Differentiation of Gaeumannomyces graminis from other turf-grass fungi by amplification with primers from ribosomal internal transcribed spacers

A region comprising the 5.8S RNA gene and internal transcribed spacers 1 and 2 of the take-all patch fungus, Gaeumannomyces graminis var. avenae , was cloned and sequenced using primers from the flanking 17S and 26S ribosomal RNA genes. The sequenced region showed 99% similarity between the two G. graminis isolates, and 70–80% similarity between these two isolates and several other species of fungi. From the sequence, oligonucleotide primers were selected which permitted specific amplification of DNA from G. graminis vars. avenae and graminis using the polymerase chain reaction (PCR). The assay could detect DNA of G. graminis strains obtained from a wide variety of hosts, but did not amplify DNA from many other fungi, including the important turf-grass root pathogens Magnaporthe poae and Leptosphaeria korrae. The primers also did not amplify DNA from G. graminis var. tritici, M. rhizophila or Phialophora graminicola. The PCR-based assay shows promise as a diagnostic tool for the take-all pathogen in turf-grass pathology.     

Detection and identification of four common rust pathogens of cereals and grasses using real-time polymerase chain reaction

ABSTRACT Puccinia spp. are widespread pathogens of cereals and grasses that annually cause significant yield losses worldwide, especially in barley, oat, and wheat. Urediniospore morphology and early symptom development have limited usefulness for distinguishing Puccinia spp. Therefore, we developed real-time polymerase chain reaction assays for rapid detection of the four rust pathogen species, Puccinia graminis (Pers.:Pers.), P. striiformis (Westend.), P. triticina (Eriks.), and P. recondita (Roberge ex Desmaz.). Duplex assays were constructed for the nuclear rDNA gene, using the variable internal transcribed spacer 1 (ITS1) region to distinguish between species, and the conserved 28S region as an internal control. Species-specific ITS1 primer/probe sets were highly specific and could detect <1 pg of DNA. The species-specific primer/probe sets showed positive results over a linear range of DNA five orders of magnitude or greater. Specificity of the assays was tested using multiple collections representing a range of races and formae speciales within a species. Additionally, assay specificity was evaluated by testing a range of other grass rust pathogens, as well as other fungi. The 28S primer/probe combination was successful in detecting all Puccinia spp. tested within the duplex assays, validating the integrity of each assay. Finally, the assays were used to identify unknown rust fungi infecting pasture grasses.     

The use of species-specific DNA probes for the identification of Mycosphaerella fijiensis and M. musicola, the causal agents of Sigatoka disease of banana

The polymerase chain reaction (PCR)-based technique of random amplification of polymorphic DNA (RAPD) was used to differentiate DNA from species of the genus Mycosphaerella. DNA from two pathogens which cause Sigatoka leafspot diseases of banana, M. fijiensis and M. musicola , and two other Mycosphaerella species which are commonly found on banana, M. musae and M. minima , gave distinct RAPD banding patterns with all PCR primers tested. PCR, using primer RC07, amplified a 1250bp RAPD fragment from all isolates of M. fijiensis obtained from 11 geographical origins. This fragment was absent from the other species of Mycosphaerella. In Southern blots of genomic DNA, this band hybridized exclusively to DNA from M. fijiensis , and the pattern of hybridization suggested that it was binding to repeated DNA. A RAPD band amplified with primer PM06 obtained from M. musicola was also found to be species-specific. Southern analysis suggested that the fragment hybridized to a single-copy sequence in the M. musicola genome. Total genomic DNA from M. musicola was found to be a species-specific hybridization probe. Dot-blots confirmed the specificity of these probes, and could be used to identify isolates of Mycosphaerella which cause Sigatoka disease of banana in south-east Asia.     

