Graminis Isolates Research Articles

Assessing variability among culturable phylloplane basidiomycetous yeasts from Italian agroecosystems

This study analysed basidiomycetous yeasts isolated from the phylloplane of crops and spontaneous plants in Italian agroecosystems. A total of 25 species belonging to 17 genera were recognized by analysing 83 isolates from vineyards and orchards, that are not treated with synthetic fungicides, and adjacent natural areas. Rhodotorula graminis and Filobasidium magnum were the most frequent species but 13 others were represented by a single isolate (e.g., Buckleyzyma salicina, Pseudozyma prolifica, and Moniliella megachiliensis). Preliminary analysis of (GTG)5-PCR fingerprinting revealed high genetic intraspecific heterogeneity. All isolates were characterized by their production of extracellular hydrolytic enzymes and their sensitivity to six commercial fungicides used in Italy. The isolates displayed great variability in these phenotypic traits, which play an important role in the survival of yeast populations in agroecosystems. Most of them exhibited lipolytic, proteolytic, β-glucosidase and pectinolytic activities, but only three (F. magnum, Kwoniella mangroviensis and Ps. prolifica) also had cellulolytic and amylolytic activity. Most isolates were sensitive to four fungicides, and one R. graminis isolate was resistant to all six. This heterogeneity was not related to the geographical origin of the isolates. The lack of selective factors (i.e. pesticide treatments) in the sampling fields and the presence of adjacent natural areas may have favored the maintenance of an elevated level of strain diversity. This study provides new information on phylloplane basidiomycetous yeasts in agroecosystems and opens the way to further investigations into the impact of agricultural practices on the microbial diversity of these natural habitats.

First Report of Take-All Root Rot Caused by Gaeumannomyces graminis on Hybrid Bermudagrass in China.

Take-all root rot caused by Gaeumannomyces graminis (Sacc.) Arx & Olivier is a destructive disease of hybrid bermudagrass (Cynodon dactylon × C. transvaalensis Burtt-Davy) (Elliott 1991; Stephens et al. 2020). In April 2018, multiple circular to irregular patches (10 to 200 cm in diameter) were evident on 'Tifway419' bermudagrass fairway of Wohu golf course (Huangshan city, Anhui province). Patches were found at five of the 18 fairways and showed a decrease in stand density. Individual plants in patches exhibited off-color to white foliage, dark and necrotic lesions were frequently observed on roots and associated stolons. Ectotrophic runner hyphae and lobed hyphopodia are ubiquitous on the surface of necrotic roots. Two diseased samples were collected from each patch by a soil corer (10 cm in diameter), and a total of ten patches were sampled across the five symptomatic fairways. Symptomatic roots or stolons were cut into 2 to 3 cm length and surface disinfested for 3 min in 0.6% sodium hypochlorite solution. The sterilized tissues were rinsed, air dried on sterile filter paper for 5 min and placed on potato dextrose agar (PDA) amended with 50 mg L-1 each of ampicillin, streptomycin sulfate and tetracycline. Plates were incubated in the dark at 20°C for 5 days, and similar colonies were consistently recovered from 85% of the symptomatic tissues. After hyphae purification, a total of 20 fungal isolates were obtained with morphology similar to G. graminis (Hernández-Restrepo et al. 2016). DNA was extracted and amplified with primers ITS5/ITS4 (White et al. 1990). PCR products were sequenced (deposited as MZ889119 and MZ889120 in GenBank), and showed 99.43 to 99.81% homology when compared with a known G. graminis isolate (accession no. KX306505). To complete Koch's postulates, healthy Tifway419 hybrid bermudagrass was grown in 5 × 20 cm Cone-Tainers containing a sand and Pindstrup substrate (Pindstrup Mosebrug, Pindstrup, Denmark) mixture (1:1, V/V) nine weeks after transplanting from the experimental field of the Institute of Botany, Jiangsu Province and Chinese Academy of Sciences. Five sterile oat seeds infested with G. graminis were inserted 5 cm below the soil surface in the root zone of Tifway419. Cone-Tainers inoculated with five noninfested sterile oat seeds served as controls, and each treatment was replicate 3 times. All Cone-Tainers were transferred into a growth chamber with a 12-h day/night cycle at 25/20°C and 95% relative humidity. They were covered with plastic bags for the first week, and watered once daily to encourage disease development. Hybrid bermudagrass inoculated with G. graminis decreased in plant density and turfgrass quality, black and necrotic roots covered with lobed hyphopodia were observed eight weeks after inoculation, no symptoms were present on the roots and above-ground of noninfested control plants. G. graminis was consistently reisolated from symptomatic roots and stolons, and confirmed by sequencing the ITS region as mentioned above. To our knowledge, this is the first report of G. graminis causing take-all root rot of hybrid bermudagrass in China, which may influence the management of this important root disease.

