Antifungal activities of hyoscyamine and scopolamine against two major rice pathogens: <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> and <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The antifungal activities of hyoscyamine and scopolamine, major alkaloids extracted from the desert plant Hyoscyamus muticus, against two rice pathogens, Magnaporthe oryzae and Rhizoctonia solani, were studied. The minimum inhibitory concentration of hyoscyamine that resulted in distinctive inhibition (MIC₅₀) was 1 μg/ml for both fungi. Exposure to hyoscyamine caused the leakage of electrolytes from the mycelia of both fungi. Hyoscyamine (>1 μg/ml) irreversibly delayed or inhibited conidial germination and appressorium formation in M. oryzae grown on polystyrene plates. Hyoscyamine effectively inhibited the attachment of conidia to the surface of rice (Oryza sativa) leaves and inhibited appressorium formation on the leaves. A high concentration of scopolamine (1000 μg/ml) also delayed or inhibited conidial germination in M. oryzae, but conidial germination was restored after washing the conidia with water. Antifungal activity of hyoscyamine was reduced by scopolamine. Magnaporthe oryzae infection was significantly suppressed (by >95%) in leaves of intact rice plants treated with hyoscyamine (10 μg/ml). Moreover, 10 μg hyoscyamine/ml significantly reduced the disease severity index for sheath blight to ≤0.2, when compared with the disease index of control plants (>7.0). Hyoscyamine (>20 μg/ml) completely inhibited sclerotial germination and development of R. solani by delaying the initiation, maturation, and melanization of the sclerotia. These results suggest that tropane alkaloids may be useful for controlling blast and sheath blight diseases of rice and for studying the mechanisms that regulate conidial germination in M. oryzae and sclerotial germination and development in R. solani.     

Phytotoxic activity of middle-chain fatty acids I: effects on cell constituents

Phytotoxic activity of several middle-chain fatty acids, especially pelargonic acid (C9 acid) was investigated. C9–C11 acids caused strong non-selective damage to plants such as crabgrass, cucumber, velvetleaf, and tobacco, while C6 and C14 fatty acids had almost no activity. Middle-chain fatty acids caused a strong and rapid electrolyte leakage. They reached highest conductivity in 3 h in the case of cucumber cotyledons. Middle-chain fatty acids caused a decrease of the amount of polar lipids, particularly MGDG and PG, and chlorophylls. They also caused an increase of free fatty acids in 24 h after treatment. These results suggested that middle-chain fatty acids caused severe damage to cell membranes and thylakoid membranes of treated leaves. C6 volatile compounds such as cis-3-hexenal, trans-2-hexenal, and cis-3-hexenol were generated in less than 1 h after spraying pelargonic acid to tobacco leaves. The application of pelargonic acid was thought to be the trigger for linolenic acid degradation in the thylakoid membranes.     

Long‐term survival of blast pathogen in infected rice residues as major source of primary inoculum in high altitude upland ecology

Magnaporthe oryzae is the fungal plant pathogen that causes rice blast. The sources of primary inoculum and overwintering mode of the fungus remain largely unknown. The effect of rice residues on the onset of blast epidemics and the potential for survival of M. oryzae in the residues were studied in upland conditions in Madagascar. Blast disease was observed in a 3‐year field experiment in three treatments: with either infected or uninfected rice residues on the soil surface, or without rice residues. Leaf blast incidence was significantly higher in the treatment with infected rice residues than in the two other treatments at the early stages of the epidemic. In a second set of trials, the survival of M. oryzae on rice residues was monitored. Infected rice stems were placed by lots in three places: on the mulch of rice residues, under the mulch, and buried at a depth of 10 cm in the soil. Each month, samples were taken from the field and tested for sporulation. The survival of the blast fungus decreased rapidly on the stems buried in the soil but remained high for the other conditions. Sporulation of the fungus was observed on stems left on the mulch for up to 18 months. It is concluded that under field conditions, the presence of infected rice residues could initiate an epidemic of blast. The results of this study may help in designing effective management strategies for rice residues infected by M. oryzae.     

