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Phomopsins are mycotoxins mainly infesting lupines, with phomopsin A (PHOA) being the main mycotoxin. PHOA is produced by Diaporthe toxica, formerly assigned as toxigenic Phomopsis leptostromiformis, causing infections in lupine plants and harvested seeds. However, Diaporthe species may also grow on other grain legumes, similar to Aspergillus westerdijkiae as an especially potent ochratoxin A (OTA) producer. Formation of PHOA and OTA was investigated on whole field peas as model system to assess fungal growth and toxin production at adverse storage conditions. Field pea samples were inoculated with the two fungal strains at two water activity (aw) values of 0.94 and 0.98 and three different levels of 30, 50, and 80% relative air humidity.After 14 days at an aw value of 0.98, the fungi produced 4.49 to 34.3 mg/kg PHOA and 1.44 to 3.35 g/kg OTA, respectively. Strains of D. toxica also tested showed higher PHOA concentrations of 28.3 to 32.4 mg/kg.D. toxica strains did not grow or produce PHOA at an aw values of 0.94, while A. westerdijkiae still showed growth and OTA production.Elevated water activity has a major impact both on OTA and, even more pronouncedly, on PHOA formation and thus, proper drying and storage of lupins as well as other grain legumes is crucial for product safety.

The fungus, Diaporthe toxica, anamorph Phomopsis sp., previously classified as P. leptostromiformis, is a plant endophyte and occasional pathogen, causing Phomopsis stem blight. This disease is damaging not only to lupins but also to the animals grazing on infected plants, due to the toxic secondary metabolites called phomopsins. The aim of this work was to validate markers for resistance to Phomopsis stem blight in narrow-leafed lupins and identify novel germplasm with increased levels of resistance to the disease. Plant inoculations were performed using ten isolates of D. toxica, originating from Australia and Poland. The European core collection of L. angustifolius was evaluated both in a controlled environment and with field experiments to classify the accessions based on their resistance to the disease. Simultaneously, the accessions were assayed with disease resistance markers to identify donors of hypothetical resistance alleles. We have found that the European lupin germplasm collection preserves wild and domesticated donors of at least two resistance genes to Phomopsis stem blight, including Phr1 and PhtjR. Molecular markers PhtjM7, InDel2, and InDel10, tagging PhtjR gene, were applicable for marker-assisted selection targeting the European gene pool with an expected accuracy of 95%. None of diagnostic markers for the Phr1 locus was found useful for European breeding programs; two existing markers Ph258M1 and Ph258M2 were unreliable, due to a high percentage of false-positive results (up to 58%) and a high recombination rate between markers (~ 30%).

Narrow-leafed lupin (Lupinus angustifolius L.) is a grain legume crop that is advantageous in animal nutrition due to its high protein content, however, livestock grazing on stubble may develop a lupinosis disease that is related to toxins produced by a pathogenic fungus, Diaporthe toxica. Two major unlinked alleles, Phr1 and PhtjR, confer L. angustifolius resistance to this fungus. Besides the introduction of these alleles into modern cultivars, the molecular mechanisms underlying resistance remained unsolved. In this study, resistant and susceptible lines were subjected to differential gene expression profiling in response to D. toxica inoculation, spanning the progress of the infection from the early to latent phases. High-throughput sequencing of stem transcriptome and PCR quantification of selected genes were performed. Gene Ontology term analysis revealed that an early (24 h) response in the resistant germplasm encompassed activation of genes controlling reactive oxygen species and oxylipin biosynthesis, whereas in the susceptible germplasm, it comprised induction of xyloglucan endotransglucosylases/hydrolases. During the first five days of the infection, the number of genes with significantly altered expressions was about 2.6 times higher in resistant lines than in the susceptible line. Global transcriptome reprogramming involving the activation of defense response genes occurred in lines conferring Phr1 and PhtjR resistance alleles about 4&ndash, 8 days earlier than in the susceptible germplasm.

Over the last 60 years the Australian lupin industry has emerged to become the largest producer in the world, accounting for 85% of global lupin seed market. This progress was achieved by the rapid domestication process of the narrow-leafed lupin (Lupinus angustifolius L.) as a grain legume crop. Narrow-leafed lupin improvement has been based on the identification of donors carrying desirable alleles conferring particular agronomic traits and, subsequently, their orchestrated transfer by classical genetic approaches into domesticated germplasm. These traits include, among others, reduced pod shattering, low alkaloid content, seed water permeability, early flowering, and resistance to diseases caused by pathogenic fungi: anthracnose (Colletotrichum lupini) and Phomopsis stem blight (Diaporthe toxica). Moreover, some of these traits are related with recessive alleles requiring additional breeding effort. To facilitate selection of desirable genotypes in the progenies and cross derivatives, molecular markers linked to particular trait loci were developed and implemented in Australian breeding program.