Wheat stem rust recorded for the first time in decades in Ireland.

Wheat stem rust, caused by the fungus Puccinia graminis f. sp. tritici (Pgt), occurs in most wheat‐growing areas worldwide, and, in western Europe since 2013, has started to re‐emerge after many decades of absence. Following this trend across western Europe, in 2020, we also detected and recorded wheat stem rust for the first time in five decades in experimental plots across five locations in Ireland. To examine the potential origin of the Irish Pgt infection in 2020, we carried out transcriptome sequencing on 12 Pgt‐infected wheat samples collected across Ireland and compared these to 76 global P. graminis isolates. This analysis identified a close genetic relationship between the Irish Pgt isolates and those from Ethiopia collected in 2015 after a severe stem rust epidemic caused by the TKTTF Pgt race, and with the UK‐01 Pgt isolate that was previously assigned to the TKTTF race. Subsequent pathology‐based race profiling designated two Irish isolates and recent UK and French Pgt isolates to the TKTTF Pgt race group. This suggests that the Irish Pgt occurrence most probably originated from recent long‐distance windborne dispersal of Pgt urediniospores from neighbouring countries in Europe where we confirmed the Pgt TKTTF race continues to be prevalent. The identification of wheat stem rust in Ireland at multiple locations in 2020 illustrates that the disease can occur in Ireland and emphasizes the need to re‐initiate local monitoring for this re‐emergent threat to wheat production across western Europe.

First detailed report on Puccinia graminis f. sp. avenae virulence structure and Pg resistance genes effective in Poland

Occurrence of stem rust, caused by Puccinia graminis f. sp. avenae, on oat fields in Europe may lead to significant yield losses. The last P. graminis virulence research in this region had been carried out in the years 1988 to 1996, therefore the aim of this study was the update of pathogen’s virulence structure as well as the evaluation of Pg race-specific resistance genes and their potential for use in practical breeding in central and eastern European growing conditions. To assess the pathotype structure of P. graminis in Poland and evaluate the potential of Pg resistance genes, 148 isolates were collected during 2017–2020. Their virulence structure was determined in laboratory conditions on 12 Pg differentials as well as oat lines ‘Pg-a’, ‘Alpha’, ‘Omega’ and ‘Wisconsin X-1588-2’. In total, 57 pathotypes were detected, of which the most dominant race was SSK. High frequency was also recorded for TSK, TKK and TTK. Puccinia graminis isolates were able to overcome from 5 to 11 Pg resistance genes with an average virulence complexity of 8.6. There was no isolate virulent to Pg12, ‘Pg-a’, ‘Alpha’ and ‘Omega’, hence these genes can be used in the breeding programmes. Gene Pg10 was also relatively effective, overcome by 33 out of 148 P. graminis isolates. The virulence profile observed may be representative of eastern Europe and beyond.

First report of Meloidogyne graminis on golf courses turfgrass in Brazil.

