Genetic and Pathogenic Characterization of Different Stagonospora sp. Isolated from Bindweed

Genetic variation among 38 isolates of Stagonospora sp. and 10 isolates of Septoria sp. from bindweed was studied using (a) restriction fragment length plymorphism (RFLP) analysis of the internal transcribed spacer (ITS) region, and (b) random amplified polymorphic DNA (RAPD) PCR analysis. RFLP analysis revealed three types of fragment patterns among the isolates. A total of 26 distinct groups, based on common fragment patterns, were identified using cluster analysis of the RAPD-PCR data. When the grouping results of the two methods were compared, the fragment pattern types and clusters were generally in agreement. The degree of pathogenicity of six genetically characterized isolates of Stagonospora sp. was assessed on three ecotypes of field bindweed (Convolvulus arvensis). Disease symptoms were observed with all isolates on all ecotypes, but only Stagonospora convolvuli strain LA39, a potential biocontrol agent, showed a high degree of pathogenicity on all ecotypes. A mixture of two Stagonospora sp. enhanced the mean necrotic leaf area on bindweed from 33.9 and 39.0% (when applied alone) to 64.9% applied together at the same final concentration of 5 X 106 spores ml -1 . Molecular methods were used to identify the two pathogens. Both were present on the same plant when applied together, but never found in the same lesion.     

Elsinochrome A production by the bindweed biocontrol fungus Stagonospora convolvuli LA39 does not pose a risk to the environment or the consumer of treated crops

Biological control as an alternative to chemical pesticides is of increasing public interest. However, to ensure safe use of biocontrol methods, strategies to assess the possible risks need to be developed. The production of toxic metabolites is an aspect which has so far largely been neglected in the risk assessment and the registration process for biocontrol products. We have evaluated the risks of elsinochrome A (ELA) and leptosphaerodione production by the fungus Stagonospora convolvuli LA39, an effective biocontrol agent used against bindweeds. The toxicity of the two metabolites to bacteria, protozoa, fungi and plants was evaluated in in vitro assays. The most sensitive bacteria and fungi were already affected at 0.01-0.07 microM ELA, whereas plants were far less sensitive. Leptosphaerodione was less toxic than ELA. Subsequently, it was investigated whether ELA is present in the applied biocontrol product or LA39-treated bindweed and crop plants. In plants ELA was never detected and in the biocontrol product the ELA concentration was far too low to have toxic effects even on the most sensitive organisms. We conclude that the production of ELA by biocontrol strain LA39 does not pose a risk to the environment or to the consumer.     

Detection of QTLs for Stagonospora glume blotch resistance in Swiss winter wheat

Stagonospora nodorum is the causal agent of the Stagonospora glume blotch disease in hexaploid wheat. The Swiss winter bread wheat cv. 'Arina' has a highly effective, durable and quantitative glume blotch resistance. We studied 240 single seed descent (SSD)-derived lines of an 'Arina × Forno' F_5:7 population to identify and map quantitative trait loci (QTLs) for glume blotch resistance under natural infestation. Using composite interval mapping (CIM) and LOD>4.5, we detected two chromosomal regions on chromosome arms 3BS and 4BL which were specifically associated with glume blotch resistance. These identified QTLs were designated QSng.sfr-3BS and QSng.sfr-4BL, respectively. QSng.sfr-3BS peaked at the locus Xgwm389 in the telomeric region of the short arm of chromosome 3B and explained 31.2% of the observed phenotypic variance for the resistance within the population. The responsible QSng.sfr-3BS allele originated from the resistant parent 'Arina'. The QTL QSng.sfr-4BL (19.1%) mapped to chromosome arm 4BL ('Forno' allele) very close to two known genes, TaMlo and a catalase (Cat). Both QTL alleles combined could enhance the resistance level by about 50%. Additionally, they showed significant epistatic effects (4.4%). We found PCR-based microsatellite markers closely linked to QSng.sfr-3BS (gwm389) and QSng.sfr-4BL (gwm251) which make marker-assisted selection (MAS) for Stagonospora glume blotch resistance feasible. We also found one resistance QTL, QSng.sfr-5BL, on the long arm of chromosome 5B which overlapped with QTLs for plant height as well as heading time.Communicated by H. C. Becker     