小麦叶锈菌生理小种MFR的分子鉴定研究

 用AFLP方法对来自中国和墨西哥的23个小麦叶锈菌生理小种进行分析,共筛选了64对引物,获得一对引物(M₀₅/E₀₃)可在MFR小种中扩增出一条特异性DNA片段,进行回收、克隆、测序,结果表明该片段具有325个碱基。根据特异性片段序列设计出SCAR标记引物,对60个叶锈菌生理小种分离物进行回检结果表明,研制的SCAR标记能够准确区分MFR生理小种。本实验结果为小麦锈菌生理小种分子检测体系的建立奠定了基础     

Specific detection of<Emphasis Type="Italic">Pythium aphanidermatum</Emphasis> from hydroponic nutrient solution by booster PCR with DNA primers developed from mitochondrial DNA

Pythium aphanidermatum causes damping-off and root rot of vegetable crops in hydroponic systems. A DNA probe was isolated and modified from a library ofHindIII-digested mitochondrial DNA ofP. aphanidermatum that strongly hybridized to DNA ofP. aphanidermatum and weakly hybridized to DNA ofPythium deliense. Cross-hybridizing sequences were absent from DNA of plants and other related fungi. The probe detected as little as 5 ng ofP. aphanidermatum DNA and 250 ng ofP. deliense DNA in slot-blot assays.P. aphanidermatum was detected by a hybridization assay of total DNA extracted directly from infected roots. A pair of oligonucleotide primers P1 and RP2, which allowed amplification of a specific 0.65 kb DNA fragment ofP. aphanidermatum using polymerase chain reaction (PCR), was designed from a specific DNA probe. Specific amplification of this fragment fromP. aphanidermatum was highly sensitive, detecting template DNA as low as 0.1 pg total DNA by booster PCR. Specific booster PCR amplification using P1 and RP2 was successful in detectingP. aphanidermatum in naturally infected nutrient solution and roots of vegetables in a field hydroponic system. http://www.phytoparasitica.org posting Sept. 22, 2002.     

Production and Characterization of a Monoclonal Antibody Raised Against Surface Antigens from Mycelium of Gaeumannomyces graminis var. tritici: Evidence for an Extracellular Polyphenol Oxidase

ABSTRACT A murine monoclonal antibody (MAb) of immunoglobulin class M (IgM) was raised against surface antigens from Gaeumannomyces graminis var. tritici and, by enzyme-linked immunosorbent assay, recognized isolates of G. graminis var. tritici, G. graminis var. avenae and G. graminis var. graminis. Characterization of the antigen by heat and protease treatments showed that the epitope recognized by the MAb was a protein. Antigen production was detected only in live mycelia. Immunofluorescence studies showed that the antigen was associated with both the broad melanized macrohyphae and hyaline mycelia of G. graminis var. tritici. Secretion of antigen into an aqueous minimal medium was promoted only by exposure of live mycelia to certain phenolic substrates, including monophenols ortho-, para-, and meta-cresol; 3,4,5-trihydroxybenzoic acid (gallic acid); and phenolic amino acid L-3-(3,4-dihydroxyphenyl) alanine (L-DOPA). Antigen secretion was not promoted by 3-(4-hydroxyphenyl) alanine (L-tyrosine). The MAb reacted strongly with purified enzyme laccase (polyphenol oxidase, EC 1.10.3.2) but did not recognize purified tyrosinase (monophenol oxidase, EC 1.14.18.1). Moreover, chemicals that bind to copper and inhibit copper-containing enzymes such as laccase completely inhibited antigen secretion in response to L-DOPA. The MAb was tested for specificity against a wide range of fungi, common yeast species, and gram positive and negative bacteria. It did not recognize antigens from a broad range of unrelated fungi, including Gliocladium roseum, Fusarium sp., Phoma exigua, Phialophora fastigiata, Penicillium crustosum, Pythium ultimum, Rhizopus stolonifer, Rhizoctonia carotae, R. oryzae, R. tuliparum, and Trichoderma viride, nor did it recognize surface antigens from yeasts or bacteria. The MAb cross-reacted with antigens from Botrytis spp., Chaetomium globosum, R. cerealis, and R. solani. However, secretion of antigen by R. solani and R. cerealis was not promoted by L-DOPA, and secretion by C. globosum in response to the phenolic amino acid was significantly less compared to G. graminis var. tritici.     

