Mannitol is required for asexual sporulation in the wheat pathogen Stagonospora nodorum (glume blotch)

The physiological role of the mannitol cycle in the wheat pathogen Stagonospora nodorum (glume blotch) has been investigated by reverse genetics and metabolite profiling. A putative mannitol 2-dehydrogenase gene (Mdh1) was cloned by degenerate PCR and disrupted. The resulting mutated mdh1 strains lacked all detectable NADPH-dependent mannitol dehydrogenase activity. The mdh1 strains were unaffected for mannitol production but, surprisingly, were still able to utilize mannitol as a sole carbon source, suggesting a hitherto unknown mechanism for mannitol catabolism. The mutant strains were not compromised in their ability to cause disease or sporulate. To further our understanding of mannitol metabolism, a previously developed mannitol-1-phosphate dehydrogenase (gene mpd1) disruption construct [Solomon, Tan and Oliver (2005) Mol. Plant-Microbe Interact. 18, 110-115] was introduced into the mutated mdh1 background, resulting in a strain lacking both enzyme activities. The mpd1mdh1 strains were unable to grow on mannitol and produced only trace levels of mannitol. The double-mutant strains were unable to sporulate in vitro when grown on minimal medium for extended periods. Deficiency in sporulation was correlated with the depletion of intracellular mannitol pools. Significantly sporulation could be restored with the addition of mannitol. Pathogenicity of the double mutant was not compromised, although, like the previously characterized mpd1 mutants, the strains were unable to sporulate in planta. These findings not only question the currently hypothesized pathways of mannitol metabolism, but also identify for the first time that mannitol is required for sporulation of a filamentous fungus.     

Bacteria associated with Stagonospora (Septoria) nodorum increase pathogenicity of the fungus

In studies with a laboratory isolate of the fungal pathogen Stagonospora ( Septoria ) nodorum three different isolates of bacteria were closely associated with the fungus. Bacteria were also closely associated with fresh isolates of S. nodorum obtained from artificially and naturally infected field material. Although a range of bacteria was isolated, only one type of bacterium was found to be associated with each isolate of S. nodorum . In co-inoculation studies with pycnidiospores of the fungus on detached leaves, some of the bacterial isolates significantly increased the pathogenicity of the fungus, particularly Xanthomonas maltophilia , Sphingobacterium multivorum , Enterobacter agglomerans and Erwinia amylovora . Evidence is presented indicating that one of the ways that the 'helper bacteria' may assist in the establishment of infections is by the production of lipases that were not detected in germinating fungal spores.     

Mannitol 1-phosphate metabolism is required for sporulation in planta of the wheat pathogen Stagonospora nodorum

An expressed sequence tag encoding a putative mannitol 1-phosphate dehydrogenase (Mpd1) has been characterized from the fungal wheat pathogen Stagonospora nodorum. Mpd1 was disrupted by insertional mutagenesis, and the resulting mpd1 strains lacked all detectable NAD-linked mannitol 1-phosphate dehydrogenase activity (EC 1.1.1.17). The growth rates, sporulation, and spore viability of the mutant strains in vitro were not significantly different from the wild type. The viability of the mpd1 spores when subjected to heat stress was comparable to wild type. Characterization of the sugar alcohol content by nuclear magnetic resonance spectroscopy revealed that, when grown on glucose, the mutant strains contained significantly less mannitol, less arabitol, but more trehalose than the wild-type strains. The mannitol content of fructose-grown cultures was normal. No secreted mannitol could be detected in wild type or mutants. Pathogenicity assays revealed the disruption of Mpd1 did not affect lesion development, however the mutants were unable to sporulate. These results throw new light on the role of mannitol in fungal plant interactions, suggesting a role in metabolic and redox regulation during the critical process of sporulation on senescing leaf material.     

Stagonospora nodorum: cause of stagonospora nodorum blotch of wheat

Stagonospora nodorum is an important pathogen of wheat and related cereals, causing both a leaf and glume blotch. This review summarizes recent advances in our understanding of taxonomy, control and pathogenicity of this species.
Taxonomy:   Stagonospora (syn. Septoria ) nodorum (Berk.) Castell. and Germano [teleomorph: Phaeosphaeria (syn. Leptosphaeria ) nodorum (Müll.) Hedjar.], kingdom Fungi, phylum Ascomycota, subphylum Euascomycota, class Dothideomycetes, order Pleosporales, family Phaeosphaeriaceae, genus Phaeosphaeria , species nodorum .
Host range:   Wheat, Triticum aestivum , T. durum , Triticale, are the main hosts but other cereals and wild grasses have been reported to harbour S. nodorum. Disease symptoms are lens-shaped necrotic lesions on leaves, girdling necrosis on stems (especially the nodes, hence ' nodorum ') and lesions on glumes. Mature lesions produce pycnidia scattered throughout the lesions, especially as tissue senesces.
Useful websites:   http://ocid.nacse.org/research/deephyphae/htmls/asco_taxlist_spat.html (taxonomic information), http://ohioline.osu.edu/ac-fact/0002.html (disease information), http://wwwacnfp.murdoch.edu.au/  (ACNFP homepage), http://www.broad.mit.edu/annotation/fungi/stagonospora_nodorum/index.html (genome sequence homepage), http://cogeme.ex.ac.uk/efungi/ (genome sequence annotation and analysis).     

