Characterization of Polymyxa species by restriction analysis of PCR-amplified ribosomal DNA

A molecular method is described to aid identification of the obligate parasite Polymyxa and discriminate between species ( P. betae and P. graminis ) and isolates. DNA was extracted from zoospores, resting spores and roots infected with P. betae and P. graminis and compared with that from negative control plants that were not inoculated with Polymyxa but were grown at the same time under the same conditions. The ribosomal internal transcribed spacers and 5.8S rDNAs were amplified by the polymerase chain reaction and digested with restriction enzymes to detect molecular differences between the species and isolates. There were differences between P. betae and P. graminis and two subgroups within P. graminis but so far this has not been correlated with any other biological property.     

我国禾谷多粘菌地理分布和生理分化研究

 调查我国26个省、市、自治区,93个县市,183个取样点,1 373个大、小麦根样本。根据休眠孢子出现频率(有休眠孢子样本数/总样本数)和每克麦根休眠孢子数,将我国禾谷多粘菌的分布区分为3个,多菌区包括江、浙、沪、皖、赣、湘、鄂和鲁的大部分和川、陕小部分,冀个别地区;少菌区包括豫大部和晋、甘小部或个别地区;其余地区为无菌区。分布区主要在长江、黄河和淮河中下游流域。禾谷多粘菌的分布范围比大麦黄花叶病等3种病毒病的分布更广泛。本研究还探讨了我国各地禾谷多粘菌的分离物存在生理分化现象     

Specific polyclonal antibodies for the obligate plant parasite Polymyxa— a targeted recombinant DNA approach

Highly specific rabbit polyclonal antibodies for the obligate sugar-beet root parasite, Polymyxa betae , were produced using a novel recombinant DNA approach. Parasite cDNA was selectively isolated from infected roots, expressed in vitro , and the purified protein used to raise antibodies. This produced clean, precisely targeted antibodies, and allowed for rigorous screening of candidate genes and their products at the molecular level prior to animal immunization. This approach selects for genes whose products are highly expressed by the parasite in planta , and five such candidate genes from Polymyxa betae were identified and cloned. Polyclonal antiserum developed using the product of one such gene was found to react specifically with P. betae in sugar-beet roots and with the closely related Polymyxa graminis in barley roots, and to cross-react with Plasmodiophora brassicae in cabbage roots, without the need for further purification. No cross-reaction was detected with protein extracts from potato roots infected by the plasmodiophoromycete Spongospora subterranea . In all cases, there was no interaction with proteins from host plants, or from other microorganisms found in association with uninoculated sugar-beet, barley, cabbage and potato roots.     

A rapid method for direct detection of Polymyxa DNA in soil

Polymyxa spp. are vectors for a number of economically important soilborne plant viruses. The development of a technique to detect virus and vectors directly in soil would be useful for epidemiological studies and assessment of disease risk prior to planting. A rapid method was developed to extract and quantify Polymyxa spp. DNA from soils. DNA was extracted from three soils infested with Polymyxa betae and three infested with P. graminis using an EDTA lysis buffer in combination with a MagneSil™ DNA extraction kit and Kingfisher™ magnetic particle processor. Primers and probes designed to correspond to sequences within the internal transcribed spacer region 2 (ITS2) of ribosomal DNA enabled recovery and amplification of P. betae and P. graminis DNA using real-time PCR and TaqMan chemistry. For the P. graminis- infested soils, the purity of DNA obtained was sufficient to allow Polymyxa DNA to be amplified without dilution to remove inhibitors, but with P. betae- infested soils, amplification was only achieved if the DNA was diluted 1:10. Using TaqMan PCR, a standard curve was constructed from uninfested soil spiked with known numbers of P. betae cystosori; the quantity of P. betae inoculum from naturally infested soil was then extrapolated from the curve. This technique offers a sensitive method of extracting, detecting and quantifying Polymyxa spp. DNA in soil.     