Enhanced field control of wheat take-all using cold tolerant isolates of Gaeumannomyces graminis var. graminis and Phialophora sp. (lobed hyphopodia)

Cold tolerant isolates of Gaeumannomyces graminis var. graminis ( Ggg ) and Phialophora sp. (lobed hyphopodia), which produced at least comparable growth rates at 5°C to those of pathogenic G. graminis var. tritici ( Ggt ), were shown to control take-all disease in wheat effectively in 2 years of field experiments in New South Wales, Australia. The addition of oat inoculum of these fungi at the rate of 60 kg/ha to the seeding furrow significantly ( P  ≤ 0.05) reduced disease and increased grain yields by 33–45% compared to the Ggt alone treatment. The use of 30 kg/ha of oat inoculum also significantly ( P  ≤ 0.05) reduced disease and increased grain yields by 21–44%. These high levels of take-all control were obtained consistently from four field experiments on three different soil types with different pHs. A treatment inoculated with Ggg alone showed no disease symptoms and produced grain yields similar to that of untreated wheat. This fungus is, therefore, non-pathogenic to wheat. At high rates of inoculation of Ggg and Phialophora sp. (lobed hyphopodia), 65–80% of tillering wheat plants (GS 32) had root systems colonized by these fungi. In contrast, two Pseudomonas spp. and an isolate each of Ggg and Phialophora sp. (lobed hyphopodia), which did not grow at 5°C, were ineffective in controlling take-all. Take-all assessments during heading (GS 61-83) were highly correlated ( R ²=0.6047, P ≤0.0005) with the relative yield increase or decrease of inoculated treatments compared to the Ggt alone treatment. The use of a Ggg isolate (90/3B) and a Phialophora sp. (lobed hyphopodia) isolate (KY) for take-all control has been patented. These fungi are being developed for commercial use.     

Selective detection of Pseudomonas syringae pv. tomato using dot blot hybridization and real-time PCR

Two rapid detection methods based on dot blot hybridization with a nonradioactive DNA probe and molecular beacon-PCR were developed for the specific detection of Pseudomonas syringae pv . tomato , the causal agent of bacterial speck of tomato. A 1378 bp DNA fragment (Acc. No. AM039892), obtained from the extension of a 255 bp fragment generated by a RAPD protocol, was used to find a suitable combination of primers specific for the tomato pathovar. A 138 bp fragment from the genome of P. syringae pv. tomato DC 3000 was used as DNA probe. In dot blots of DNA extracted from either pure cultures or artificially contaminated seeds washes, the probe recognized specifically the tomato pathovar. A molecular beacon was designed from the same region for the specific detection and quantification of P. syringae pv . tomato by real-time PCR. A highly significant correlation was observed between the amount of target DNA and the cycle threshold (Ct). Using a fast protocol for DNA extraction, from pure cultures and from washes of artificially contaminated seeds, the limit of detection was about 1 × 10² CFU. The diagnostic tools developed proved highly specific for P. syringae pv. tomato and simple to use. They can therefore be applied to large-scale testing of tomato seeds and seedlings for the assessment of their phytosanitary condition in nurseries.     

Molecular Detection of Diaporthe phaseolorum and Phomopsis longicolla from Soybean Seeds