Effects of panicle development stage and temperature on rice panicle blast infection by Magnaporthe oryzae and visualization of its infection process

Panicle blast, caused by the fungus Magnaporthe oryzae (syn. Pyricularia oryzae), directly contributes to yield loss in the field. The effects of panicle development stage and temperature on panicle blast were studied and the infection process of M. oryzae in panicles was visualized. Rice panicles at different development stages from three rice cultivars were inoculated with a conidial suspension in vitro. The rice cultivar Lijiangxintuanheigu was highly susceptible to panicle blast at 5 days postinoculation (dpi) when the pulvinus distance was 15–20 cm. Nanjing 9108 was moderately susceptible to panicle blast when the pulvinus distance was 8–10 cm, but Yliangyou 800 was resistant. The effect of temperature on panicle blast was determined under 22–35 °C temperature treatments. Inoculated panicles placed at temperatures of 28 and 30 °C showed the highest lesion grade based on lesion length at 5 dpi. The infection process of M. oryzae in rice panicles was observed by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). M. oryzae initially formed the appressorium to invade through the epidermis of rice panicles at 24 hours postinoculation (hpi). As the disease progressed, the invasive hyphae formed dense mycelial networks in the inner parenchyma cells at 60 hpi. Our results will contribute to the understanding of panicle development stage and temperature effects on panicle blast and improve resistance evaluation methods. Additionally, visualization of the infection process by CLSM and TEM are valuable methods to observe M. oryzae invasive hyphae inside rice panicle cells.     

Antagonistic activity against rice blast disease and elicitation of host-defence response capability of an endophytic Streptomyces albidoflavus OsiLf-2

Rice blast, caused by the fungus Magnaporthe oryzae, can result in notable yield losses in rice production. The objective of this study was to investigate the potential of a rice endophytic isolate, Streptomyces albidoflavus OsiLf-2, on the control of rice blast and the possible mechanisms involved. In vitro assays displayed a variety of antagonistic effects of OsiLf-2 against different physiological races of M. oryzae, with peak mycelial growth inhibition ranging from 74.1% to 83.0%. In vivo tests of OsiLf-2 showed 18.0% and 19.6% reduction in disease index in greenhouse and field conditions, respectively. The stable active metabolites in its cell-free culture filtrate inhibited the mycelial growth, spore germination and appressorial formation of M. oryzae in a dose-dependent manner. They also possessed strong antifungal capacities toward various phytopathogens in vitro. OsiLf-2 secreted multiple antimicrobial compounds, cell wall degradation enzymes, siderophore, plant hormones, and 1-aminocyclopropane-1-carboxylate deaminase, which might function in direct or indirect resistance to M. oryzae. In addition, a variety of defence responses were induced in OsiLf-2-treated rice, including hydrogen peroxide (H₂O₂) accumulation, callose deposition, defence-related enzymes activation, and elevated expression of salicylic acid (SA) and jasmonic acid (JA) pathways genes, which might contribute in resisting pathogen attack. The significant biological control activity and host defence-stimulation ability of OsiLf-2 suggest that this endophytic actinobacterial strain could be a promising candidate in the management of rice blast disease.     

In vitro and in vivo antifungal activities of decursin and decursinol angelate isolated from Angelica gigas against Magnaporthe oryzae, the causal agent of rice blast

Blast is considered the most important fungal disease of rice due to its wide distribution and destructiveness under favorable conditions. Development of new effective and environmentally benign agents against the causal pathogen, Magnaporthe oryzae, is of great interest. In the course of a search for natural antifungal compounds in medicinal plants, we found that the methanol extract of Angelica gigas roots showed a potent control efficacy against rice blast caused by M. oryzae. We isolated antifungal coumarins from the extract, and they were identified as decursin and decursinol angelate. Antifungal activities of these compounds, along with kasugamycin, were tested on M. oryzae in vivo and in vitro. In an in vivo assay, the three compounds effectively suppressed the development of rice blast at concentrations more than 100 μg/mL. Coumarins showed relatively weak inhibitory effect on fungal mycelial growth when compared to kasugamycin. However, they strongly inhibited M. oryzae spore germination, which was not observed in kasugamycin treatments. This is the first report demonstrating that decursinol angelate can provide control against rice blast and that the two coumarins inhibit M. oryzae spore germination. In addition, the wettable powder formulation of the crude extract of A. gigas prohibited the development of blast symptoms on rice plants more effectively than liquid concentrate formulation of kasugamin, a commercial fungicide. Based on our study, we propose that coumarin compounds as well as the A. gigas root crude extract can be used as natural, benign fungicides for controlling rice blast.     