Analysis of the resistance-breaking ability of different beet necrotic yellow vein virus isolates loaded into a single Polymyxa betae population in soil

The genome of most Beet necrotic yellow vein virus (BNYVV) isolates is comprised of four RNAs. The ability of certain isolates to overcome Rz1-mediated resistance in sugar beet grown in the United States and Europe is associated with point mutations in the pathogenicity factor P25. When the virus is inoculated mechanically into sugar beet roots at high density, the ability depends on an alanine to valine substitution at P25 position 67. Increased aggressiveness is shown by BNYVV P type isolates, which carry an additional RNA species that encodes a second pathogenicity factor, P26. Direct comparison of aggressive isolates transmitted by the vector, Polymyxa betae, has been impossible due to varying population densities of the vector and other soilborne pathogens that interfere with BNYVV infection. Mechanical root inoculation and subsequent cultivation in soil that carried a virus-free P. betae population was used to load P. betae with three BNYVV isolates: a European A type isolate, an American A type isolate, and a P type isolate. Resistance tests demonstrated that changes in viral aggressiveness towards Rz1 cultivars were independent of the vector population. This method can be applied to the study of the synergism of BNYVV with other P. betae-transmitted viruses.     

Differences in temperature requirements between Polymyxa sp. of Indian origin and Polymyxa graminis and Polymyxa betae from temperate areas

The temperature requirements of three single cystosorus strains of Polymyxa sp. from India were studied at 15–18, 19–22, 23–26 and 27–30 °C (night-day temperature), and compared with the temperature requirements of three strains of P. graminis from Belgium, Canada and France and two strains of P. betae from Belgium and Turkey. Sorghum was used as the host-plant for the Indian strains; the strains of P. graminis and P. betae from temperate areas were cultivated on barley and sugar beet, respectively. The cystosori germination and the development of plasmodia, zoosporangia and cystosori of Polymyxa sp. from India were optimal at 27–30 °C. Infection progression was slower at 23–26 °C than at 27–30 °C. At 19–22 °C, infection was insignificant. No infection occurred below 19 °C. In contrast, the infection of barley with P. graminis strains from temperate areas was optimal at 15–18 °C, but at 19–22 °C the progression appeared inconsistent and infection stayed low. Above 22 °C, infection was insignificant. P. betae strains showed consistent infection in the range of 15–18 °C to 27–30 °C. Plasmodia formation and cystosori detection of the Belgian strain were slightly advanced at 23–26 °C compared to 19–22 °C but clearly restrained at 27–30 °C. Fungus development of the P. betae strain from Turkey was almost as high at 27–30 °C as at the lower temperatures. These results strengthen the case for distinguishing between Polymyxa sp. from India and P. graminis or P. betae from temperate areas.     

禾谷多粘菌分离、培养、接种及用于大麦抗黄花叶病的鉴定

 通过禾谷多粘菌Polymyxa graminis L.休眠孢子分离接种感病大麦品种,并进行砂培养,获得13个纯化了的禾谷多粘菌分离物,且其中3个带有大麦黄花叶病毒(BaYMV)。用分别带有BaYMV和大麦温和花叶病毒(BMMV)的英国禾谷多粘菌分离物的游动孢子接种13个中国大麦品种,以及用BaMMV摩擦接种36个中外大麦品种,抗性鉴定结果游动孢子接种与摩擦接种一样,均与田间鉴定结果一致,且大麦对BaYMV的抗性与对BaMMV的抗性一致,从而这2种接种方法可用于大麦品种(系)和育种中间体对BaYMV抗性的快速鉴定和筛选。游动孢子或休眠孢子接种方法还可有效地鉴定大麦对禾谷多粘菌的抗性。     

Production of monoclonal antibodies to potato virus YNTN strain and their use for strain differentiation

Four mouse monoclonal antibodies (MAbs) against potato virus Y (PVY) were produced. MAb 4C1 reacted with four isolates of PVY^NTN and only very weakly with one isolate of the necrotic strain of PVY (PVY^N). It did not react with other isolates of the ordinary strain of PVY tested. MAb 2C9 reacted with all isolates tested and can be used to produce a specific diagnostic kit for routine PVY detection. Other MAbs had different specificities and reacted with isolates of various strains of PVY. MAbs did not react with seven other members of the Potyvirus group including potato virus A. A MAb-based ELISA, using MAb 4C1, was devised and shown to detect PVY^NTN specifically.     