ABSTRACT Species-specific detection of Diaporthe phaseolorum and Phomopsis longicolla from soybean seeds was accomplished using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and TaqMan chemistry. To use these detection systems, fungal DNA was released from soybean seed coats using an ultrasonic processor to break the cells. DNA fragment lengths ranged from 200 to 1,200 base pairs (bp), with the majority of fragments <500 bp. Based on DNA sequences of the internal transcribed spacer (ITS) regions of ribosomal DNA, three TaqMan primer/probe sets were designed. Primer/probe set PL-5 amplified a 96-bp fragment within the ITS1 region of P. longicolla, D. phaseolorum var. caulivora, D. phaseolorum var. meridionalis, and D. phaseolorum var. sojae. Set PL-3 amplified a 86-bp DNA fragment within the ITS2 region of P. longicolla. Set DPC-3 amplified a 151-bp DNA fragment within the ITS2 region of D. phaseolorum var. caulivora. TaqMan primer/probe sets were able to detect as little as 0.15 fg (four copies) of plasmid DNA. When using PCR-RFLP for Diaporthe and Phomopsis detection, the sensitivity was as low as 100 pg of pure DNA. Among 13 soybean seed lots from Italy and the United States, the total Diaporthe and Phomopsis detected using a traditional seed-plating technique ranged from 0 to 32%. P. longicolla was most prevalent, followed by D. phaseolorum var. sojae. D. phaseolorum var. caulivora, which only occurred in 0.5% of the Italian seed lots, was not detected in the U.S. seed lots. D. phaseolorum var. meridionalis was not detected in either the U.S. or Italian seed lots. Using TaqMan primer/probe set PL-3, the frequency of P. longicolla was 18% in seed lot I3, similar to the frequency obtained from PCR-RFLP and potato dextrose agar plating detection. The frequencies of D. phaseolorum and P. longicolla in each seed lot obtained by the different detection methods were comparable with respect to total infection and individual species detection. However, TaqMan detection provided the fastest results of all the methods tested.     

Serological Methods for Detection of Polymyxa graminis, an Obligate Root Parasite and Vector of Plant Viruses

A purification procedure was developed to separate Polymyxa graminisresting spores from sorghum root materials. The spores were used as im-munogen to produce a polyclonal antiserum. In a direct antigen coating enzyme-linked immunosorbent assay (DAC ELISA), the antiserum could detect one sporosorus per well of the ELISA plate. In spiked root samples, the procedure detected one sporosorus per mg of dried sorghum roots. The majority of isolates of P. graminis from Europe, North America, and India reacted strongly with the antiserum. Interestingly, P. graminis isolates from the state of Rajasthan (northern India), from Pakistan, and an isolate from Senegal (West Africa) reacted weakly with the antiserum. The cross-reactivity of the serum with P. betae isolates from Belgium and Turkey was about 40% of that observed for the homologous isolate. There was no reaction with common fungi infecting roots or with the obligate parasite Olpidium brassicae. However, two isolates of Spongospora sub-terranea gave an absorbance similar to that observed with the homologous antigen. The DAC ELISA procedure was successfully used to detect various stages in the life cycle of P. graminis and to detect infection that occurred under natural and controlled environments. A simple procedure to conjugate antibodies to fluorescein 5-isothiocyanate (FITC) is described. Resting spores could be detected in root sections by using FITC-labeled antibodies. The potential for application of the two serological techniques for studying the epidemiology of peanut clump disease and for the characterization of Polymyxa isolates from various geographical origins is discussed.     

桃潜隐花叶类病毒RT-PCR和分子杂交检测技术的建立

本研究从武汉周边采集表现典型花叶症状的桃样品,提取总RNA为模板,采用RT-PCR方法对这些样品进了分析,结果显示所分析的5个样品(P1～P5)均获得了预期大小约为337bp的目标扩增条带,表明样品均带有PLMVd.通过回收RT-PCR产物,用生物素标记制备探针,分别采用DNA斑点杂交、RNA斑点杂交和组织印迹杂交3种杂交方法对这些样品进行检测比较,3种杂交方法中,组织印迹杂交操作步骤相对简单快捷,适合于对PLMVd进行大田快速检测.     