Plant-parasitic nematodes of the genus Meloidogyne, known as root-knot nematodes (RKN), have an important economic impact on golf course turfgrasses. The most prevalent RKN species associated with grasses are M. chitwoodi, M. graminicola, M. graminis, M. incognita, M. marylandi, M. microtyla, M. minor, M. naasi and M. sasseri. In 2010, slight thickening of the roots and RKN females with unusual features were observed in turfgrass roots on golf courses in Araras, São Paulo state, Brazil. This population (MgARA) was maintained in the lab and studied including morphological, morphometrical, biochemical and molecular markers. Morphology and morphometry were variable and not useful for identification, although perineal pattern morphology showed highly similarity with M. graminis description. Concerning to biochemical characterisation, the esterase phenotype Mg1, characterised by a very slow and fainter band, was detected in some protein homogenates. Regarding to molecular analysis, D2-D3 region of 28S rDNA gene and cytochrome oxidase subunit II region from mitochondrial DNA were amplified by PCR and sequenced. Phylogenetic analysis revealed that the Brazilian isolate, found associated with turfgrass, grouped with M. graminis isolates (98–99% bootstrap; variation of 8–11 and 0–24 bp, respectively), close to M. marylandi, supporting its identification as M. graminis. This is the first report of M. graminis on golf courses in Brazil.

Ribotypes of Polymyxa graminis in Wheat Samples Infected with Soilborne Wheat Viruses in China.

Polymyxa graminis is an obligate parasite and important vector of more than 14 soilborne plant viruses that pose a significant threat to cereal crops in Europe, North America, and Asia. Different ribotypes or formae speciales of P. graminis have been recognized and these may be associated with different cereal hosts or with transmission of different viruses. Two soilborne viruses infecting winter wheat in China have been reported and well studied (Wheat yellow mosaic virus [WYMV, genus Bymovirus] and Chinese wheat mosaic virus [CWMV, genus Furovirus]) but there has been no reported characterization of P. graminis isolates associated with them. In this study, the ribosomal DNA internal transcribed spacer (ITS) regions of P. graminis were examined from 63 wheat samples with apparent virus symptoms obtained from 16 sites within six Chinese provinces. Their associations with soilborne viruses were investigated. Ribotype I (P. graminis f. sp. temperata) and ribotype II (P. graminis f. sp. tepida) were confirmed in winter wheat regions of China for the first time. All 63 wheat root samples were infected with ribotype I of P. graminis and 11 were also infected with ribotype II. There was no obvious association between the ribotypes and infection by either WYMV or CWMV (or double infection). Phylogenetic analysis of the P. graminis ITS1-5.8S-ITS2 sequences revealed that ribotype I in China belongs to previously reported subgroup Ib, whereas ribotype II belongs to IIa. There was considerable sequence variation (pairwise distances from 0.0219 to 0.0319) between Chinese ribotype I isolates of different regions and previously reported ribotype I isolate Ken5 (accession number HE860055.1).

Exploring the Serbian GenBank barley (Hordeum vulgare L. subsp. vulgare) collection for powdery mildew resistance

Unlocking resistance genes in genbank collections are of prime importance for securing sustainable crop production. In this regard, the Serbian GenBank barley collection, comprising 93 local landraces and 36 commercial cultivars and elite barley breeding lines, was screened for novel resistances to powdery mildew (Blumeria graminis f. sp. hordei) using a set of 28 isolates with a wide spectrum of virulences/avirulences. No line was resistant to all the isolates, but one and three accessions showed resis- tance to 27 and 26 isolates, respectively. Twenty landraces (21.51 %) and ten cultivars (27.78 %) exhibited resistance to 50 % of the isolates. Infection type 2 was most frequent among resistant accessions. Nine B. graminis isolates were sufficient for gene postulation in 73 barley lines. In total, thirty-five different resistance spectra were recorded and the following known resistance genes were postulated namely, Mlra, Mlh, Mla12, Mla7(Mlu), Mlg, MlLa, Mla6, Mla7, Mlt, Mla22, Mlat, Mla1, Mlk. The majority of resistance profiles was constituted by only one line. Unidentified genes alone or in combination were proposed for twenty landraces and six cultivars. This report demonstrated that the barley collection held at the Serbian GenBank could be exploited as a new source for powdery mildew resistance.