Purification and serology of virions of impatiens necrotic spot tospovirus

Impatiens necrotic spot tospovirus (INSV) virions were purified using a procedure devised for tomato spotted wilt tospovirus (TSWV) from systemically infectedNicotiana benthamiana plants grown at 33 °C day/26 °C night and a photoperiod of 14 hours. With plants grown at 24/18 ° C purification was unsuccessful. In SDS-PAGE the protein pattern of INSV was similar to that reported for TSWV, except the appearance of a single G2 protein band. A polyclonal antiserum, prepared against virions, reacted in Western blots with INSV nucleoprotein and glycoproteins but only with TSWV glycoproteins. In DAS ELISA the antiserum reacted with both INSV and TSWV infected plant sap and, after absorption with TSWV, only with INSV. In TAS ELISA the antiserum trapped both INSV and TSWV nucleoproteins and glycoproteins as detected by specific monoclonal antibodies, and, after absorption with TSWV, only the homologous proteins. This appears to be the first report of the purification of INSV virions and the production of an antiserum reacting with both nucleoprotein and glycoprotein antigens.     

The host range of Polymyxa betae in Britain

The host range of Polymyxa betae on common arable weed species in Britain was determined by growing plants in naturally infested soil and examining their root systems for the presence of resting spores (cystosori). Of the 24 species tested, only Atriplex patula and Chenopodium album of the Chenopodiaceae, and Silene alba of the Caryophyllaceae, were found to be heavily infected. S. alba is a newly recorded host species for Polymyxa. The host specificity of isolates of P. betae from Beta vulgaris, C. album and A. patula was investigated by observing which of 11 test plants could be infected by the isolates obtained from this soil. Three main biotypes of P. betae appeared to be distinguishable: one which was able to infect all chenopodiaceous species; one which had a narrower host range; and one which was able to infect S. alba. The role of weed species in the epidemiology of rhizomania is discussed.     

Evaluation of antisera raised against Phytophthora fragariae for detecting the red core disease of strawberries by enzyme-linked immunosorbent assay (ELISA)

Antiserum was raised against pooled mycelial suspensions from five isolates (designated Pf 1, Pf 2, Pf 3, Pf 10 and Pf 11) representing five physiologic races of Phytophthora fragariae. In enzyme-linked immunosorbent assay (ELISA), this antiserum detected homologous soluble antigens at protein concentrations as low as 2 ng/ml.
Fungal antigens could also be detected in extracts of strawberry plants infected with P. fragariae. Root extracts prepared from the alpine strawberry Fragaria vesca and F. ananassa cv. Cambridge Favourite infected with any of the five isolates studied produced strong reactions in ELISA. In F. vesca , ELISA-positive material was detectable 6-8 days after inoculation before macroscopic symptoms appeared. The cultivar Red Gauntlet, which is resistant to Pf 1, 2 and 3 but susceptible to Pf 10 and 11, reflected this differential response in ELISA; the absorbance produced by extracts of plants infected with virulent isolates was significantly higher than that obtained with the corresponding extracts of plants inoculated with a virulent isolates. The recently introduced cultivars Hapil, Ostara and Providence were found to be susceptible to all isolates in this study: the corresponding root extracts were also positive in ELISA.
The antiserum also detected P cactorum infections. Nevertheless, the ELISA test described should prove valuable in screening certified strawberry stocks.     

Genetic diversity and identification of race 3 of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lactucae</Emphasis> in Taiwan

Sorghum is an important drought tolerant crop cultivated for food and fodder purposes. Anthracnose disease caused by Colletotrichum graminicola is a major constraint in sorghum productivity in India. Certain antagonistic fungi, that were isolated in the previous study from the rhizosphere and rhizoplane of perennial grasses in India, were studied for their antagonism in vitro to C. graminicola, root colonization ability and rhizosphere competence. Out of 138 isolates tested, 89 were antagonistic. Fifteen fungal isolates with greater than 70 % in vitro inhibition zone to the pathogen tested positive for root and rhizosphere colonization abilities. Three isolates – Chaetomium globosum isolate 57, Trichoderma harzianum isolate 184 and Fusarium oxysporum (NSF isolate 9) with prominent biocontrol potentials were tested for the control of sorghum anthracnose in greenhouse and field. Chaetomium globosum, Trichoderma harzianum and Fusarium oxysporum isolates decreased seedling mortality, and incidence and severity of disease at different growing stages. They promoted plant growth (dry biomass- 45.3, 40.0 and 46.7 %) and increased yield (grain biomass- 33.3, 23.8 and 49.2 %) respectively, over control in field. The population of the above fungi in soil was moderately high at harvest stage. The present investigation revealed that fungal isolates from rhizosphere and rhizoplane of perennial grasses could be employed to manage anthracnose and enhance plant growth and yield potentialities in sorghum, at the same time.     