Biology of the Gaeumannomyces graminis Arx & Olivier/Phialophora radicicola Cain Complex on Roots of the Gramineae

Biological characteristics of the varieties of Gaeumannomyces graminis Arx & Olivier and Phialophora radicicola Cain are reviewed. They include: 1) pathogenicity to roots of cereals; 2) growth on living stem tissues; 3) type of fungal growth-cessation structure formed on or in the host. Together, these provide a simple means of characterizing isolates and standard methods for assessing them are presented.
The role of pigmented cells and tissues (growth-cessation structures) in host penetration is questioned, since they are seldom associated with a progressive infection by G. graminis or P. radicicola . However, they are a taxonomic aid, especially as they are now known to form on cellophane overlying agar.
New evidence is presented on factors affecting conidiation by these fungi. Flooding colonies with distilled water induced formation of conidia by G. graminis var. tritici Walker, P. radicicola var. radicicola (British isolates but not the type) and P. radicicola var. graminicola Deacon, whereas none formed on corresponding unflooded agar plates. Conidia were usually much larger and germinated readily when they formed in response to flooding; many of these fungi therefore have 2 conidial types, distinct in both morphology and behaviour. Preliminary results suggest that biotin and the form of nitrogen also affect the type of conidium formed by P. radicicola var. radicicola .     

Using allele-specific oligonucleotide probes to characterize benzimidazole resistance in Rhynchosporium secalis

Benzimidazole fungicides are important mixture components in strategies to combat fungicide resistance in Rhynchosporium secalis Davis. To monitor the performance of these strategies, a rapid, accurate assay has been developed to detect point mutations in the β-tubulin gene which confers resistance of benzimidazoles. The β-tubulin gene of a benzimidazole-resistant strain of R. secalis has been cloned and sequenced. Except for the difference in the position of one of its six introns, this gene showed a strong homology with other β-tubulin genes from filamentous fungi. Resistance was related to a point mutation in codon 198 which caused a glutamic acid to glycine change in resistant field strains, but glutamic acid to lysine in a laboratory mutant. A DNA fragment surrounding codon 198 was amplified directly from diseased lesions using a ‘nested’ set of PCR primers. Combining PCR amplificiation of a target DNA sequence with hybridization of Allele-Specific Oligonucleotide probes (ASO_s, 15-mers) allowed accurate detection of benzimidazole resistance. Only two probes, one sensitive and one resistant, were sufficient to monitor current field populations. Detection was achieved using either ³²P-labelled probe, or non-radioactively using a biotin-labelled probe coupled to streptavidin/alkaline phosphatase. This rapid method using ASOS can detect benzimidazole resistance within 48 h compared with 6–8 weeks by conventional assay procedures.     

The classification of isolates of Gaeumannomyces graminis from wheat, rye and oats using restriction fragment length polymorphisms in families of repeated DNA sequences

Isolates of the take-all fungus, Gaeumannomyces graminis var. avenae , which affects oats, wheat and other grasses, and of G.g. var. tritici , which preferentially affects wheat, rye and barley, contain a high proportion of repeated sequences. Total DNA from 57 fungal isolates collected from many locations and different cereal hosts, and scored for virulence on wheat, rye and oats, revealed many restriction fragment length polymorphisms. These RFLP s were observed either by staining the DNA directly, by hybridization to radioactively labelled total fungal DNA , or by hybridization with labelled wheat ribosomal DNA . With only a few exceptions, the isolates with the same preferred cereal hosts showed more similar patterns of restriction fragments than isolates that had different pathogenicity properties on cereal hosts, irrespective of the geographical origins of the isolates. This was even the case for R isolates of G.g. var. tritici that were virulent on wheat and rye compared with N isolates that were virulent only on wheat. Isolates were identified by hybridizing DNA from infected root samples with ³²P-labelled total fungal DNA . The restriction fragment polymorphisms involving families of repeated sequence can therefore be used as a predictive assay for host preference of an isolate, and have probably arisen by host selection of fungal lineages. The variation between isolates in different pathogenicity groups suggests that there is little gene flow between isolates that can infect different hosts, even though they can coexist in the same field.     