Transgenic rice plants that over-express the mannose-binding rice lectin have enhanced resistance to rice blast

Mannose-binding rice lectin (MRL), which is almost identical to the salt-induced protein SalT, binds to mannose and glucose residues. Expression of the MRL gene in response to infection with Magnaporthe oryzae, the rice blast fungus, was stronger in the incompatible interaction than in the compatible. Transgenic rice plants that constitutively over-expressed MRL strongly suppressed the growth of invasive hyphae of the fungus on leaf sheaths and the development of typical susceptible-type lesions on leaf blades, but did not affect penetration by the fungus in comparison with the wild-type. On a polycarbonate plate, purified recombinant MRL inhibited conidial attachment and appressorium formation but not conidial germination. These results suggest that MRL may play an essential role in disease resistance by suppressing development of M. oryzae in situ.     

Antifungal activity of <Emphasis Type="Italic">Biscogniauxia</Emphasis> sp. culture filtrates against the rice pathogen <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

An ethyl acetate extract of a culture filtrate (ECF) from an unidentified fungal isolate O821 was evaluated for antifungal activity against the rice pathogen Magnaporthe oryzae. The O821-ECF significantly inhibited spore germination, appressorium formation, and mycelial growth of M. oryzae, and its antifungal activity was heat-stable. It also significantly suppressed the number and size of blast lesions. In an analysis of the ITS sequence of this isolate, it shared similarities with species of the fungus Biscogniauxia. These results suggest that isolate O821 of the genus Biscogniauxia produces a heat-stable antifungal compound(s) in its culture filtrate.     

Investigation of rice blast development in susceptible and resistant rice cultivars using a gfp‐expressing Magnaporthe oryzae isolate

In this study, an isolate of Magnaporthe oryzae expressing the green fluorescent protein gene (gfp) was used to monitor early events in the interaction of M. oryzae with resistant rice cultivars harbouring a blast resistance (R) gene. In the resistant cultivars Saber and TeQing (Pib gene), M. oryzae spores germinated normally on the leaf surface but produced morphologically abnormal germ tubes. Germling growth and development were markedly and adversely affected in leaves of these resistant cultivars. Penetration of host cells was never seen, supporting the idea that disruption of germling development on the leaf surface might be one of the resistance mechanisms associated with Pib function. Thus, this particular R gene appeared to function in the absence of host penetration by the fungal pathogen. Confocal laser scanning microscopy of M. oryzae‐infected susceptible rice cultivars showed the dimorphic growth pattern that is typically observed during the biotrophic and necrotrophic stages of leaf colonization in susceptible cultivars. The suitability of the gfp‐expressing M. oryzae isolate for further research on R‐gene function and identification of resistant genotypes in rice germplasm collections is discussed.     

Synergistic induction of wheat stem rust appressoria by chemical and topographical signals

The leaf alcohols cis -3-hexen-1-ol and trans -2-hexen-1-ol have been previously shown to induce the wheat stem rust (Puccinia graminis f. sp. tritici) to differentiate appressoria in a complex axenic culture medium. In this paper, the possible role of these chemicals in appressorium induction during infection has been analysed further. The inductive potential of ethylene and three cutin monomers were also tested because they have been found to induce appressoria in other fungi. Both hexenols were found to be inductive in aqueous solution in the absence of media constituents. 0.5–1 m trans -2-hexen-1-ol was the most inductive resulting in a maximum of 51% appressorium differentiation. trans -2-hexen-1-ol was also shown to be inductive in vapour form. In aqueous solution, trans -2-hexen-1-ol acted synergistically with topographical signals by increasing the rate of appressorium induction. Combining the two signals also increased the total number of appressoria differentiated (88%). The other chemicals tested were non-inductive although a biochemical domain rich in exposed cutin was localized around stomatal apertures of wheat leaves. The characteristic bipolar morphology of appressoria formed over stomata was found to be determined by topographical signals. Overall, our data support a role for both chemical signals (hexenols or their analogues) and topographical signals being involved in appressorium induction by wheat stem rust.     

