带来一系列问题[1].探索该病生物防治途径,克服农药残留和病原菌抗药性十分重要.芽孢杆菌是土壤和植物微生态区系的优势生物种群,具有优良生防特性[2],采用芽孢杆菌作为烟草根黑腐病生防因子尚未见报道.本研究从烟草根际土壤中分离筛选对烟草根黑腐病病菌有较强拮抗作用的芽孢菌株,研究其抑菌活性、控病作用及其分类学地位.     

土壤中烟草根黑腐病菌的实时定量PCR检测技术研究

 Thielaviopsis basicola is a soil-borne plant pathogen which causes root rot disease in tobacco plants. Detection and monitoring of T. basicolain soil is of great significance to control this disease. Based on the differences in internal transcribed spacer (ITS) sequences of T. basicola and other fungal pathogens, a specific primer pair Tb1/Tb2 for T. basicolawas developed. The results showed that the primer pair gave a single amplicon of 330 bp from T. basicola and revealed no undesirable cross-reaction with other seven soil-borne pathogen isolates and three tobacco rhizosphere dominant fungi isolates. With a series of 10-fold genomic DNA dilutions of T. basicola, the detection limit of 1 pg/μL in conventional PCRand100 fg/μL in real-time quantitative PCR was achieved. With DNA from the soil inoculated with different numbers of T. basicola conidia, the detection limit was 10 conidia per reaction in conventional PCR and 0.4 conidia per reaction in real-time quantitative PCR.     

Thielaviopsis basicola,a factor in the cherry replant problem in the Netherlands

Samenvatting Het aaltljePratylenchus penetrans kan bijdragen tot het optreden van herbeplantingsmoeilijkhenden bij kers op lichte grond. Op zwaardere gronden komt dit aaltje veelal niet in schadelijke aantallen voor; toch kunnen ook hier ernstige herbeplantingsmoeilijkheden optreden. Bodemmoeheidsverschijnselen bij meerjarige gewassen zijn meestal specifiek voor verwante soorten, bijv. voor steenrespectievelijk pitvruchten. In potproeven is het bestaan van deze specificiteit en van factoren die naast aaltjes verantwoordelijk zijn voor moeheidsverschijnselen bij appel en kers nog eens aangetoond.Op wortels van kersezaailingen, die geteeld waren op grond afkomstig uit een kerseboomgaard, werden chlamydosporen vanThielaviopsis basicola (Berk. & Br.) Ferr. gevonden. Met deze schimmel werden inoculatieproeven gedaan, waaruit duidelijk de pathogeniteit ten opzichte van kers bleek. Bovendien zijn er aanwijzingen dat de teelt van kers tot een toeneming van de besmettingsgraad van de grond leidt (tabel 1). Appel bleek resistent te zijn tegen de schimmel.Deze gegevens wijzen er op datT. basicola een rol speelt bij het herbeplantingsprobleem van kers. Nader onderzoek zal echter moeten uitwijzen hoe groot de betekenis van deze factor onder praktijkomstandigheden is.     

Aerial Transmission of Thielaviopsis basicola, a Pathogen of Corn-Salad, by Adult Shore Flies

ABSTRACT Chlamydospores of Thielaviopsis basicola were consistently observed in frass excreted by adults and larvae of shore flies that were collected in the immediate vicinity of naturally infected corn-salad plants obtained from a commercial greenhouse production facility. Approximately 95% of the adult flies and 85% of the larvae were internally infested with the pathogen. Pathogen-free adult shore flies were subsequently shown to acquire the pathogen by ingestion after feeding on naturally infected plants. Viable propagules of the pathogen were excreted by these internally infested adults and were capable of transmitting the pathogen to healthy seedlings, which subsequently became infected.     

Ultrastructure of the Penetration and Infection of Pansy Roots by Thielaviopsis basicola

ABSTRACT Transmission electron microscopy was used to study the penetration and infection of pansy roots by Thielaviopsis basicola. Events observed in 7- to 10-day-old roots produced on moist filter paper differed slightly from those in roots from 4-week-old plants washed free of potting media prior to inoculation. By 3 h postinoculation (PI), epidermal cells of roots produced on filter paper exhibited aggregated cytoplasm and papilla formation in response to germ tube tips. The presence of callose in papillae was demonstrated using immunogold labeling. Papilla formation was not effective in preventing host cell penetration. A slender infection hypha emerged from a germ tube tip and grew through a papilla. Its tip then expanded to form a globose infection vesicle. By 6 h PI, infection hyphae emerged from infection vesicles, and invaded host cells showed signs of necrosis. By 8 h PI, infection hyphae had grown into cortical cells in spite of papilla formation in these cells. By 24 h PI, distinctive intracellular hyphae were present in necrotic cortical cells. In washed roots, most epidermal cells failed to respond to invasion. Hyphae simply grew through these cells and contacted cortical cells that exhibited aggregated cytoplasm and papillae formation. Infection structures similar to those produced in epidermal cells from roots grown on filter paper then formed in cortical cells of washed roots. The fact that T. basicola formed infection structures only in cells that responded to invasion suggests that T. basicola has a more complex relationship with its host than would be expected in a nectrotrophic pathogen. We believe that T. basicola is best described as a necrotrophic hemibiotroph.     