In planta-polymerase-chain-reaction detection of the wilt-inducing pathotype ofFusarium oxysporumf.sp.cicerisin chickpea (Cicer arietinumL.)

A 1.6 kb fragment of random amplified polymorphic DNA (RAPD-PCR, polymerase chain reaction), which was specific for race 5, a wilt-inducing isolate ofFusarium oxysporumf.sp.ciceris(Foc), was cloned and sequenced. This fragment was not detected in RAPD-PCR reactions with DNA from yellowing-inducing pathotypes ofFoc, or from other fungi tested. Specific PCR primers were designed from the sequence data and used to detect the presence of the fungus in genomic DNA isolated from symptomless chickpea plants, 16 days after inoculation. A single, 1.5 kb PCR product was only observed in PCR reactions with DNA from plants infected with a wilt-inducing isolate. No products were observed in reactions with DNA from plants infected with yellowing-inducing pathotypes, or from DNA isolated from uninfected chickpea cultivar controls. Southern hybridization demonstrated homology between the second PCR product and the original specific wilt-associated RAPD fragment. PCR products were detected with DNA extracted from roots and stem tissue, but no fungal DNA was detected in leaf tissue of the same infected plants. In a blind trial, the specific primers correctly identified the fungal pathotype in four different, wilt-infected chickpea cultivars.     

Rusted Root of Ginseng (Panax quinquefolius) Is Caused by a Species of Rhexocercosporidium

ABSTRACT Rusted root (also known as rusty root) of ginseng (Panax quinquefolius) was first described over 70 years ago, but the causal agent has not been clearly established. The disease is characterized by slightly raised reddish-brown to black root lesions of varying size. The lesions, regardless of size, remain superficial; however, peridermal tissue is ruptured and sloughed off, giving the root a scabbed appearance. Culture-independent techniques were used to demonstrate that a fungal internal transcribed spacer (ITS) region DNA fragment was strongly associated with diseased but not healthy root tissue. The fragment ( approximately 650 bp in length) was cloned. Restriction enzyme digests of cloned DNA indicated that the 650-bp fragment represented a single taxon. BLAST analysis following sequencing of the fragment found that the nearest matches in GenBank were anamorphic genera associated with discomycetes, in particular Rhexocercosporidium spp. This putative identification was supported further by isolating fungi from diseased tissue using a semiselective agar medium. With this procedure, a Rhexocercosporidium-like fungus was isolated; DNA extracted from fungal cultures and amplified using ITS oligonucleotide primers was found to be identical to similarly amplified DNA from the 650-bp bands. However, the isolates were distinct, with respect to growth rate on agar media and ITS sequence, from Rhexocercosporidium carotae, the only described species in this genus. The ability to reproduce symptoms on ginseng roots was confirmed in pathogenicity tests. Oligonucleotide primers based on ITS sequences were designed to amplify DNA of Rhexocercosporidium spp. Polymerase chain reaction assays on DNA extracted from naturally infected root tissue showed that the fungus was present in nearly all symptomatic roots but was infrequent in healthy-appearing roots. The most probable cause of rusted root of ginseng is a previously undescribed species of Rhexocercosporidium.     

Molecular variation in populations of Erysiphe graminis on barley, oats and rye

DNA isolated from the formae speciales of Erysiphe graminis that grow on barley, wheat, rye and oats was studied using restriction endonucleases and DNA/DNA hybridization procedures. DNA fragments were purified by molecular cloning and a few containing repeated sequences were used to demonstrate the many variations in restriction fragments both within and between the four formae speciales. In an analysis of six single-colony isolates of the barley mildew pathogen collected from different UK sites in different years, more than a quarter of the fragments scored varied among isolates. One isolate, with an uncommon pathogenicity character, differed from the remainder in the distribution of DNA bands. Isolates of rye mildew were also distinct from one another but isolates of oat mildew from a population of similar size appeared to belong to a single clone.
It is concluded that the chromosomes of E. graminis contain many families of dispersed repeated sequences and that there may be extensive polymorphism for restriction endonuclease cleavage sites associated with these repeats. Such unselected polymorphisms could be useful in helping to understand and discriminate among the factors affecting population structure in the pathogen as it responds to different agricultural practices.     