Diagnostics of phytopathogen fungi Septoria tritici and Stagonospora nodorum by fluorescent amplification-based specific hybridization (FLASH) PCR

A PCR system in the fluorescent amplification-based specific hybridization (FLASH) format was developed for the detection and identification of two important wheat pathogenic fungi Septoria tritici (teleomorph of Mycosphaerella graminicola and Stagonospora nodorum (teleomorph of Phaeosphaeria nodorum), which cause spots on leaves and glumes, respectively. The pathogen detection system is based on the amplification of a genome fragment in the internal transcribed spacer 1 (ITS 1) region and a site encoding the 5.8S ribosomal RNA. The forward primers to ITS1 and a universal reverse primer and a Beacon type probe to the 5.8S ribosomal RNA region were chosen to provide the detection of the products in the FLASH format. This system was tested on different isolates of the pathogens, and on infected soil, leaf, and seed samples.     

Incidence and effects of Septoria nodorum on wheat cultivars

Wheat cultivars were grown in two field trials in which half the plots were artificially inoculated with Septoria nodorum. There were differences between cultivars in the degree of 5. nodorum infection on flag leaves and on ears. Hybrid 46, Maris Huntsman and Maris Widgeon were among those with the least infection, and Maris Ranger and Maris Templar among those with the most. Severity of infection was positively correlated with both short stature and earliness of maturity. Infection on leaves was positively correlated with infection on ears. Inoculation reduced yield by decreasing two of its components, grain number per ear and 1000-grain weight. A very early maturing cultivar, Sterling, sustained no yield loss despite severe infection; for the remaining cultivars, yield loss was positively correlated with degree of infection.     

Predisposing effects of Eyespot (Pseudocercosporella herpotrichoides) on Septoria nodorum infection of winter wheat

All isolates of PeniciUium simplicissimum, P. verrucosum var. cyclopium, P. brevicompactum, P. multicolor, P. oxalicum, P. paxilli, Botrytis cinerea, and of a Gliocladium sp. obtained from necrotic virus-tested narcissus twin-scales previously dtoped in benomyl were tolerant to 1000 μg/ml of this fungicide in agar. Every necrotic twin-scale examined was infected with at least one of these species. The first two species were the most frequently isolated and a similar range of species infected different narcissus clones or different cultivars. It is argued that the use of benomyl during the twin-scaling programme should be discontinued as soon as a suitable alternative fungicide is found.     

Components of partial resistance to Septoria nodorum in winter wheat

Components of partial resistance to Septoria nodorum were investigated in 10 cultivars of winter wheat having similar field resistances. The components measured were infection frequency, latent period, size, shape and rate of growth of lesions, spore production and its rate of increase. Latent period was found to be lognormally distributed. Some of the components of resistance were found to be significantly different between cultivars. Cluster analysis also showed that cultivars could be distinguished on the basis of their components of resistance. Principal components analysis indicated that resistance could be broken down into four underlying factors, three of which could be readily interpreted. The measurements of the components of resistance were combined in a model, the r-index, based on Van der Plank's r. The amount of variation between cultivars cast some doubt on the predictive value of the index but all the cultivar values were well within the range bounded by two ‘synthetic’ cultivars made up of combinations of either the most resistant or the most susceptible components. It is considered that the r-index has potential in screening for field resistance. The possibility of incorporating the most resistant-type components into one cultivar is discussed. The use of cultivar mixtures containing cultivars having similar field resistances is also discussed in the light of the variability found in this study.     

A bioassay for predicting the resistance of wheat leaves to Septoria nodorum

Plants of the winter wheat cv. Bounty were inoculated with Septoria nodorum and then assayed for ergosterol. A detached leaf technique was used in which leaf segments were incubated on agar containing benzimidazole. Four levels of a conidial suspension inoculum were applied using a measured droplet technique. Ergosterol in plant extracts was measured using HPLC and its identity confirmed by co-injection with pure ergosterol as an internal standard. The extracts were also assayed by silica-gel thin layer chromatography with the sterol being visualised with rhodamine; the presence of ergosterol in the material from the two higher disease level treatments was confirmed by analysing the eluted spots in a spectrophotometer. Additional confirmation of ergosterol was obtained using gas-liquid chromatography comparing a mixture of known plant sterols with a sample from the diseased leaf tissue. The ergosterol assay was found to be very sensitive and offers a high level of reproducibility. It would therefore appear that such assays could be of value in breeding programmes when it is necessary to screen wheat cultivars for their reaction to S. nodorum or other fungal pathogens.     