黑龙江省水稻种质抗瘟性及稻瘟病菌致病性分析

为明确黑龙江省水稻种质抗性及稻瘟病菌的致病性,以黑龙江省8个水稻品种、24个单基因系作为供试材料,120株稻瘟病菌株作为接种体,采用喷雾接种法测定了各供试水稻的抗瘟性及稻瘟病菌的致病性。结果表明,水稻品种对2010年和2011年菌株的抗性频率分别在31.67%~68.33%和21.67%~55.00%之间,2010年最好的抗性品种为松粳12,2011年最好的抗性品种为五优稻4和东农425;松粳12东农425组合联合抗病性最好。水稻单基因系对2010年和2011年菌株的抗性频率分别在10.00%~90.00%和5.00%~86.67%之间,抗性最好的单基因系分别为IRBLzt-T(Pi-zt)和IRBLz5-CA(Pi-z5);松粳12、东农425和龙粳22的基因聚合效果最好。2010年和2011年菌株对抗瘟基因群的致病率分别在8.33%~95.83%和25.00%~95.83%之间;无毒基因总出现频率分别为461和412次。研究表明,水稻种质抗性受菌株致病性影响较大,但高抗种质相对稳定,基因聚合方式更适宜当地品种抗性改良。     

Races of <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> in Australia and genes with resistance to these races revealed through host resistance screening in monogenic lines of <Emphasis Type="Italic">Oryza sativa</Emphasis>

Rice blast is the most serious disease threat to rice production worldwide. It is difficult to control due to the complex diversity and wide geographic distribution of the causal pathogen Magnaporthe oryzae. In Australia, rice blast occurs in northern Australia but remains exotic to the main south-eastern rice growing area; however, there is the potential for rice blast to threaten this area; in addition, rice production is currently expanding from south-eastern Australia into northern Australia, which makes rice blast a major concern and challenge to rice industry in Australia. Prior to this study, there was lack of information on the race status of M. oryzae present in Australia and on how to manage the disease through host resistance. The races of rice blast isolates collected in northern Australia was characterised based on the disease reactions of eight standard rice differentials used in an international race differential system. The following studies revealed genes conferring resistance to these races through investigating the responses of 25 monogenic rice lines with targeted resistance gene against different races. The rice blast isolates were characterised into five races: IA-1, IA-3, IA-63, IB-3 and IB-59. Genes Pi40, Piz-t, Pi9, Pi5(t) and Pi12(t) exhibited resistance to all the isolates belonging to five races. In addition, two genes showed complete resistance to multiple races, viz. Pi9 that showed complete resistance to races IA-1, IA-3, IA-63 and IB-3 and Pita2 that had complete resistance to races IA-3, IB-3 and IB-59. This study provides information about the races of M. oryzae in Australia. Genes identified conferring resistance to multiple races will not only streamline the identification via molecular markers of imported rice varieties with resistance to rice blast in Australia, but will also allow the Australian rice breeding program to develop new varieties with broad-spectrum resistance to rice blast and pyramid multi-gene resistance into Australian rice varieties.     

Site‐directed mutagenesis of the cytochrome b gene and development of diagnostic methods for identifying QoI resistance of rice blast fungus

BACKGROUND: It is possible that a single nucleotide polymorphism (SNP) (G143A mutation) in the cytochrome b gene could confer resistance to quinone outside inhibiting (QoI) fungicides (strobilurins) in rice blast fungus because this mutation caused a high level of resistance to fungicides such as azoxystrobin in Pyricularia grisea Sacc. and other fungal plant pathogens. The aim of this study was to survey Magnaporthe oryzae B Couch sp. nov. isolates in Japan for resistance to QoIs, and to try to develop molecular detection methods for QoI resistance. RESULTS: A survey on the QoI resistance among M. oryzae isolates from rice was conducted in Japan. A total of 813 single‐spore isolates of M. oryzae were tested for their sensitivity to azoxystrobin using a mycelial growth test on PDA. QoI fungicide resistance was not found among these isolates. The introduction of G143A mutation into a plasmid containing the cytochrome b gene sequence of rice blast fungus was achieved by site‐directed mutagenesis. Molecular diagnostic methods were developed for identifying QoI resistance in rice blast fungus using the plasmid construct. CONCLUSION: As the management of rice blast disease is often dependent on chemicals, the rational design of control programmes requires a proper understanding of the fungicide resistance phenomenon in field populations of the pathogen. Mutation of the cytochrome b gene of rice blast fungus would be specifically detected from diseased leaves and seeds using the molecular methods developed in this study. Copyright © 2009 Society of Chemical Industry     

