Leaf Blotch Disease Research Articles

Sensitivity of Mycosphaerella graminicola isolates to demethylation-inhibiting (DMI) fungicides

Mycosphaerella graminicola (Fuckel) J. Schrot. (anamorph Zymoseptoria tritici (Desm.) Quaedvlieg & Crous) causes Septoria leaf blotch disease of wheat. The disease can be responsible for yield losses of 30–50% and, when severe, requires management with a fungicide. Single picnidia isolates collected from different locations of Lithuania were tested in vitro for sensitivity to demethylation-inhibiting (DMI) fungicides epoxiconazole, cyproconazole and prothioconazole. Fungicide concentrations were chosen from the proposed Fungicide Resistance Action Committee (FRAC) methods. Microtitre plates with 96-wells were inoculated with suspensions of pycnidiospores taken from single pycnidia of leaves. In total, 196 isolates from 34 locations were tested over the three experimental years (2009–2011). The results showed reduced sensitivity of M. graminicola isolates to epoxiconazole and cyproconazole. The average EC 50 values over three experimental years for epoxiconazole varied between 1.02 and 3.15 mg l -1 , for cyproconazole – 1.51 and 10.66 mg l -1 . The sensitivity to prothioconazole of 64 isolates from 13 locations (2011) ranged from 0.43 to 32.28 mg l -1 . Results of field experiments (2007– 2011) showed that fungicides epoxiconazole and prothioconazole significantly reduced the area under the disease progress curve (AUDPC) values of Septoria leaf blotch compared with untreated plots in winter wheat. In most cases, a single fungicide application significantly increased grain yield and a thousand grain weight weight.

First Report of Raspberry leaf blotch virus in Raspberries in Finland.

Raspberry (Rubus idaeus L.) is a valuable and widely grown softfruit that is a host for 40 viruses and virus-like agents, of which many are not characterized at the molecular level. Recently, Raspberry leaf blotch virus (RLBV, putative emaravirus species) was described from raspberries (cv. Glen Ample) in the United Kingdom and Serbia. Plants displayed conspicuous yellow blotches on leaves and abnormal development of leaf hairs in the corresponding areas of the abaxial side (3). Similar symptoms were observed in 'Glen Ample' grown in protective plastic tunnels and open fields in the main berry growing area in eastern Finland in June 2011. In three farms, leaves were sampled from symptomatic and symptomless plants of 'Glen Ample' and also cv. Polka displaying no symptoms. Total RNA was extracted using CTAB reagent. Equal amounts of RNA were pooled from 13 samples and subjected to small-RNA (sRNA) deep sequencing (Fasteris SA, Plan-les-Ouates, Switzerland) to detect viruses without advance information (2). Contigs were built on 21- to 24-nt sRNA reads using Velvet. Contigs larger than 50 nt were used to search homologous sequences in GenBank by BLAST and significant similarity (up to 99%) was observed with RLBV RNA3 and RNA4. Mapping sRNA reads to the genome of RLBV (1) by MAQ resulted in significant coverage of RNA1 (16%), RNA2 (37%), RNA3 (46%), RNA4 (65%), and RNA5 (27%). cDNA was synthesized on RNA of one symptom-expressing plant using random hexamer primers and the cDNA tested by PCR with a forward primer (RLBV-F 5'-TCAAATCCACTTGCATAGAACC-3', nt 723 to 744) and reverse primer (RLBV-R1 5'-CCTCAAACCTTGCAAACACA-3', nt 1,318 to 1,337) designed according to the nucleocapsid (NP) gene of the Scottish RLBV isolate (3). The sequence of the amplified partial NP gene (576 nt; GenBank Accession No. JQ684678) was 92.8% and 94.8% identical to the Scottish isolate at nt and amino acid levels, respectively. The forward primer RLBV-F and a new reverse primer (RLBV-R2 5'-GCCGAAAGTCAAACCTGGTG-3', nt 943 to 962) were used to test additional plants for RBLV and to make a probe (198 nt) to detect RLBV using digoxigen-labeled sense and antisense RNA probes, as described for European mountain ash ringspot associated virus (1). RLBV was detected in all tested plants of 'Glen Ample' with yellow leaf blotch symptoms in the three farms, but not in any symptomless plants of 'Glen Ample' and 'Polka.' The sense probes gave strong signals, in contrast to the antisense probes, which gave only weak or no detectable signals in the virus-positive plants, consistent with the negative RNA strand of RLBV being encapsidated in virus particles. The results show RLBV is associated with severe, distinct, and characteristic symptoms in raspberries of cv. Glen Ample grown in plastic tunnels and open fields in Finland and has an apparent negative impact on plant growth and yield. Our observations in 2011 also suggest that the incidence of diseased plants is much higher in plastic tunnels than in open fields, perhaps because the conditions for the vector of RLBV (raspberry leaf and bud mite, Phyllocoptes gracilis Nalepa) (1) are more favorable in plastic tunnels. These results clarify the etiology of raspberry leaf blotch disease in Finland and emphasize the need to inspect raspberry planting materials for RLBV for better control of the disease.