Calystegines in Calystegia sepium do not inhibit fungal growth and invertase activity but interact with plant invertase

Abstract: Calystegines are alkaloidal glycosidase inhibitors. They accumulate predominantly in young and meristemic parts of Calystegia sepium (Convolvulaceae). C. sepium, bindweed, infests meadows and cereal fields and is difficult to control chemically. Fungal pathogens against C. sepium are established as mycoherbicides. Stagonospora convolvuli LA39 attacks C. sepium and does not affect crop plants, but young plants of C. sepium are less susceptible to the fungus. The interaction of Stagonospora convolvuli with calystegines was investigated. Further, endophytic fungi of several classes were isolated from wild-grown Calystegia sepium leaves, and selected strains were tested for interaction with calystegines. Fungal growth on agar containing calystegines was not affected considerably. Plants in climate chambers were infected with an endophyte, Phomopsis, and with the fungal pathogen, Stagonospora convolvuli. Calystegine levels were measured in infected and non-infected plant tissues. Accumulation depended on developmental stage of the plant tissue and was not influenced by infection. Acid invertase was measured from fungal mycelia and from infected and non-infected plant tissues. Fungal acid invertase activity was not inhibited by 10 mM calystegine B₂, while invertase from C. sepium leaves was inhibited. It is concluded that calystegines do not inhibit fungal development and sucrose consumption under the conditions of the present investigation, but may act by redirection of plant carbohydrate metabolism.     

Isolation and characterization of EST‐derived microsatellite loci from the fungal wheat pathogen Phaeosphaeria nodorum

Eleven polymorphic microsatellite loci and one minisatellite locus originating from expressed sequence tag (EST) libraries of Phaeosphaeria (syn. Stagonospora) nodorum were isolated and characterized. The satellite markers were used to genotype isolates from field populations collected in China, North America and South Africa. The number of alleles per locus ranged from two to 15. Genotype diversity ranged from 87.5 to 95.3 and gene diversity from 0.1 to 0.8. The variable levels of polymorphism within and among populations of P. nodorum renders these 12 satellite loci ideal markers for population genetic analysis of P. nodorum.     

Biological Control of Hedge Bindweed (Calystegia sepium) with Stagonospora convolvuli Strain LA39 in Combination with Competition from Red Clover (Trifolium pratense)

In a maize cropping system where a living green cover suppresses many weeds, Calystegia sepium is able to escape control. In this paper we report the potential for biological control of C. sepium by using the bindweed pathogen Stagonospora convolvuli strain LA39 as a mycoherbicide in combination with competition by the green cover plant Trifolium pratense. In a greenhouse experiment, competition from shoots of T. pratense caused a strong reduction of the biomass of C. sepium, and combined competition from shoots and roots had the same effect. In a second, factorial greenhouse experiment, competition by T. pratense again reduced C. sepium biomass. However, S. convolvuli did not influence the number of leaves or the biomass of C. sepium in the greenhouse even though severe necrosis was observed on inoculated bindweed leaves. In contrast, in a 2-year field study, S. convolvuli caused severe disease and a strong reduction of C. sepium ground coverage in maize. Underseeding with T. pratense had no effect on disease severity, but T. pratense reduced ground coverage by C. sepium at one of eight samplings in the first year. In conclusion, S. convolvuli is useful in the field and, as shown in the greenhouse, a competitive green cover might improve biological control of C. sepium.     