Antifungal activity of a novel compound purified from the culture filtrate of <Emphasis Type="Italic">Biscogniauxia</Emphasis> sp. O821 against the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Rice blast is a devastating fungal disease resulting in major losses to rice crops. Owing to continuous acquisition of resistance by the causal fungus, several fungicide chemicals are no longer effective. Therefore, there is a need to identify natural components and develop new agents to control fungal pathogens. We previously demonstrated that the culture filtrate of Biscogniauxia sp. O821 inhibited infection behavior of Magnaporthe oryzae and subsequent blast lesion formation. In the present study, we isolated a new compound, (3aS,4aR,8aS,9aR)-3a-hydroxy-8a-methyl-3,5-dimethylenedecahydronaphto[2,3-b]furan-2(3H)-one (HDFO), from the culture filtrate of Biscogniauxia sp. O821 and determined its molecular weight as 248. The HDFO structure was determined by electrospray ionization-mass spectrometry and nuclear magnetic resonance spectroscopy after purification with column chromatography and high-performance liquid chromatography. The structure of this antifungal compound was similar to that of alantolactone and isoalantolactone. The growth inhibition zone against M. oryzae in presence of HDFO was observed at Rf 0.5–0.6 on a thin layer chromatography plate. HDFO inhibited conidial germination of M. oryzae in a dose-dependent manner (1–200 ppm). Furthermore, blast lesion formation was significantly suppressed by HDFO at over 5 ppm. These results suggest that HDFO from the culture filtrate of Biscogniauxia sp. O821 can protect rice from rice blast disease caused by M. oryzae. This is the first report that HDFO produced by Biscogniauxia sp. can serve as an antifungal compound against M. oryzae.     

The DNA damage signal transducer ortholog <Emphasis Type="Italic">Mop53BP1</Emphasis> is required for proper appressorium differentiation and pathogenicity in <Emphasis Type="Italic">Pyricularia oryzae</Emphasis>

Appressorium differentiation, one of the most important steps in pathogenesis by the rice blast fungus, Pyricularia oryzae, is strongly coordinated with the cell cycle. In this study, we identified an ortholog gene of 53BP1, which encodes a signal transducer protein that participates in G2-M cell cycle checkpoint in higher eukaryotes, in the genome of P. oryzae and characterized the phenotype of deletion and overexpression mutants. Deletion mutants showed no significant deficiency in vegetative growth compared to wild-type and complemented strains, even on the media containing DNA-damaging agents. However, these null mutants had abnormal appressoria and formed more appressoria per conidium than in the wild type and were unable to penetrate the epidermis of rice leaves. eGFP-fused Mop53BP1 and qRT-PCR analyses revealed that Mop53BP1 is expressed during the first hours of appressorium formation. In addition, in overexpression mutants, Mop53BP1 localized to nuclei during all stages of appressorium maturation and penetration, and the mutants were resistant to the microtubule inhibitor benomyl, suggesting that Mop53BP1 is nuclear protein and may have some role related to microtubules.     

Transmission of rice blast from seeds to adult plants in a non‐systemic way

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is a serious threat to rice production worldwide. In temperate regions, where rice is not cultivated for several months each year, little is known about the initial onset of the disease in the field. The main overwintering and primary inoculum sources reported are infested residues and seeds, but the subsequent steps of the disease cycle are largely unknown, even though a systemic infection has been proposed but not demonstrated. The present work follows rice blast progression in infected seeds from germination to seedling stage, with direct and detailed microscopic observations under both aerobic conditions and water seeding. With the use of GFP‐marked M. oryzae strains, it was shown that spores are produced from contaminated seeds, infect emerging seedling tissues (coleoptile and primary root) and produce mycelium that colonizes the newly formed primary leaf and secondary roots. Using different rice cultivars exhibiting distinct levels of resistance/susceptibility to M. oryzae at the 2/4‐leaf stage, it was observed that resistance or susceptibility of a considered genotype is already established at the seedling stage. The results also showed that when plants are inoculated either at ripening stage (mature panicles), heading stage (flowering/immature panicles) or even before heading (flag leaf fully developed), they produce infested seeds. These seeds produce contaminated seedlings that mostly die and serve as an inoculum source for healthy neighbouring plants, which gradually develop disease symptoms on leaves. The possible rice blast disease cycle was reconstructed on irrigated rice in temperate regions.     