Multiple Alternaria species groups are associated with leaf blotch and fruit spot diseases of apple in Australia

Leaf blotch and fruit spot of apple caused by Alternaria species occur in apple orchards in Australia. However, there is no information on the identity of the pathogens and whether one or more Alternaria species cause both diseases in Australia. Using DNA sequencing and morphological and cultural characteristics of 51 isolates obtained from apple leaves and fruit with symptoms in Australia, Alternaria species groups associated with leaf blotch and fruit spot of apples were identified. Sequences of Alternaria allergen a1 and endopolygalacturonase gene regions revealed that multiple Alternaria species groups are associated with both diseases. Phylogenetic analysis of concatenated sequences of the two genes resulted in four clades representing A. arborescens and A. arborescens‐like isolates in clade 1, A. tenuissima/A. mali isolates in clade 2, A. alternata/A. tenuissima intermediate isolates in clade 3 and A. longipes and A. longipes‐like isolates in clade 4. The clades formed using sequence information were supported by colony characteristics and sporulation patterns. The source of the isolates in each clade included both the leaf blotch variant and the fruit spot variant of the disease. Alternaria arborescens‐like isolates were the most prevalent (47%) and occurred in all six states of Australia, while A. alternata/A. tenuissima intermediate isolates (14%) and A. tenuissima/A. mali isolates (6%) occurred mostly in Queensland and New South Wales, respectively. Implications of multiple Alternaria species groups on apples in Australia are discussed.

First Report of Leaf Blotch on Sorghum Caused by Bipolaris spicifera in Turkey.

In July 2009, a leaf blotch disease was observed on sorghum in Sakarya Province, Turkey. Disease incidence and severity were rated at 45% and 25 to 75%, respectively. Typical symptoms included elliptical, straw-colored, necrotic lesions with darker margins. The lesions eventually coalesced, resulting in drying of leaves. A fungus was isolated from the lesions on potato dextrose agar (PDA) incubated under near ultraviolet light for 12 h daily at 22°C for 2 weeks. Flexuous conidiophores of the fungus were solitary or produced in small groups. They were geniculate with numerous well-defined scars, medium to dark brown, ≥300 μm long, and 4 to 9 μm thick. Five to eight or more conidia were produced on the apices of the conidiophores. The conidia were straight, oblong or cylindrical, rounded at the ends, golden brown at maturity except for a small area just above the dark scar, pseudoseptate, and 20 to 31 × 7.5 to 12.5 μm (1). The causal fungus was identified as Bipolaris spicifera (Bain) Subram. (teleomorph Cochliobolus spicifer Nelson). The identification was confirmed with specific PCR primers (Bipol-1 F: 5'-CAGTTGCAATCAGCGTCAGT-3', R: 5'-AAGACAAAAACGCCCAACAC-3', Bipol-2 F: 5'-GTGTTGGGCGTTTTTGTCTT-3', R: 5'-CCTACCTGATCCGAGGTCAA-3', Bipol-3 F: 5'-GATGAAGAACGCAGCGAAAT-3', R: 5'-AAGACAAAAACGCCCAACAC-3'). These primers were designed by the authors using Primer3 primer design software and sequences of B. spicifera found in GenBank. PCR products were amplified from nuclear DNA of the cultured fungus and were sequenced after cleaning with a Beckman 8000 CEQ DNA sequencer. Sequences amplified using Bipol-1 and Bipol-2 showed 99 to 100% similarity with B. spicifera sequences from GenBank. The DNA sequence amplified using Bipol-2 was deposited in GenBank (Accession No. HQ538774). B. spicifera has been reported previously as pathogenic in Africa, North and South America, Asia, Australia, Oceania, and the West Indies on Agrostis, Avena, Cymbopogon, Cynodon, Dactylis, Desmostachya, Eleusine, Holcus, Hordeum, Oryza, Panicum, Pennisetum, Phleum, Poa, Saccharum, Sorghum, Triticum, and Zea spp. (3). Pathogenicity tests were conducted on sorghum (Sorghum bicolor (L.) Moench) and Sorghum × sudangrass hybrid cultivars. From PDA cultures, conidia were collected in sterile distilled water with a concentration of 3% Tween 20. Twenty-five plants (five per pot) were inoculated by spraying the conidia (105 ml-1) onto 21-day-old plants, which were then maintained at 25 ± 2°C in a greenhouse following 48 h in a humid chamber. The test was repeated once. Control plants were sprayed with sterile distilled water. Typical symptoms (2) were obtained from all inoculated plants 7 days after inoculation. No symptoms developed on the control plants. The pathogen was reisolated from inoculated leaves to fulfill Koch's postulates. To our knowledge, this is the first report of B. spicifera on sorghum in Turkey.