Comparative studies on the activities of chitinase, β-1,3-glucanase, peroxidase and phenylalanine ammonia lyase in the leaves of triticale and wheat infected with Stagonospora nodorum

The activities of chitinase, β-1,3-glucanase, peroxidase and phenylalanine ammonia lyase, constitutive and induced by Stagonospora nodorum were examined in the 10 – 14 day old seedlings of three triticale and two wheat cultivars under controlled environmental conditions and in flag leaves of two triticale cultivars in the field. Two S. nodorum isolates of different virulence were used. Both the constitutive and induced activities in triticale and wheat depended on genotype and in triticale the effect of growth conditions was also evidenced. The constitutive activities of chitinase, β-1,3-glucanase and peroxidase were several fold lower in flag triticale leaves in plants from the field than in the seedlings, growing under controlled conditions, but induction in the infected flag leaves was significantly more pronounced. In triticale genotypic differences in the response to infection were revealed only upon inoculation by S. nodorum isolate of higher virulence. The enzymatic activities increased several fold during successive days after the infection except for phenylalanine ammonia lyase. Induction of this enzyme was only transient and the activity decreased 48 or 96 h after infection when the activities of other enzymes were rising. In flag leaves in the field this activity was differentiated only after infection with more a virulent strain. A tendency appeared in triticale seedlings for association of the resistance to the pathogen with lower enzymatic constitutive activities. This relationship became more evident in triticale infected by S. nodorum and may imply that although the investigated enzymes are certainly involved in general, non-specific defense mechanism, they do not decide on the resistance to pathogen at least in the early stages of infection and cooperate with other factors in the complex pathogen-plant interaction. One can also assume that the enzymatic activities are associated with severity of infection rather than resistance to pathogen.     

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.     

ISSR fingerprinting for the assessment of the bindweed biocontrol agent Stagonospora convolvuli LA39 after field release

AIMS: To develop a molecular identification method based on ISSR fingerprints to monitor the fungal leaf pathogen Stagonospora convolvuli LA39 used to biologically control bindweeds after a field release. METHODS AND RESULTS: The developed method proved to be suitable to clearly distinguish LA39 from resident Stagonospora spp. and was applied in two field experiments. First, the environmental persistence of LA39 was assessed in an overwintering experiment. LA39 could be re-isolated from infected bindweed 1 year after field application, but with very low frequency of occurrence. Secondly, LA39 was applied in an area with natural bindweed infestation and re-isolated from infected bindweed. The dispersal of LA39 during one season was poor (4-5 m). CONCLUSIONS: ISSR fingerprinting has been shown to be a valuable tool to monitor the environmental fate of S. convolvuli in the field. It is concluded that an LA39-based mycoherbicide will have minimal environmental impact caused by the restricted mobility, poor proliferation and poor persistence over seasons of LA39. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies about the dispersal and survival of biocontrol agents after field release as well as the development of methods needed for this purpose are indispensable for a comprehensive risk assessment for biocontrol agents.     

Study of Genetic Variability Among Monopycnidial and Monopycnidiospore Isolates Derived from Single Pycnidia of Stagonospora ssp. and Septoria tritici with the use of RAPD-PCR, MP-PCR and rep-PCR Techniques

Thirty-six isolates of Stagonospora avenae f. sp. triticea, S. nodorum and Septoria tritici recovered from asexual fruiting bodies - pycnidia and their spores were assessed for DNA polymorphism with the use of three molecular techniques: microsatellite-primed polymerase chain reaction (MP-PCR), primers correspond to dispersed repetitive elements (rep-PCR) and random amplified polymorphic DNA (RAPD-PCR). These polymerase chain reaction (PCR)-based techniques were simultaneously evaluated for their capacity to detect genetic variation at DNA level. The most polymorphic DNA profiles of monopycnidial and monopycnidiospore isolates were detected with diverse microsatellite motifs used for PCR priming. The lowest similarity values 0.86, 0.76 and 0.84 were identified among monopycnidiospore isolates derived from the same pycnidium of S. avenae f. sp. triticea, S. nodorum and S. tritici, respectively. The above, rather low similarities, found for isolates recovered from single pycnidia, supported a hypothesis that heterokaryosis resulted from high mutation rate of microsatellites and transposons activity. This would have fundamental consequences for the genetic status of asexual populations of Stagonospora spp. and S. tritici. The data produced by this study indicate that more attention should be paid to asexual reproduction as a possible source of genetic variability among populations of the pathogens.