Reassessment of the Role of Saprophytic Activity in the Ecology of Thielaviopsis basicola

ABSTRACT The ability of Thielaviopsis basicola to survive saprophytically in soil was investigated using root tissue from susceptible hosts as organic substrates. Inoculum densities were lower in soils amended with root tissue than in nonamended controls after 2 and 4 weeks of incubation. The greatest decrease occurred in soils containing the highest concentration of root tissue or in soils in which root tissue included the soluble components of the living root. Reproduction by T. basicola also was examined in axenic media containing either killed root pieces or various carbohydrates as the sole carbohydrate source. T. basicola utilized killed root tissue as a carbohydrate source in axenic media, particularly in cultures in which root tissue included the soluble components. Enzymatic activities of T. basicola, however, did not result in maceration of the root tissue. T. basicola utilized sucrose and cellobiose, but did not utilize structural carbohydrates such as cellulose, hemicellulose, or pectin. Based on the absence of significant saprophytic ability, T. basicola should be classified ecologically as an obligate parasite.     

The effect of benomyl on tobacco leaf necrosis induced by Thielaviopsis basicola

Tobacco leaf discs inoculated with a standardized suspension of endoconidia ofThielaviopsis basicola (Berk. & Br.) Ferr. were floated on tap water and on different concentrations of benomyl suspension. Lesion rating was 0.5, 0.25 and trace on 0.5-, 1- and 5-ppm benomyl concentration, respectively, as compared with 4.0 for the check. No lesions were formed on 10- and 50-ppm concentrations. Benomyl protected tobacco leaf discs systemically against lesion formation when applied before the penetration of the germ tubes. It also suppressed the development of mycelium on the necrotic spots.The bearing of these results on the relation between leaf and root necrosis, is discussed.Samenvatting Schijfjes tabaksblad dreven op leidingwater met verschillende concentraties benomylsuspensie. De bladeren waren geïnoculeerd met een gestandaardiseerde endoconidiënsuspensie vanThielaviopsis basicola. De verhouding van de lesies in de schijfjes op water met resp. 0.5, 1 en 5 ppm benomyl was 0.5, 0.25 en zeer weinig, vergeleken met 4.0 bij de controle. Er werden geen lesies gevormd op een benomylconcentratie van 10 en 50 ppm. Benomyl beschermde de schijfjes tabaksblad systemisch tegen lesievorming, als het vóór het binnendringen van de kiembuizen werd toegediend. Het onderdrukte ook de myceliumontwikkeling op de necrotische plekken.De betekenis van deze resultaten voor de relatie tussen blad- en wortelnecrose wordt besproken.Contribution 53, Delhi Research Station.     

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.     

The occurrence in Denmark of black root rot of pea caused by Thielaviopsis basicola

Thielaviopsis basicola has been shown to be a root pathogen of pea of considerable importance in Denmark. The fungus is only found in fields with one or more previous pea crops in the field history. In the dry and warm growing season of 1989 the fungus was found in 0·6% and 3·2% of the fields in two separate areas in Denmark. In the fields where T. basicola was detected the average disease severity index in plant samples was 51·8, whereas the average disease severity index in plant samples without the fungus was 27·0. The average yield of green peas was reduced from 5167 to 4171 kg/ha when T. basicola was present. For detection and isolation of T. basicola it is important to use a technique combining microscopic examination, a semi-selective medium and a dilution plate method.     

烟草根黑腐病菌致病力分化及品种抗性差异研究

 烟草根黑腐病由根串珠霉（Thielaviopsis basicola (Berk.et Br.)Ferr.）引起,主要为害烟株的根部,可使根部组织呈特异性黑色坏死而导致烟苗死亡或地上部分生长不良。该病是世界性的烟草病害,在各产烟国家如美国、日本、加拿大等国普遍发生,在我国主要分布于河南、山东、安徽、云南等地     