The effect of isoprothiolane on the emergence of infection pegs from appressoria of Pyricularia oryzae

The effect of isoprothiolane^a (di-isopropyl 1,3-dithiolan-2-ylidenemalonate), a systemic fungicide used for rice blast control, on the infection process of Pyriculuria oryzae was studied by micromanipulation in a scanning electron microscope. Whether or not infection pegs emerged from the appressoria was examined by inverting each appressorium contact surface. Infection pegs were observed on more than 80% of the appressoria, 48 h after inoculation, on both untreated rice leaves and cellophane film. Isoprothiolane, approximately 10 μg ml^?1 in the ambient water of inoculated conidia, reduced the emergence of infection pegs on rice leaves and cellophane film by 96 and 100%, respectively.     

Management of rice blast (Pyricularia oryzae): implications of alternative hosts

Rice blast (Pyricularia oryzae) has become a serious disease on commercial rice (Oryza sativa) crops in northern Australia and is present there on wild rice (O. australiensis). Characterisation of the host range of P. oryzae is fundamental to both reducing disease spread and to preventing development of epidemics via better management of Poaceae inoculum reservoirs in Australia. Studies on response of three different wild O. australiensis sources toward four isolates of P. oryzae showed all genotypes very susceptible to three isolates [WAC13466 (race IA-1), BRIP53376 (race IB-3), NT2014a (race unknown)], but resistant to isolate BRIP39772 (race IA-3). Studies to investigate levels of blast disease development following inoculation on a range of Poaceae hosts showed both P. oryzae isolates (WAC13466, BRIP53376) were highly virulent on barley (disease index, DI?=?100%), and on Phalaris and O. australiensis (DI?=?70%). However, isolate BRIP53376 showed a significantly higher level of aggressiveness (DI ~80%) on ryegrass, wild oat and rice. Of the two wheat cultivars tested, only one cultivar showed disease and only from WAC13466 (DI ~30%). Sweet corn and goosegrass were also susceptible to both blast isolates, but DI was <50%. That P. oryzae was virulent across these diverse Poaceae hosts, highlights, for Australia, the possibility for these species in, first, harbouring P. oryzae isolates highly virulent to commercial rice, second, fostering spread of rice-attacking P. oryzae strains into regions currently free of rice blast, and third, potential for these alternative host species to encourage development of more virulent host-specific strains of P. oryzae. The current study is an important step towards facilitating improved crop protection in the medium and long term from reducing P. oryzae disease epidemics via a better understanding and management of inoculum reservoirs in Australia.

     

Induced Resistance to Rice Blast by Antagonistic Bacterium, <Emphasis Type="Italic">Serratia marcescens </Emphasis>Strain B2

An antagonistic bacterium, Serratia marcescens strain B2, controlled rice blast after being sprayed onto rice phylloplane, as did the bacterial suspension when poured into rhizosphere soil of rice plants. Three days after root treatment, rice blast conidia were sprayed onto rice foliage. A week after pathogen inoculation, rice blast was suppressed and lesions caused by the pathogen decreased in size. Brown deposits were observed around sites of pathogen infection after root treatment. Induced resistance was not associated with an increase in the activitiy of peroxidase, phenylalanine ammonia lyase, tyrosine ammonia lyase, β-1,3-glucanase, β-1,4-glycosidase, N-acetylhexosaminidase or chitinase. However, lipoxygenase levels were elevated after the root treatment with strain B2 following inoculation with the pathogen. Strain B2 was not detected in rice foliage after root treatment. These data suggest that strain B2 induced resistance against rice blast caused by Pyricularia oryzae. Received 1 November 2001/ Accepted in revised form 25 January 2002