Polymyxa graminis Isolates from Australia: Identification in Wheat Roots and Soil, Molecular Characterization, and Wide Genetic Diversity.

Polymyxa graminis is an obligate parasite of roots and an important vector of viruses that damage cereal crops in different parts of the world. In 2011 and 2012, P. graminis was identified infecting 11 wheat root samples from three widely dispersed locations in southwest Australia. Its presence was detected by polymerase chain reaction (PCR) and confirmed by DNA sequencing of the transcribed regions of its ribosomal RNA genes (rDNA) and observing sporosori of characteristic morphology and size in stained wheat roots. Also, when soil samples were collected from two locations where P. graminis was found and wheat bait plants grown in them, P. graminis was detected in their roots by PCR. Ribosomal DNA sequences of six southwest Australian isolates were obtained from wheat roots, and one northeast Australian isolate from barley roots. When these seven P. graminis sequences were compared with others from GenBank by phylogenetic analysis, three southwest Australian isolates were classified as P. graminis f. sp. temperata (ribotypes Ia and Ib), and three as f. sp. tepida (ribotypes IIa and IIb). P. graminis f. sp. temperata and tepida both occur in temperate growing regions of other continents and are associated with transmission of soil-borne viruses to cereal crops. The P. graminis isolate from northeast Australia was sufficiently distinct from the five existing sequence groups for it to be placed into a newly proposed grouping, ribotype VI, which also included an isolate from tropical West Africa. However, when randomly collected wheat leaf samples from 39 field crops from 27 widely dispersed locations, 21 individual wheat plant samples collected from low lying areas within 21 fields at 11 locations, and wheat bait plants growing in five soil samples from two locations were tested by reverse transcription (RT)-PCR for the presence of Soil-borne wheat mosaic virus, Soil-borne cereal mosaic virus, Wheat spindle streak mosaic virus, and furoviruses in general, no virus infection was detected. These findings suggest at least three P. graminis introductions into Australia, and the occurrence of f. sp. temperata (ribotype I) and f. sp. tepida (ribotype II) suggests that, if not already present, soil-borne cereal viruses are likely to become established should they become introduced to the continent in the future.

Virulence Structure of the Powdery Mildew (Blumeria graminis) Population Occurring on Triticale (x Triticosecale) in Poland

AbstractPowdery mildew, caused by Blumeria graminis is an important disease of cereals in many production regions. Until end of the last century triticale had been regarded as a species characterized by high level of resistance for this disease. However, after several years of intensive production on a big area in Poland, Germany and other European countries it start to be susceptible for many pathogens including B. graminis. Because of this, virulence structure of this pathogen population on triticale in Poland was evaluated across 2008–2010. Leaf samples with symptoms of the powdery mildew disease were collected randomly from nineteen localities. As a total, 1402 B. graminis isolates were collected: 23–25 isolates per locality in each year. Standard differential set of 28 genotypes was used: twenty‐one wheat with known resistance genes and seven triticale. Based on the obtained results it was possible to observe significant differences in virulence structure between years and localities. No virulence's against Pm21 (Yangmai5), and Pm3d + 4b (Kadett) were found in any year. All tested isolates were virulent on Moreno and Lamberto cultivars. In a total, 36% of tested isolates possessed 9, 11 or 12 virulence's per genotypes. Twenty five percent of tested isolates were virulent to 5 triticale cultivars. Correlation between pathotypes frequency and sampling region were not found what suggest that local epidemics play the most important role in triticale growing regions in Poland.