New capabilities for Mycosphaerella graminicola research

Mycosphaerella graminicola is a major pathogen of wheat worldwide, causing Septoria leaf blotch disease. Targeted gene disruption in M. graminicola, by Agrobacterium tumefaciens-mediated transformation, has become an established functional genomics tool for M. graminicola research in recent years. However, in order to advance research into this economically important pathogen, further functional genomics tools need to be developed. Here, we report three new capabilities for M. graminicola research: (i) two selectable markers have been shown to work robustly in M. graminicola, namely G418 and the fungicide carboxin; (ii) the generation of a strain of M. graminicola in which the KU70 (MUS-51) homologue has been disrupted; in this strain, homologous recombination efficiencies increased to more than 95%, whilst maintaining wild-type growth in vitro and full pathogenicity on wheat leaves; (iii) the ability to efficiently target and generate precise mutations of specific genes in the genomic context in M. graminicola. In addition, the insertion of the E198A mutation into the beta-tubulin gene (MgTUB1), conferring resistance to the fungicide benomyl, suggests that this mutant allele may provide an additional selectable marker. The collective use of these tools will permit further advancements in our knowledge of the biology and pathogenicity of this important plant pathogen.

Occurrence of leaf blotch disease (<i>Botryosphaeria</i> Sp.) in <i>Ficus religiosa</i> in Sri Lanka

This paper reports the occurrence of a leaf blotch disease in Ficus religiosa in Sri Lanka. The disease first appears as small, irregular, yellow specks in leaves at later stages of maturity and with time the infected areas become necrotic and gradually enlarge. At advanced stages the whole leaf turns yellow with brown blotches and is shed. The causal agent was identified as Botryosphaeria sp. The necrotic blotches are associated with erumpent fruiting bodies of the fungus, ascomata. Ascomata are solitary or botryose and are found on both sides of leaves. Asci are with numerous pseudoparaphyses and ascospores were observed. Colony developed on water agar medium showed hyaline, aseptate conidia and mature brown conidia. In the presence of leaf blight caused by Glomerella cingulata, the leaf blotch symptoms appeared much later, about 10-12 weeks after the new flush. The diseased leaves do not last long as F. religiosa, being a deciduous species, sheds leaves seasonally. However, the fallen leaves, unless eliminated, may provide a good source of inoculum for infection of the new flush. Commercial fungicide, BULLET 50, inhibited the in vitro growth of Botryosphaeria sp. This is the first report on a blotch disease attacking leaves of F. religiosa in Sri Lanka. Key words: sacred fig; fungal disease DOI: 10.4038/cjsbs.v38i2.1858 Cey. J. Sci. (Bio. Sci.) 38 (2): 51-56, 2009