拮抗放线菌TA21对烟草根黑腐病菌的抑制及其控病作用

菌株TA21是一株从重庆烟区土壤中筛选出的对烟草根黑腐病菌Thielaviopsis basicola有较强拮抗作用的放线菌,为了明确其抑菌效果和控病作用及其在烟草根黑腐病生防中的应用潜力,本文进行了菌株TA21抑菌活性检测和温室控病测试;并通过形态学、生理生化和分子分类的方法确定其分类地位。室内测定试验表明,菌株TA21对烟草根黑腐病菌的抑菌带宽达12.5mm;温室控病试验发现其对烟草根黑腐病防治效果达85.3%。无菌滤液试验表明,在试验浓度范围内无菌滤液均能有效抑制烟草根黑腐病菌菌丝生长、减少孢子萌发。根据菌株形态特征、培养性状、生理生化特性、细胞组分及16S rDNA序列分析,确定菌株TA21为链霉菌属吸水链霉菌Streptomyces hygroscopicus。     

Effect of Temperature on and Histopathology of the Interaction Between Meloidogyne incognita and Thielaviopsis basicola on Cotton

ABSTRACT Controlled environments were used to study the relationship between the root-knot nematode (Meloidogyne incognita) and Thielaviopsis basicola on cotton. Temperature treatments were continuous 20, 24, and 28 degrees C or two cyclic linear regimes with ranges of 14 to 32 or 18 to 28 degrees C over 24 h. Cotton seeds were planted in fumigated soil infested with T. basicola, M. incognita, or both. After 42 days, pathogen effects on plant growth and pathogen development were evaluated. Histology was conducted on roots collected 14, 28, and 42 days after planting in the continuous 24 degrees C treatment. Reductions in plant height-to-node ratio and total fresh weight were observed for soils infested with both pathogens compared with the control or with soils infested with either pathogen, except for M. incognita-infested soil at 28 degrees C. T. basicola reduced root galling and reproduction of the nematode at all temperatures. Vascular discoloration caused by T. basicola was greater in the presence of M. incognita compared with that by T. basicola alone. At 2 and 4 weeks, histological studies showed that plants grown in all T. basicola-infested soils contained chlamydospore chains on the root surface and in cortical cells. The fungus was not observed inside the vascular cylinder. Roots from 4-week-old plants from soils infested with T. basicola and M. incognita showed fungal sporulation in vascular tissue and localized necrosis of vascular tissue adjacent to the nematodes. At 6 weeks, plants grown in soil infested with T. basicola alone exhibited no remaining cortical tissue and no evidence of vascular colonization by the fungus. Six-week-old plants grown in T. basicola + M. incognita-infested soils exhibited extensive vascular necrosis and sporulation within vascular tissue. These studies suggest that coinfection expands the temperature ranges at which the pathogens are able to cause plant damage. Further, M. incognita greatly increases the access of T. basicola to vascular tissue.     

Thielaviopsis basicola (Berk. et Br.) Ferraris als veroorzaker van een voor Nederland nieuwe ziekte van de tabak

Summary Tobacco black root rot, caused byThielaviopsis basicola (Berk. etBr.) Ferraris has been reported for the first time in the Netherlands. It occurred in tobacco seed beds in the Maas and Waal district, especially in the variety Kentucky Italiana.A part of the literature on this subject is briefly discussed and methods for control are given.     

Incidence of Chalara elegans in groundnut seed samples and seed transmission of blackhull

The incidence of Chalara elegans ( =Thielaviopsis basicola ) was determined in seven commercial groundnut seed samples in the Vaalharts area of South Africa. It was demonstrated that 1.19% of the seeds were infected by the pathogen. Surface contamination of visually healthy seeds from the same samples was confirmed by testing seed washings for viable propagules of C. elegans on raw carrot dises. Naturally infected seed planted in autoclaved soil in the greenhouse resulted in 34.4% severely diseased plants. These results indicate that seed-borne inoculum may be an important factor in the transmission of this disease.     

Control of groundnut blackhull and its causal fungus Chalara elegans with fungicides

Fungicides were tested for activity against Chalara elegans, in culture, in artificially infested soil columns and in field experiments. Propiconazole and etaconazole were the only two fungicides which performed well in all tests, and provided effective control of blackhull of groundnuts in the field. The most effective treatments in the field were etaconazole foliar spray and propiconazole granular treatment applied over the plant row at planting, providing respectively 64% and 46% disease reduction in comparison with the untreated controls.     

Interaction of root-lesion nematodes, Pratylenchus thornei and P. crenatus, with the fungus Thielaviopsis basicola and a grey sterile fungus on roots of pea (Pisum sativum)

Pratylenchus thornei invaded excised pea roots in agar in greater numbers and penetrated the cortex more deeply than P. crenatus . Both species fed on the roots ectoparasitically and displaced root cells into the surrounding medium. The cytoplasm of cortical cells near cither nematode became granulated, with enlarged vacuoles and nuclei. P. thornei also caused these responses in the endodermis. Infection of the root surface with a grey sterile fungus inhibited invasion by P. crenatus and P. thornei . Infection by Thielaviopsis basicola inhibited P. thornei invasion but encouraged penetration by P. crenatus and the hyphae were found deeper in the cortex when P. crenatus was present.