Biology of Gaeumannomyces graminis var. graminis isolates from rice and grasses and epidemiological aspects of crown sheath rot of rice

A collection of Gaeumannomyces graminis var. graminis isolates obtained from symptomatic rice and grass plants in central and northern Brazil were studied in regard to pathogenicity on rice and cultural characteristics. For all isolates, only one type of lobed hyphopodia was observed both in the field and artificially inoculated plants. Perithecia were formed on artificial media and inoculated leaf sheaths. Hyphopodia were formed from ascosporic germ tubes. The hyphae under moist stress conditions produced dark brown chlamydospores that were initially hyaline. Large variation in the number of chlamydospores produced and duration of perithecia formation was observed among the isolates. Pathogenicity assays showed that 60-day old rice plants were more susceptible than 35-day old plants. The isolates from rice and grasses varied significantly in regard to disease severity on both rice seedlings and adult rice plants. In general, the isolates from rice were more aggressive than the isolates from grasses. Spontaneous infection of rice plants by ascosporic inoculum from perithecia on rice stubbles was observed in the greenhouse, suggesting their role as a source of primary inoculum in the field, which deserves further investigation.

Ribosomal DNA analyses reveal greater sequence variation in Polymyxa species than previously thought and indicate the possibility of new ribotype‐host‐virus associations

Polymyxa species transmit viruses to many important crops. They are poorly understood obligate parasites occupying a distinct position in the Tree of Life. To better understand the potential for spread of Polymyxa-vectored diseases, ribosomal DNA was analysed from isolates covering a wide range of geographical locations, virus associations and hosts. Internal transcribed spacer 2 structure analysis indicated that Polymyxa graminis isolates could represent many species and there was more sequence variation within the known subgroups (ribotypes) than previously described. In cereal crops and soils from temperate climates Polymyxa isolates were usually ribotype I or II, but their host specificities or preferences were unclear. For the first time, there was evidence that ribotype I (in addition to ribotype II) could transmit SBWMV/SBCMV. Different ribotypes often occurred together in the same soil or plant. New hosts were identified for particular ribotypes, including the first detection of the sugar beet-infecting Polymyxa betae, in wheat. Unexpectedly, ribotype III-like sequences, usually restricted to crops in the tropics, were found in wheat from the USA. P. betae isolates showed limited variation (≤ 2%) and the recent change in susceptibility of sugar beet varieties to BNYVV in the USA is unlikely to be due to changes in P. betae.

Phylogeography and virulence structure of the powdery mildew population on its 'new' host triticale.

BackgroundPowdery mildew, caused by the obligate biotrophic fungus Blumeria graminis, is a major problem in cereal production as it can reduce quality and yield. B. graminis has evolved eight distinct formae speciales (f.sp.) which display strict host specialization. In the last decade, powdery mildew has emerged on triticale, the artificial intergeneric hybrid between wheat and rye. This emergence is probably triggered by a host range expansion of the wheat powdery mildew B. graminis f.sp. tritici. To gain more precise information about the evolutionary processes that led to this host range expansion, we pursued a combined pathological and genetic approach.ResultsB. graminis isolates were sampled from triticale, wheat and rye from different breeding regions in Europe. Pathogenicity tests showed that isolates collected from triticale are highly pathogenic on most of the tested triticale cultivars. Moreover, these isolates were also able to infect several wheat cultivars (their previous hosts), although a lower aggressiveness was observed compared to isolates collected from wheat. Phylogenetic analysis of nuclear gene regions identified two statistically significant clades, which to a certain extent correlated with pathogenicity. No differences in virulence profiles were found among the sampled regions, but the distribution of genetic variation demonstrated to be geography dependent. A multilocus haplotype network showed that haplotypes pathogenic on triticale are distributed at different sites in the network, but always clustered at or near the tips of the network.ConclusionsThis study reveals a genetic structure in B. graminis with population differentiation according to geography and host specificity. In addition, evidence is brought forward demonstrating that the host range expansion of wheat isolates to the new host triticale occurred recently and multiple times at different locations in Europe.