The economics of fungicide use in winter wheat in southern Sweden

In southern Sweden, fungicide treatment of winter wheat is prevalent and recommended almost routinely against leaf blotch diseases. However, yield increases and hence the resulting net returns from fungicide use are highly variable within and between years. These variations raise questions about whether, when and how fungicides should be used. To help answer these questions, a thorough economic evaluation of fungicide use was carried out, based on results from untreated plots and fungicide-treated plots in trials in farmers' fields, 1983–2007. Scenarios with varying grain prices and costs of fungicide treatment were evaluated and examined. Doubling and tripling the grain price led to the largest impact on the net return from fungicide treatment, followed by increasing cost of the fungicide. Other costs were of minor importance. The mean net return from fungicide use was no more than 12 € ha −1 over the 25 years (2008 grain prices and costs used in calculations). Furthermore, the mean net return was negative in 10 years and less than 50% of the entries were profitable to treat in 11 years. Changes over time and changes in controllable factors (e.g. fungicide and cultivar choice, crop rotation, techniques) and uncontrollable factors (e.g. emerging and new diseases, price relations) influenced the profitability of fungicide use. Fungicide use was in fact more profitable (mean net return 21 compared with 3 € ha −1) during the latter part of the period (1995–2007) than in the earlier part (1983–1994). Improved decision support systems in a holistic framework based on sound economics are urgently needed.

Assessing the Accuracy of Simulation Model for Septoria Leaf Blotch Disease Progress on Winter Wheat.

A mechanistic model, PROCULTURE, for assessing the development of each of the last five leaf layers and the progress of Septoria leaf blotch, caused by Septoria tritici (teleomorph Mycosphaerella graminicola), has been applied on susceptible and weakly susceptible winter wheat (Triticum aestivum) cultivars in two locations (Everlange and Reuland) in Luxembourg over a 3-year period (2000 to 2002). A double performance assessment of PROCULTURE was conducted in this study. First, the capability of PROCULTURE to correctly simulate S. tritici incidence was checked. Second, the model's ability to accurately estimate disease severity was assessed on the basis of the difference between simulated and observed levels of disease development at each leaf layer. The model accurately predicted disease occurrence in the 2000 and 2002 seasons, on susceptible and semi-susceptible cultivars, with a probability of detection (POD) exceeding 0.90. However, in 2001, even though the POD never fell below 0.90, the false alarm ratio (FAR) was too high to consider the simulations satisfactory. Concerning the evaluation of disease severity modeling, statistical tests revealed accurate simulations performed by PROCULTURE for susceptible cultivars in 2000 and 2002. By contrast, for weakly susceptible cultivars, the model overestimated disease severity, especially for the upper leaves, for the same period.

Mycosphaerella species occurring on Eucalyptus globulus in Portugal

Mycosphaerella leaf disease (MLD) is caused by species of Mycosphaerella and several anamorphic form genera that have been connected to Mycosphaerella. Until recently, MLD of eucalypts was largely ignored in Portugal. However, serious damage to Eucalyptus globulus has been reported since 1999 when frequent and severe defoliation of young trees was observed. The severity of this disease prompted a preliminary study of the Mycosphaerella species associated with major symptoms of a leaf blotch disease in commercial plantations of E. globulus in Portugal, which is presented here. The species were identified by molecular methods based on the ITS1-5.8S-ITS2 cluster, together with morphological characters. In addition to confirming the species previously recorded, Mycosphaerella vespa is reported for the first time from Portugal, while the status of Mycosphaerella grandis remains to be resolved.

Impact of temperature and precipitation on yield and plant diseases of winter wheat in southern Sweden 1983–2007

Weather factors are driving forces in plant disease development and differ between years and locations. Results from long-term fungicide field trials 1983–2007 and disease surveys 1988–2007 in winter wheat in southern Sweden were used to evaluate relationships between yield, the yield increase obtained by fungicide treatment, thousand grain weight (TGW), disease severity and disease incidence, and the independent variables air temperature and precipitation as monthly means. These two weather variables explained more than 50% of the variation between years regarding yield increase, TGW, LBDs (Leaf Blotch Diseases, including Septoria tritici blotch, Stagonospora nodorum blotch and tan spot), brown rust, yellow rust and eyespot, but less than 50% of the variation in yield and powdery mildew. Precipitation in May was the factor most consistently related to LBD disease intensity, and adding another two weather factors further improved the degree of explanation. Weather factors in the preceding growing season influenced growth stage, powdery mildew and brown rust. Mild winters and springs favoured the biotrophs such as powdery mildew, brown rust and yellow rust. Statistically significant correlations between incidence and severity were found for LBDs, brown rust and eyespot, but not for yellow rust and powdery mildew. Regression models with disease incidence as dependent variable generally had a higher degree of explanation and lower P-value than models with disease severity as dependent variable. Our results confirm that weather data can be successfully used in wheat disease prediction models.