Characterization of fluorescent Pseudomonas spp. associated with roots and soil of two sorghum genotypes

Sorghum is used as bioenergy feedstock, animal feed, and food. Economical methods for disease prevention and control are valuable for producers. Fluorescent Pseudomonas spp. were isolated from sorghum roots and surrounding soil with the goal of finding isolates that significantly inhibited sorghum fungal pathogens. Fluorescent pseudomonads were collected from seedlings of sorghum cultivars RTx433 and Redlan and wheat cultivar Lewjain, grown in two soils. Lewjain is known to support growth of producers of the antibiotic, 2,4-diacetylphloroglucinol (2,4-DAPG). Isolates from all three plants were assessed for hydrogen cyanide (HCN) and extracellular protease production, and for a 2,4-DAPG gene, phlD. Both soil type and plant type affected HCN- and protease-production, but phlD was not affected. Subsets of phlD ⁺ isolates were chosen to determine phlD genotypes and to conduct in vitro inhibition assays against sorghum pathogens. Most isolates from sorghum and wheat were genotype D, previously associated with superior root colonization. phlD ⁺ sorghum isolates were co-cultured with five sorghum pathogens. One isolate from each sorghum line exhibited inhibition to all five pathogens but more Redlan isolates were inhibitory to the virulent pathogen, Fusarium thapsinum, than RTx433 isolates. Nearly all inhibitory isolates from either sorghum cultivar were from one soil type. This is consistent with what had been previously observed in field studies: that soil type played a significant role in determining characteristics of fluorescent Pseudomonas spp. isolated from roots or soil, but sorghum genotype also had a considerable effect.     

Host Range of Tropical and Sub-tropical Isolates of Polymyxa graminis

The host range of Polymyxa graminis isolates originating from peanut clump-infested areas in India (Andhra Pradesh and Rajasthan), Pakistan and Senegal was studied on monocotyledonous and dicotyledonous cultivated species, using known quantities of sporosori as inoculum. Profuse multiplication occurred only on some graminaceous species, but the various isolates showed different host specificity. All the isolates produced high infection on sorghum and pearl millet, and all but one isolate from Rajasthan infected maize. Wheat, rye and barley were susceptible to some of the tested isolates. The isolates from Rajasthan and Pakistan produced moderate to severe infection on at least one of these species. On rice, groundnut and sugar beet, only traces of infection by some isolates were detected, whereas no infection was observed on mustard and sunflower. Differences of susceptibility in Pennisetum spp. and Sorghum spp. were demonstrated. The variations in host specificity among isolates from peanut clump-infested areas may result from an adaptation of P. graminis populations to various biotopes. The implications of these results for the management of peanut clump disease are discussed. A comparison of the host ranges of isolates of P. graminis and P. betae from temperate areas demonstrated that distinct types of Polymyxa might be identified based on their relative ability to multiply on susceptible species. Nevertheless, overlapping in the host ranges among the different Polymyxa types, characterised by distinct ecological and genomic features, raises doubts about the host range as a classification criterion for the Polymyxa genus.     

Host Specialization in the Charcoal Rot Fungus, Macrophomina phaseolina

ABSTRACT To investigate host specialization in Macrophomina phaseolina, the fungus was isolated from soybean, corn, sorghum, and cotton root tissue and soil from fields cropped continuously to these species for 15 years in St. Joseph, LA. Chlorate phenotype of each isolate was determined after growing on a minimal medium containing 120 mM potassium chlorate. Consistent differences in chlorate sensitivity were detected among isolates from different hosts and from soil versus root. To further explore genetic differentiation among fungal isolates from each host, these isolates were examined by restriction fragment length polymorphism and random amplified polymorphic DNA (RAPD) analysis. No variations were observed among isolates in restriction patterns of DNA fragments amplified by polymerase chain reaction covering the internal transcribed spacer region, 5.8S rRNA and part of 25S rRNA, suggesting that M. phaseolina constitutes a single species. Ten random primers were used to amplify the total DNA of 45 isolates, and banding patterns resulting from RAPD analysis were compared with the neighbor-joining method. Isolates from a given host were genetically similar to each other but distinctly different from those from other hosts. Chlorate-sensitive isolates were distinct from chlorate-resistant isolates within a given host. In greenhouse tests, soybean, sorghum, corn, and cotton were grown separately in soil infested with individual isolates of M. phaseolina that were chosen based on their host of origin and chlorate phenotype. Root colonization and plant weight were measured after harvesting. More colonization of corn roots occurred when corn was grown in soil containing corn isolates compared with isolates from other hosts. However, there was no host specialization in isolates from soybean, sorghum, or cotton. More root colonization in soybean occurred with chlorate-sensitive than with chlorate-resistant isolates.     