Resistance to powdery mildew in Spanish barley landraces is controlled by different sets of quantitative trait loci

Twenty-two landrace-derived inbred lines from the Spanish Barley Core Collection (SBCC) were found to display high levels of resistance to a panel of 27 isolates of the fungus Blumeria graminis that exhibit a wide variety of virulences. Among these lines, SBCC145 showed high overall resistance and a distinctive spectrum of resistance compared with the other lines. Against this background, the main goal of the present work was to investigate the genetic basis underlying such resistance using a doubled haploid population derived from a cross between SBCC145 and the elite spring cultivar Beatrix. The population was genotyped with the 1,536-SNP Illumina GoldenGate Oligonucleotide Pool Assay (Barley OPA-1 or BOPA1 for short), whereas phenotypic analysis was performed using two B. graminis isolates. A major quantitative trait locus (QTL) for resistance to both isolates was identified on the long arm of chromosome 6H (6HL) and accounted for ca. 60% of the phenotypic variance. Depending on the B. graminis isolate tested, three other minor QTLs were detected on chromosomes 2H and 7H, which explained less than 5% of the phenotypic variation each. In all cases, the alleles for resistance derived from the Spanish parent SBCC145. The position, the magnitude of the effect observed and the proportion of phenotypic variation accounted for by the QTL on 6HL suggest this is a newly identified locus for broad-based resistance to powdery mildew.

First Report of Polymyxa graminis f. sp. temperata, a Vector of Soilborne Cereal Viruses in the Czech Republic.

Polymyxa graminis L. is an eukaryotic, obligate, biotrophic parasite of plant roots (1), which belongs to a poorly studied, discrete, taxonomic unit informally called the 'plasmodiophorids'. P. graminis has the ability to acquire and transmit a range of soilborne viruses that belong to at least three separate genera and can cause economically significant diseases in cereal crops. For example, the winter barley disease caused by Barley yellow mosaic virus (BaYMV) and/or Barley mild mosaic virus (BaMMV) is widespread in Europe, Japan, and China; yield losses of >50% may occur when susceptible barley cultivars are grown on severely infested soils (2). Monitoring for P. graminis was started in the Czech Republic in May 2008. Fifty-six soil samples were collected from different localities of cereal production (wheat and barley) in the Královéhradecký Region (eastern Czech Republic). Soil from each sample was placed in five replicate pots (12 × 12 cm) in a greenhouse at 22 to 25°C. Seeds of barley cv. Florian were sown into the soil (10 seeds per pot). Negative control soil (noninfested soils from the Czech Republic) and positive control soil (known P. graminis-infested soil from Germany) were also planted to barley in five replicate pots. After 90 days, plants were collected and the roots were washed thoroughly in sterilized water and examined with a light microscope without staining. The fungus was identified as P. graminis on the basis of morphology of resting spores (cystosori) and sporangia and the size of individual cystosori (4 to 5 μm in diameter) according to Thouvenel et al. (3). Cystosori of P. graminis were observed in the roots of plants grown in 20 of the 56 soil samples, especially the samples from Ceské Mezirici. The presence of P. graminis in the roots of plants grown in the soil samples and the positive control sample versus the absence of the vector in roots of plants in the negative control soil was verified by PCR assay with DNA extracts and the Psp1 and Psp2rev primers according to Legrève et al. (Page 40 in: Proceedings of the Fifth Symposium of the International Working Group on Plant Viruses with Fungal Vectors, 2003). The PCR assay included denaturation at 94°C for 2 min, then 35 cycles including denaturation of 30 s at 94°C, annealing at 60°C for 1 min, and elongation at 72°C for 35 s. A final elongation was completed at 72°C for 7 min. To characterize the P. graminis isolates, the amplified PCR product (a DNA fragment of 472 bp) was sequenced and blasted for each of the samples that tested positive. These sequences were aligned with a known sequence (GenBank Accession No. AM259276) for P. graminis. The sequences from P. graminis on barley were 100% homologous to the published sequence of P. graminis f. sp. temperata. To our knowledge, this is the first report of P. graminis f. sp. temperata in the Czech Republic.