Dayra, A New Bread Wheat Cultivar for Warm Humid Climate of Khazar, Iran

Line N-78-14 with the pedigree of SHA4/CHIL CM91099-25Y-OM-3N-1Y-OYZ-O10M-OY-3M-O10 was obtained from CIMMYT, Mexico in 1996 through High Rainfall Wheat Screening Nursery. Preliminary evaluation of the genotypes in International Nursery in Gorgan and Sari Research Stations during 1996-97 cropping season, resulted in selection of this line as one of the most high yielding genotypes. Selected lines were evaluated in preliminary regional wheat yield trials of north warm and humid climate in 1997-98 in four locations of Gorgan, Sari, Gonbad and Moghan. Line N-78-14 together with other selected lines were tested then in advanced regional wheat yiled trial (ARWYT-N) in 1998-79, and later in ellite regional wheat yield trials (ERWYT-N) for three years in four locations. In all these experiments line N-78-14 was the most high yielding line compared to the check cultivar and other lines. Evaluation of line N-78-14 for resistance to different diseases in special disease nurseries in different locations under artifical inoculations indicated that in most locations, this line was resistant or moderately resistant to yellow rust and leaf rust while tolerated well fusarium head blight and septoria leaf blotch diseases. Line N-78-14 has been evaluated as a good baking quality wheat containing high molecular weight glutenine allels 2*, 7+9 and 5+10. Due to high yield, yield stability and compatibility, resistance to diseases and good baking quality, this line was introduced as Darya in 2006 for cultivation in warm humid climate regions of Khazar, Iran.

Yield and disease control in winter wheat in southern Sweden during 1977–2005

Fungicides are routinely used to prevent yield losses in winter wheat in southern Sweden. Yield and 1000 grain weight (TGW) data from 432 trials in farmers' fields were evaluated to review long-term yields (1977–2005) and control of eyespot and Leaf Blotch Diseases (LBDs, including Septoria tritici blotch, Stagonospora nodorum blotch and tan spot), powdery mildew, brown rust and yellow rust. Regression analyses revealed that control of LBDs explained 74% of the yield increase achieved by fungicide treatment at GS 45–61, followed by powdery mildew (20%), brown rust (5%) and yellow rust (1%). Yield of both untreated and fungicide-treated plots increased from approx. 6000 to 12 000 kg ha −1 over the period 1983–2005. Single eyespot treatment improved yield by ∼320 kg ha −1 yr −1 during the period 1977–2002, mainly due to occasional years with severe eyespot. Single leaf disease treatment at GS 45–61 increased mean yield by 10.3% or 810 kg ha −1 yr −1 (9.9% or 660 kg ha −1 yr −1 for 1983–1994 and 10.7% or 970 kg ha −1 yr −1 for 1995–2005) due to increased TGW and grain numbers, especially in high-yielding stands. Additional extra early treatment at GS 30–40 against LBDs increased yield by ∼250 kg ha −1 yr −1. Estimated variance in yield and TGW was higher between years than within years, while that in yield increase and plant diseases was lower between years than within. The results confirm potential and limits of fungicides and the need for supervised control strategies including factors affecting disease, yield and interactions.

A signaling-regulated, short-chain dehydrogenase of Stagonospora nodorum regulates asexual development.