抗苯并咪唑的小麦赤霉病菌β-tubulin基因序列分析与特性研究

 本研究用真菌U-微管蛋白基因的通用寡聚核苷酸引物B1和B3,扩增并克隆了一段821 bp的小麦赤霉病菌Fusarium graminearum的U-微管蛋白基因片段,并进行了序列测定。根据该序列设计了F.graminearum U-微管蛋白基因的特异性测序引物,测定了赤霉病菌对多菌灵不同抗感菌株的U-微管蛋白基因核苷酸序列,结果表明不同F.graminearum菌株的U-微管蛋白的165,198,200和257位氨基酸未发生突变,在克隆的片段内也未发现核苷酸突变引起的氨基酸改变。表明该菌对多菌灵产生抗性的分子机制与目前已知的其它真菌有所不同,有待进一步研究。     

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.     

Mechanisms influencing the evolution of resistance to Qo inhibitor fungicides

Fungicides inhibiting the mitochondrial respiration of plant pathogens by binding to the cytochrome bc1 enzyme complex (complex III) at the Qo site (Qo inhibitors, QoIs) were first introduced to the market in 1996. After a short time period, isolates resistant to QoIs were detected in field populations of a range of important plant pathogens including Blumeria graminis Speer f sp tritici, Sphaerotheca fuliginea (Schlecht ex Fr) Poll, Plasmopara viticola (Berk & MA Curtis ex de Bary) Berl & de Toni, Pseudoperonospora cubensis (Berk & MA Curtis) Rost, Mycosphaerella fijiensis Morelet and Venturia inaequalis (Cooke) Wint. In most cases, resistance was conferred by a point mutation in the mitochondrial cytochrome b (cyt b) gene leading to an amino-acid change from glycine to alanine at position 143 (G143A), although additional mutations and mechanisms have been claimed in a number of organisms. Transformation of sensitive protoplasts of M fijiensis with a DNA fragment of a resistant M fijiensis isolate containing the mutation yielded fully resistant transformants, demonstrating that the G143A substitution may be the most powerful transversion in the cyt b gene conferring resistance. The G143A substitution is claimed not to affect the activity of the enzyme, suggesting that resistant individuals may not suffer from a significant fitness penalty, as was demonstrated in B graminis f sp tritici. It is not known whether this observation applies also for other pathogen species expressing the G143A substitution. Since fungal cells contain a large number of mitochondria, early mitotic events in the evolution of resistance to QoIs have to be considered, such as mutation frequency (claimed to be higher in mitochondrial than nuclear DNA), intracellular proliferation of mitochondria in the heteroplasmatic cell stage, and cell to cell donation of mutated mitochondria. Since the cyt b gene is located in the mitochondrial genome, inheritance of resistance in filamentous fungi is expected to be non-Mendelian and, therefore, in most species uniparental. In the isogamous fungus B graminis f sp tritici, crosses of sensitive and resistant parents yielded cleistothecia containing either sensitive or resistant ascospores and the segregation pattern for resistance in the F1 progeny population was 1:1. In the anisogamous fungus V inaequalis, donation of resistance was maternal and the segregation ratio 1:0. In random mating populations, the sex ratio (mating type distribution) is generally assumed to be 1:1. Therefore, the overall proportion of sensitive and resistant individuals in unselected populations is expected to be 1:1. Evolution of resistance to QoIs will depend mainly on early mitotic events; the selection process for resistant mutants in populations exposed to QoI treatments may follow mechanisms similar to those described for resistance controlled by single nuclear genes in other fungicide classes. It will remain important to understand how the mitochondrial nature of QoI resistance and factors such as mutation, recombination, selection and migration might influence the evolution of QoI resistance in different plant pathogens.