Identification of quantitative trait loci for resistance to powdery mildew in a Spanish barley landrace

The Spanish landrace-derived inbred line SBCC97, together with other lines from the Spanish Barley Core Collection, displays high resistance to powdery mildew, caused by the fungus Blumeria graminis f. sp. hordei. The objective of this study was to map quantitative trait loci (QTLs) for resistance to powdery mildew in a recombinant inbred line population derived from a cross between SBCC97 and the susceptible cultivar ‘Plaisant’. Phenotypic analysis was performed using four B. graminis isolates, and genetic maps were constructed with mainly simple sequence repeat (SSR) markers, following a sequential genotyping strategy. Two major QTLs with large effects were identified on chromosome 7H, and they accounted for up to 45% of the total phenotypic variance. The alleles for resistance at each QTL were contributed by the Spanish parent SBCC97. One locus was mapped to the short arm of chromosome 7HS, and was flanked by the resistance gene analogue (RGA) marker S9202 and the SSR GBM1060. This corresponded to the same chromosomal region in which a major race-specific resistance gene from Hordeum vulgare ssp. spontaneum, designated as mlt, had been identified previously. The second QTL was linked tightly to marker EBmac0755, and it shared its chromosomal location with the qualitative resistance gene Mlf, which has only been described previously in the wild ancestor H. spontaneum. This is the first report of these two QTLs occurring together in cultivated barley, and it paves the way for their use in barley breeding programs that are designed to transfer resistance alleles into elite cultivars.

Pm23: a new allele of Pm4 located on chromosome 2AL in wheat

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is one of the major diseases of common wheat (Triticum aestivum) worldwide. The powdery mildew resistance gene Pm23, identified in the common wheat Line 81-7241 and originally assigned to wheat chromosome 5A, was relocated on chromosome 2AL with the aid of molecular markers. Mapping of microsatellite markers in two wheat crosses segregating for Pm23 and Pm4b, respectively, in combination with the reported mapping of Pm4a, indicated that the three genes were all linked to the marker Xgwm356 with a distance of 3-5 cM. Allelism between Pm4b and Pm23 was then confirmed, when the progenies of a cross between VPM1 (Pm4b) and Line 81-7241, were shown to be all resistant to a B. graminis isolate avirulent to the both parents. Pm23 is therefore a new allele of the Pm4 locus, and was redesignated as Pm4c.

Multilocus phylogenetic analyses within Blumeria graminis, a powdery mildew fungus of cereals

Blumeria graminis, a powdery mildew fungus, is an important plant pathogen that causes serious damage to a variety of cereal crops. In spite of the importance of the pathogen, information on phylogenetic structure within B. graminis is scarce. In this study we conducted phylogenetic analyses of B. graminis based on the DNA sequences of four different DNA regions (ITS, 28S rDNA, chitin synthase 1, and β-tubulin). The analyses revealed that the protein-coding regions have higher amounts of phylogenetic signals than rDNA regions and are useful for phylogenetic analyses of B. graminis. The present phylogenetic analyses revealed nine distinct groups in the B. graminis isolates used in this study, a result which was commonly supported by all trees constructed from the four DNA regions. Isolates from a single host genus belonged to a single group except for isolates from Lolium and Bromus, in which the isolates were split into two and three groups, respectively. Isolates from Agropyron, Secale and Triticum formed a distinct clade ( Triticum clade) with identical or similar DNA sequences. The Hordeum clade was a sister of the Triticum clade, and Poa and Avena clades were distantly related to the Triticum and Hordeum clades. This phylogenetic relationship of B. graminis is well concordant with the level of reproductive isolation between formae speciales and also with phylogeny inferred from a cytological study. Shimodaira–Hasegawa and Templeton tests using sequences of four different DNA regions significantly rejected the tree topology of plants. Therefore, possibility of co-speciation between B. graminis and its host plants was obscure in this study.