The fungus Stagonospora nodorum is a causal agent of leaf and glume blotch disease of wheat. It has been previously shown that inactivation of heterotrimeric G protein signaling in Stagonospora nodorum caused development defects and reduced pathogenicity [P. S. Solomon et al., Mol. Plant-Microbe Interact. 17:456-466, 2004]. In this study, we sought to identify targets of the signaling pathway that may have contributed to phenotypic defects of the signaling mutants. A comparative analysis of Stagonospora nodorum wild-type and Galpha-defective mutant (gna1) intracellular proteomes was performed via two-dimensional polyacrylamide gel electrophoresis. Several proteins showed significantly altered abundances when comparing the two strains. One such protein, the short-chain dehydrogenase Sch1, was 18-fold less abundant in the gna1 strain, implying that it is positively regulated by Galpha signaling. Gene expression and transcriptional enhanced green fluorescent protein fusion analyses of Sch1 indicates strong expression during asexual development. Mutant strains of Stagonospora nodorum lacking Sch1 demonstrated poor growth on minimal media and exhibited a significant reduction in asexual sporulation on all growth media examined. Detailed histological experiments on sch1 pycnidia revealed that the gene is required for the differentiation of the subparietal layers of asexual pycnidia resulting in a significant reduction in both pycnidiospore size and numbers.

Operational application and improvements of the disease risk forecast model PROCULTURE to optimize fungicides spray for the septoria leaf blotch disease in winter wheat in Luxembourg

Abstract. The model PROCULTURE has been developed by the Université Catholique de Louvain – UCL (Belgium) to simulate the progress of the septoria leaf blotch disease on winter wheat during the cropping season. The model has been validated in Luxembourg for four years at four distinct representative sites. It is able to identify infection periods due to the causal agent Mycosphaerella graminicola on the last five leaf layers by combining meteorological data with phenological data from PROCULTURE's crop growth model component. The meteorological forcing consists of hourly time-series of air temperature, relative humidity and cumulative rainfall since the time of sowing, retrieved from automatic weather stations for hindcast and numerical weather prediction model outputs for the forecast periods. In order to improve the model, leaf wetness – which is one of the most important drivers for the spread of the disease – shall be added as an additional predictor. Therefore leaf wetness sensors were set up at four test sites during the 2007 growing season. To get a continuous spatial coverage of the country, it is planned to couple the PROCULTURE model offline to 12-hourly operational weather forecasts from an implementation of the Weather Research and Forecasting (WRF) model for Luxembourg at 1 km resolution. Because the WRF model does not provide leaf wetness directly, an artificial neural network (ANN) is used to model this parameter.

First Report of Cercospora concors Causing Cercospora Leaf Blotch of Potato in Inner Mongolia, North China.

Cercospora leaf blotch disease of potato (Solanum tuberosum L.) caused by Cercospora concors (Casp.) Sacc (synonym Mycovellosiella concors (Casp.) Deighton) occurs worldwide but mainly has been reported in the cool and temperate climates of Europe, Asia, North America, and eastern Africa. Cercospora leaf blotch is usually a minor disease and may go unnoticed since it commonly occurs simultaneously with other potato leaf diseases such as late blight (caused by Phytophthora infestans) and early blight (caused by Alternaria solani) (2). Symptoms of Cercospora leaf blotch first appear on lower leaves as small, yellowish green, irregular blotches and later may appear on middle and upper leaves. As the leaves expand, the blotches enlarge and become purplish brown or black. Conidiophores and conidia form on the underside of the lesions, giving the lesions a mildewed appearance similar to late blight. Necrotic lesions are distinguished from those caused by the early blight pathogen A. solani by the lack of concentric rings (1). In more severe epidemics of Cercospora leaf blotch, potato leaves may be killed, stem lesions become dark and entire plants die, but no resulting yield loss from the disease has been documented. Potato tubers are not infected. From August to September of 2005, yellow-brown lesions appeared on the upper side of potato leaves (cv. Zihuabai, certified virus free) and gray mildew developed on the underside of leaves in potato field trials conducted in Jining County, 41°N, 113°E of Inner Mongolia, North China. The infections were observed mostly on lower and middle leaves of plants; 20 to 30% of plants were infected. In the laboratory, the mildew was scraped with a sterile scalpel and examined microscopically. The conidiophores were irregular in width, grayish, and highly branched. The conidia were numerous, light to dark, straight or slightly bent, cylindrical or obclavate, with conspicuous scars, and zero to six septa. The mature spores were from 16 to 59 μm long and 4 to 6 μm wide. The teleomorph of the fungus was not found. On the basis of the morphological characters, the causal agent was identified as C. concors. C. concors has been previously identified from potato leaves in the Engshi District of Hubei Province, China (3), but to our knowledge, this is the first report of the fungus causing Cercospora leaf blotch of potato in Inner Mongolia, North China.