Response of Hard Red Winter Wheat to Soilborne wheat mosaic virus Using Novel Inoculation Methods

ABSTRACT Soilborne wheat mosaic virus (SBWMV) is an agronomically important pathogen of wheat that is transmitted by the soilborne plasmodiophorid vector Polymyxa graminis. In the laboratory, attempts to generate SBWMV-infected plants are often hampered by poor infectivity of the virus. To analyze the mechanism for virus resistance in wheat cultivars, we developed novel inoculation techniques. A new technique for foliar inoculation of SBWMV was developed that eliminated wound-induced necrosis normally associated with rub inoculating virus to wheat leaves. This new technique is important because we can now uniformly inoculate plants in the laboratory for studies of host resistance mechanisms in the inoculated leaf. Additionally, wheat plants were grown hydroponically in seed germination pouches and their roots were inoculated with SBWMV either by placing P. graminis-infested root material in the pouch or by mechanically inoculating the roots with purified virus. The susceptibility of one SBWMV susceptible and three field resistant wheat cultivars were analyzed following inoculation of plants using these novel inoculation techniques or the conventional inoculation technique of growing plants in P. graminis-infested soil. The results presented in this study suggest that virus resistance in wheat likely functions in the roots to block virus infection.     

Cross-reactivity of antiserum raised against Phytophthora fragariae with other Phytophthora species and its evaluation as a genus-detecting antiserum

Antiserum (anti-PfM) raised against mycelial suspensions of Phytophthora fragariae isolates reacted strongly with antigens from several Phytophthora species. Some cross-reactions with antigens from Pythium species were decreased by fractionating on an affinity column of Sepharose 4B bound to extracts of Fragaria vesca roots infected with P. fragariae. The affinity-purified anti-PfM retained its high cross-reactivity with the various Phytophthora species tested. It also detected infection of raspberry and strawberry roots by some Phytophthora species. This antiserum could, therefore, prove useful as a broad-spectrum Phytophthora-detecting antiserum.
Anti-PfM could not be made specific for P. fragariae because it was raised against components shown to be antigenically similar in all Phytophthora species tested. However, immunoblotting with the affinity-purified anti-PfM produced distinct patterns for P. fragariae, P. erythroseptica and P. cactorum: three serotypes were identified for the latter species. This antiserum might therefore prove useful in classifying Phytophthora species.     

Development of a monoclonal antibody immunoassay for the eyespot pathogen Pseudocercosporella herpotrichoides

A double-antibody-sandwich ELISA test has been developed for the detection of Pseudocercosporella herpotrichoides using a highly specific monoclonal antibody PH-10 as the capture antibody and genus-specific rabbit polyclonal antiserum as the detector antibody. The assay recognizes extracts from plants both artificially and naturally infected with P. herpotrichoides giving at least three-fold higher absorbance values with extracts from Pseudocercosporella-infected tissue than with extracts from healthy tissues or from tissues naturally infected with Microdochium nivale, Rhizoctonia cerealis or material artificially inoculated with P. anguioides. The assay tested positively against all isolates of P. herpotrichoides , including both W-type and R-type isolates. In this assay system, extraction of the antigen from the stem bases of infected plants is a one-step process not requiring any dilution procedures. The assay can be used to detect the pathogen in presymptomatic infected seedlings. The immunogen used to generate the specific monoclonal antibody and the rabbit antiserum was a mycelial extract from which the high-molecular-weight proteins and glycoproteins had been removed by ammonium sulphate precipitation. The high-molecular-weight fraction was shown to contain cross-reactive antigens; it induced antiserum in mice that cross-reacted with the other stem-base fungi even at high dilutions. The monoclonal antibody PH-10 is an IgM antibody. Heat and periodate treatment of the antigen indicate that it is a glycoprotein and that the epitope recognized by the antibody is a protein.