Ornithine decarboxylase knockout in Tapesia yallundae abolishes infection plaque formation in vitro but does not reduce virulence toward wheat.

A knockout strain of Tapesia yallundae lacking the single ornithine decarboxylase (ODC) allele has been created by targeted gene replacement. A central region of the ODC gene was isolated by polymerase chain reaction with degenerate oligonucleotides and used to probe a lambda genomic library. The gene was sequenced, and the encoded ODC protein sequence was shown to be similar to those from other fungi. The functionality of the T. yallundae ODC was confirmed by complementation of an Aspergillus nidulans mutant (puA) strain devoid of ODC activity, restoring growth in the absence of exogenous polyamines. Transformation-mediated gene replacement was used to create strains that were auxotrophic for putrescine and lack ODC coding sequences. ODC knockout strains were unable to differentiate infection structures after in vitro induction and showed an abnormal hyphal branching phenotype. Pathogenicity studies on these mutants showed that, surprisingly, they are not reduced in virulence compared with nondisrupted transformants. This suggests that the strains carrying an ODC disruption can obtain sufficient polyamines from the host plant for normal growth and differentiation and, therefore, that fungal ODC may not be a suitable target for fungicides.     

Predisposition to Septoria nodorum as a result of take-all (Gaeumannomyces graminis) infection of wheat

In a series of experiments excised leaves from take-all infected wheat plants and from control plants were inoculated with Septoria nodorum. Larger lesions, more lesions/leaf and more pycnidia/unit area of lesion were produced from take-all plants. Significant effects of predisposition were demonstrated when only 3% of the area of the total root system was infected by take-all. Microscopical investigations revealed that germ-tubes of S. nodorum grew more rapidly on leaves from take-all plants, but the time of penetration was not affected. It is proposed that the observed effects of predisposition arose because more germ-tubes produced successful infections and host tissue was more rapidly colonised. The importance of these results for the epidemiology of glume blotch is discussed.     

Field techniques for assessing the reaction of winter wheat cultivars to Septoria nodorum

Methods of assessing the reaction of winter wheat cultivars to Septoria nodorum were compared in nine field trials. Plots were inoculated either by scattering naturally infected wheat debris or by spraying with a suspension of conidia at different growth stages, and were subsequently kept damp by several methods. Disease development was monitored by assessing the percentage cover of different organs by lesions, or by measuring the maximum height of lesions above ground. Major differences between cultivars were evident with all methods of inoculation and assessment, but time of assessment was critical for best differentiation. Inoculation with conidia soon after ear emergence, followed by assessment of lesions on flag leaves and ears, was a simple and effective method. Yield loss due to disease was measured by making comparisons either between inoculated and uninoculated plots, with various degrees of separation, or between inoculated plots with and without fungicidal treatment: the first of these methods was more effective in maintaining a disease differential. Yield loss was positively correlated with severity of disease and there was little evidence that cultivars differed in tolerance of disease. Disease was measured more easily and with less error than yield loss.     

Hydrogen peroxide production in wheat leaves infected with the fungus Septoria nodorum Berk. Strains with different virulence

The effect of two strains of the phytopathogenic fungus Septoria nodorum Berk. of different virulence on the intensity of local generation of hydrogen peroxide in common wheat leaves and the role of oxidoreductases in this process was studied. Differences in the pattern of hydrogen peroxide production in wheat plants infected with high- and low-virulence pathogen strains have been found. The low-virulent S. nodorum strain caused a long-term hydrogen peroxide (H₂O₂) generation in the infection zone, whereas the inoculation of leaves with the highly virulent strain resulted in a transient short-term increase in the H₂O₂ concentration at the initial moment of contact between the plant and the fungus. It was shown that the low level of H₂O₂ production by plant cells at the initial stages of pathogenesis facilitates S. nodorum growth and development. The decrease in the H₂O₂ concentration induced by the highly virulent S. nodorum strain is determined by inhibition of the oxalate oxidase activity in plant tissues and by the ability of the fungus to actively synthesize an extracellular catalase. The pattern of hydrogen peroxide generation at the initial stages of septoriosis may serve as an index of virulence of S. nodorum population.     