Diversity of the trifunctional histidine biosynthesis gene (his) in cerealPhaeosphaeriaspecies

Phaeosphaeria species are important causal agents of Stagonospora leaf blotch diseases in cereals. In this study, the nucleotide sequence and deduced polypeptide of the trifunctional histidine biosynthesis gene (his) are used to investigate the phylogenetic relationships and provide molecular identification among cereal Phaeosphaeria species. The full-length sequences of the his gene were obtained by PCR amplification and compared among cereal Phaeosphaeria species. The coding sequence of the his gene in wheat-biotype P. nodorum (PN-w) was 2697 bp. The his genes in barley-biotype P. nodorum (PN-b), two P. avenaria f. sp. triticea isolates (homothallic Pat1 and Pat3), and Phaeosphaeria species from Polish rye and dallis grass were 2694 bp. The his gene in heterothallic isolate Pat2, however, was 2693 bp because the intron had one fewer base. In P. avenaria f. sp. avenaria (Paa), the his gene was only 2670 bp long. The differences in the size of the his gene contributed to the variation in amino acid sequences in the gap region located between the phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase sub-domains. Based on nucleotide and deduced amino acid sequences of the his gene, Pat1 was not closely related to either PN-w or the Paa clade. It appears that rates of evolution of the his gene were fast in cereal Phaeosphaeria species. The possible involvement of meiotic recombination in genetic diversity of the his gene in P. nodorum is discussed.

Coexistence of Related Pathogen Species on Arable Crops in Space and Time

This review considers factors affecting the coexistence of closely related pathogen species on arable crops, with particular reference to data available at Rothamsted for Septoria tritici/Stagonospora nodorum (Mycosphaerella graminicola/Phaeosphaeria nodorum) (septoria leaf blotch diseases on winter wheat), Oculimacula yallundae/O. acuformis (eyespot disease of winter cereals), and Leptosphaeria maculans/L. biglobosa (phoma stem canker on winter oilseed rape). Factors affecting the short-term, medium-term, and long-term coexistence of such related pathogen species are reviewed, and their evolution from common ancestors considered. Small niche differences between the related pathogen species enable them to coexist on the same host. The niche differences result from small differences in their biology/epidemiology, leading to separation in space, time, or resource use. Changes in both natural (e.g., fluctuating temperature) and man-made (e.g., agronomic practices, pollution) factors influence the coexistence. Such factors may result in coexistence between the related species in some parts of the world, whereas in other parts only one species occurs. These principles illustrated with pathogens of arable crops are generic to other host-pathogen systems.

A New Biotype of Phaeosphaeria sp. of Uncertain Affinity Causing Stagonospora Leaf Blotch Disease in Cereals in Poland.