The use of conventional and quantitative real‐time PCR assays for Polymyxa graminis to examine host plant resistance, inoculum levels and intraspecific variation

* A real-time PCR protocol based on 18S rDNA sequences was developed to provide a specific, sensitive and quantitative assay for the root-infecting virus vector Polymyxa graminis. * The assay was calibrated with zoospore suspensions and inoculated roots and then shown to work with naturally infected plant roots and infested soil. Both the temperate P. graminis ribotypes previously described are detected but are not distinguished. DNA from related plasmodiophorids and from a range of fungi and plants was not detected. * Different genotypes of Triticum were grown in a soil infested with P. graminis and Soil-borne cereal mosaic virus (SBCMV). The genotypes differed in susceptibility to P. graminis, the least susceptible being the Triticum monococcum accession K-58505. * Conventional PCR assays and sequencing of amplified rDNA fragments showed that P. graminis isolates infecting wheat were mostly, but not exclusively, of ribotype II. Ribotype II was clearly associated with SBCMV transmission and seems to occur preferentially on wheat whereas ribotype I is mostly associated with barley.

Phylogenetic analysis of Polymyxa species based on nuclear 5.8S and internal transcribed spacers ribosomal DNA sequences

A region of the nuclear ribosomal DNA containing the internal transcribed spacer 1 (ITS1), the 5.8S DNA and the internal transcribed spacer 2 (ITS2) was sequenced in 12 Polymyxa graminis and P. betae isolates, with particular emphasis on P. graminis from peanut clump-infested areas of the Indian subcontinent and West-Africa. Four different sequences were obtained from the seven isolates on sorghum or pearl millet, which differed from four sequences previously published for Polymyxa species and obtained for P. graminis isolates on barley, oat and wheat originating from temperate areas in Europe and America (two distinct sequences), for isolates on rice from Colombia and for P. betae isolates on sugar beet from several origins. The sequence variations concerned mainly the composition and length of ITS1 and ITS2 regions. Phylogenetic trees built with the distinct sequences currently known for Polymyxa spp. using parsimony, maximum likeihood and neighbour-joining methods separated P. betae from P. graminis, Within P. graminis, the hierarchy of the clustering partially matched the host range and geographical origin of the isolates. These results confirm the great diversity within P. graminis that has already been observed in host range and temperature requirements studies, and provide new arguments for considering several taxa within the species. On the basis of the ecological requirements and rDNA sequences of distinct P. graminis isolates, five special forms are proposed: P. graminis f. sp. temperata, P. graminis f. sp. tepida, P. graminis f. sp. subtropicalis, P. graminis f. sp. tropicalis and P. graminis f. sp. colombiana.

Virulent Blumeria graminis infection induces penetration susceptibility and suppresses race-specific hypersensitive resistance against avirulent attack in Mla1 -barley

Leaves of near isogenic barley lines containing the race-specific resistance alleles Mla1 ormla1 were subjected to double inoculation treatments with virulent and avirulent Blumeria graminis isolates. Attack by the avirulent isolate alone frequently caused hypersensitive death of individual Mla1 epidermal cells, but underlying mesophyll cells survived. Virulent and avirulent B. graminis isolates were inoculated sequentially (inducer followed by challenger) in all possible combinations. As shown previously, where a papilla formed due to inducer attacks, the attacked epidermal cell, and to some extent its neighbours, showed highly increased resistance to challenger penetration. When epidermal cells died in response to avirulent inducer attack, adjacent cells also showed increased penetration resistance. By contrast, where the virulent inducer formed a haustorium, invaded epidermal cells, and to some extent their neighbours, showed induced penetration susceptibility to later challenger attack. Furthermore, when virulent inducer haustoria formed in Mla1 epidermal cells, these cells became highly susceptible to successful infection by the avirulent isolate and vigorous colonies developed. It appears that suppressive factors released during virulent inducer infection prevented the hypersensitive epidermal cell death that normally is induced by the race-specific avirulence elicitor. However, suppression ofMla1 race-specific resistance was confined to the epidermis. The presence of successful avirulent fungus infection of the epidermis often led to the death of the underlying mesophyll cells, thus generating a novel cellular response phenotype in B. graminis attacked Mla1 barley.