Identification of molecular markers for resistance to Septoria nodorum blotch in durum wheat

The development of Septoria nodorum blotch-resistant cultivars has become a high priority objective for durum wheat breeding programs. Marker-assisted selection enables breeders to improve selection efficiency. In order to develop markers for resistance to Septoria nodorum blotch, a set of F₅ recombinant inbred lines, derived from the crosses Sceptre/3–6, Sceptre/S9–10 and Sceptre/S12–1, was developed based on the F₂-derived family method. Two RAPD markers, designated UBC521₆₅₀ and RC37₅₁₀, were detected by bulked segregant analysis and located approximately 15 and 13.1 centiMorgans (cM) from the resistance gene snbTM, respectively. A SCAR marker was also successfully developed for marker-assisted selection in breeding programs based on the sequence of the RAPD marker UBC521₆₅₀. This is the first report of DNA-based markers linked to resistance for Septoria nodorum blotch in durum wheat. Received: 8 March 2000 / Accepted: 25 June 2000     

Relationship between leaf stages and epistasis for resistance to Stagonospora nodorum in durum wheat

Ten varieties and eight generations (2F1, 2F2, 2B1 and 2B2) of durum wheat derived from two crosses were evaluated for resistance to natural infection by Stagonospora nodorum blotch (SNB) at the 2-3 and 6-7 leaf stages at two sites over two years. There were significant differences in the incidence of SNB between leaf stages in most of the wheat varieties, with resistance being most evident at the 6-7 leaf stage. Separate analyses of the mean values for each generation showed that the genetic mechanism of defense against the pathogen depended upon the leaf stage. At the 2-3 leaf stage, only additive and dominance effects were implicated in the control of SNB for the two crosses at the two sites and for the two replications. For the 6-7 leaf stage, inheritance was more complicated and an epistatic effect was involved. Narrow-sense heritability values (range: 0.63-0.67) were consistent between crosses and leaf stages. These findings indicate a lack of resistance to SNB at the 2-3 leaf stage whereas resistance was observed at the 6-7 leaf stage and involved the genetic mechanisms of plant defense such as epistasis.     

QTL mapping of partial resistance in winter wheat to Stagonospora nodorum blotch.

Stagonospora nodorum blotch is an important foliar and glume disease in cereals. Inheritance of resistance in wheat appears to be quantitative. To date, breeding of partially resistant cultivars has been the only effective way to combat this pathogen. The partial resistance components, namely length of incubation period, disease severity, and length of latent period, were evaluated on a population of doubled haploids derived from a cross between the partially resistant Triticum aestivum 'Liwilla' and susceptible Triticum aestivum 'Begra'. Experiments were conducted in a controlled environment and the fifth leaf was examined. Molecular analyses were based on bulked segregant analyses using 240 microsatellite markers. Four QTLs were significantly associated with partial resistance components and were located on chromosomes 2B, 3B, 5B, and 5D. The percentage of phenotypic variance explained by a single QTL ranged from 14 to 21% for incubation period, from 16 to 37% for disease severity, and from 13 to 28% for latent period,     

Effects of inoculation with Septoria nodorum on yield components of spring wheat

Spring wheat, cv. Kolibri, was artificially inoculated with spore suspensions of Septoria nodorum, from the time of full expansion of the third leaf to the end of flowering and the effect on yield components recorded. Most components were reduced by increasing severity of infection and grain weight losses of 56 and 35% were produced on single-stemmed and tillering plants respectively, by the most severe infection. The effects on components of yield differed considerably between the main stems and the tillers and an increase in grain number on moderately infected tillers partly compensated for reductions in yield on the main stem.     

Effects of alien cytoplasm substitution on the response of wheat cultivars to Septoria nodorum

The effects of alien cytoplasm substitution on the response of wheat to Septoria nodorum were studied, using alloplasmic series of two cultivars, Chris and Selkirk. In general, cytoplasmic substitution caused unidirectional effects on Septoria-response, alloplasmic lines of both cultivars expressing lower levels of partial resistance (in leaf and head tissue) but higher levels of yield tolerance than the corresponding euplasmic line. The reduced resistance in alloplasmics was closely associated with reduced incubation periods of Septoria infection in both leaf and head tissue. Cytoplasmic substitution resulted in increased yield tolerance to Septoria-infection in both the non-tolerant Selkirk and the relatively tolerant Chris. Unlike their effects on partial resistance, specific cytoplasms exerted similar effects on tolerance in the two parental cultivars, several cytoplasms of the D plasmatype being particularly effective in increasing Septoria-tolevance. The potential for the development of Septoria-toterant cultivars by the incorporation of alien cytoplasms is discussed, in view of the observed neutral effects of D plasmatype cytoplasms on other agronomic traits.