A new Phaeosphaeria sp. biotype was isolated from winter ryes in Poland during 1995. Two isolates, Sn23-1 and Sn48-1, were obtained from diseased leaves of cvs. Motto and Dańkowskie, respectively. The rye Phaeosphaeria sp. represented by isolate Sn48-1 has similar pycnidiospore morphology and induces disease symptoms in cereals similar to Phaeosphaeria nodorum, the causal agent of Stagonospora nodorum blotch disease (4). The pathogen (Sn48-1) produces hyaline, cylindrical pycnidiospores that are mostly three-septate and measure 12.8 to 23.7 × 2.1 to 3.2 μm (average size = 16 × 2.6 μm) on water agar. A molecular comparison of several genes in isolates Sn23-1 and Sn48-1 revealed that the rye Phaeosphaeria sp. was different from P. nodorum. In the conserved alpha-box sequence (1,93 bp) of the MAT1-1 gene, a four nucleotide difference occurred between the wheat-biotype P. nodorum and isolates Sn23-1 and Sn48-1 (GenBank Accession Nos. AY072933 and AF322008). In addition, the length of the internal transcribed spacer (ITS) region of the nuclear rDNA was the same for the wheat-biotype P. nodorum and the two rye Phaeosphaeria sp. isolates. However, a six nucleotide discrepancy was found in the ITS region (GenBank Accession Nos. U77362 and AF321323). The beta-glucosidase (bgl1) and beta-tubulin (tubA) genes differ in length between the wheat-biotype P. nodorum and two rye Phaeosphaeria sp. isolates (2,3). The main difference was due to the intron sizes of these two genes. One extra nucleotide was found in the intron2 of the bgl1 gene (GenBank Accession Nos. AY683619 and AY683620) and the intron1 of the tubA gene (GenBank Accession Nos. AY786337 and AY786331), respectively, in these two rye Phaeosphaeria sp. isolates. Disease severity on the fifth leaf (GS15) of Polish wheat (Alba, Begra, and Liwilla), triticale (Bogo and Pinokio), and rye (Zduno) cultivars was assessed with one (resistant) to nine (susceptible) scales 14 days after inoculation. Aggressiveness of wheat-biotype P. nodorum isolate Sn26-1 and rye Phaeosphaeria sp. isolate Sn48-1 was significant (P < 0.01) in five cultivars except in the moderately resistant wheat cv. Liwilla. The rye Phaeosphaeria sp. isolate Sn48-1 severely affected Polish rye Zduno (8.3) and two triticale cultivars (6.5), while the infection by isolate Sn26-1 was moderate (3-4). On the contrary, the wheat-biotype P. nodorum isolate Sn26-1 was more aggressive on wheat (4.1 on moderately resistant Alba and 6.2 on highly susceptible Begra) than the rye Phaeosphaeria sp. isolate Sn48-1, which had a scale of 2.2 and 4.3, respectively. Under laboratory conditions, the rye isolate Sn48-1 was able to cross with the wheat-biotype P. nodorum isolate Sn26-1 that has an opposite mating-type (MAT1-2) gene, but few viable ascospores were produced (1).

Analysis of expressed sequence tags from the wheat leaf blotch pathogen Mycosphaerella graminicola (anamorph Septoria tritici)

Mycosphaerella graminicola is a major fungal pathogen of wheat as the causal agent of Septoria leaf blotch disease. As a first step toward a greater understanding of the mechanism of host infection we have generated, sequenced, and analyzed three M. graminicola EST libraries from conditions predicted to resemble independent phases of the host infection process, including one library generated from the fungus during interaction with its host. A total of 5180 ESTs were sequenced and clustered into 886 contigs and 2039 singletons to give a set of 2925 unique sequences (unisequences). BLASTX analysis revealed 33% of the unknown M. graminicola unisequences to be orphans. Very limited inter-library overlap of expression was seen with the majority of unisequences (contigs and singletons) being library-specific. Analysis of EST redundancy between libraries demonstrated a significant difference in gene expression in the three conditions. Comparisons made against fully sequenced genomes revealed most M. graminicola sequences to be homologous to genes present in both pathogenic and non-pathogenic Ascomycete filamentous fungi. A range of sequences having significant homology to verified pathogenicity/ virulence genes ( HvPV-genes) of either plant or mammalian fungal and Oomycete pathogens were also identified (<1e−20). The generation of, and the diversity present within, this EST collection will facilitate future efforts aimed at a more detailed study of the transcriptome of the fungus during host infection.

Jasmonic Acid and Salicylic Acid Induce Accumulation of β-1,3-Glucanase and Thaumatin-Like Proteins in Wheat and Enhance Resistance Against Stagonospora nodorum

The effect of application of jasmonic acid (JA) and salicylic acid (SA) on the induction of resistance in wheat to Stagonospora nodorum and on the induction of μ-1,3-glucanase and thaumatin-like proteins (TLPs) was studied. Western blot analysis revealed that two β-1,3-glucanases with apparent molecular masses of 31 and 33 kDa that cross-reacted with a barley glucanase antiserum were induced in wheat leaves after treatment with JA and SA. When wheat plants were treated with SA and JA, a TLP with an apparent molecular mass of 25 kDa and several other isoforms of TLP were induced. Pre-treatment of wheat plants with SA and JA significantly reduced (up to 56 %) the incidence of leaf blotch disease incited by S. nodorum compared with untreated control plants.