The hepatotoxin phomopsin A (PHO-A), a secondary metabolite mainly produced by the fungus Diaporthe toxica, occurs predominantly on sweet lupins. Along with the growing interest in sweet lupins for food and feed commodities, concerns have been raised about fungal infestations, and consequently, about the determination of PHO-A. High performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) represents the most suitable analytical technique for sensitive and selective detection of mycotoxins including PHO-A. However, isotopic labeled substances are needed as internal standards for a reliable and convenient quantification. As no isotope standard for PHO-A is currently available, a biosynthesis of fully (15)N6-labeled PHO-A was established by cultivation of D. toxica on defined media containing Na(15)NO3 and (15)N-labeled yeast extract as the only nitrogen sources. The identity of (15)N6-PHO-A was confirmed by high resolution mass spectrometry. The new (15)N6-labeled standard will facilitate the method development for PHO-A including a more accurate quantification by LC-MS/MS.

Phomopsin A (PHO-A), produced by the fungus Diaporthe toxica, is a mycotoxin known to be responsible for fatal liver disease of lupin-fed sheep. The full spectrum of the toxic secondary metabolites produced by D. toxica is still unknown. PHO-A and the naturally occurring derivatives B–E have been subject to several studies to reveal their structures as well as chemical and toxicological properties. In this work, a methylated derivative (1) of PHO-A isolated from lupin seeds inoculated with D. toxica is described. It was characterized by high-resolution mass and NMR data and shown to be the N-methylated derivative of PHO-A. 1 is cytotoxic against HepG2 cells.

L. albus is an annual grain-legume crop mainly grown for high-protein fodder worldwide but also to produce large seeds for human consumption as a snack-food. In order to make genetic gains in grain yield, assessment of the genetic variation in the germplasm and identification of loci associated with agronomic traits are essential. Phomopsis blight (PB) and Pleiochaeta root rot (PRR), caused by the fungal pathogens Diaporthe toxica and, Pleiochaeta setosa respectively, are two major yield-limiting diseases of the L. albus crop. The extent of genetic diversity in 94 accessions of L. albus comprising: Australian and exotic cultivars, advanced breeding lines, and landraces originating from 26 different countries was determined utilizing PCR-based genic, and microarray-based Diversity Arrays Technology (DArT&#8482), markers. All accessions were evaluated for resistance to PB in two plant tissues (leaves and stems) using either sprayed or injected spore inoculum. A subset of 58 accessions was further evaluated for resistance to PRR by growing seedlings in spore-infested potting mix. The combined data of 724 (50 genic- and 674 DArT) markers were used for cluster analysis. A subset of 324 markers with call rate ≥95% and predicted disease scores of different genotypes were used to identify marker loci accounting for phenotypic variation in PB and PRR resistance using linear regression analysis. Several markers showed significant association with PB or PRR resistance at P

Phomopsis blight in Lupinus albus is caused by a fungal pathogen, Diaporthe toxica. It can invade all plant parts, leading to plant material becoming toxic to grazing animals, and potentially resulting in lupinosis. Identifying sources of resistance and breeding for resistance remains the best strategy for controlling Phomopsis and reducing lupinosis risks. However, loci associated with resistance to Phomopsis blight have not yet been identified. In this study, quantitative trait locus (QTL) analysis identified genomic regions associated with resistance to Phomopsis pod blight (PPB) using a linkage map of L. albus constructed previously from an F8 recombinant inbred line population derived from a cross between Kiev-Mutant (susceptible to PPB) and P27174 (resistant to PPB). Phenotyping was undertaken using a detached pod assay. In total, we identified eight QTLs for resistance to PPB on linkage group (LG) 3, LG6, LG10, LG12, LG17 and LG27 from different phenotyping environments. However, at least one QTL, QTL-5 on LG10 was consistently detected in both phenotyping environments and accounted for up to 28.2% of the total phenotypic variance. The results of this study showed that the QTL-2 on LG3 interacts epistatically with QTL-5 and QTL-6, which map on LG10 and LG12, respectively.

Phomopsins (PHO) are mycotoxins produced by the fungus Diaporthe toxica (also referred to as Phomopsis leptostromiformis). Lupin is the most important host crop for this fungus and PHO are suspected as cause of lupinosis, a deadly liver disease, in sheep. Lupin is currently in use to replace genetically modified soy in many food products available on the European market. However, a validated method for analysis of PHO is not available until now. In this work, a dilute-and-shoot LC-MS/MS-based method was developed for the quantitative determination and identification of phomopsin A (PHO-A) in lupin and lupin-containing food. The method involved extraction by a mixture of acetonitrile/water/acetic acid (80/20/1 v/v), dilution of the sample in water, and direct injection of the crude extract after centrifugation. The method was validated at 5 and 25 µg PHO-A kg(-1) product. The average recovery and RSD obtained were 79% and 9%, respectively. The LOQ (the lowest level for which adequate recovery and RSD were demonstrated) was 5 µg PHO-A kg(-1). Identification of PHO-A was based on retention time and two transitions (789 > 226 and 789 > 323). Using the average of solvent standards from the sequence as a reference, retention times were all within ± 0.03 min and ion ratios were within ± 12%, which is compliant with European Union requirements. The LOD (S/N = 3 for the least sensitive transition) was 1 µg PHO-A kg(-1) product. Forty-two samples of lupin and lupin-containing food products were collected in 2011-2012 from grocery stores and internet shops in the Netherlands and analysed. In none of the samples was PHO-A detected.

A detached leaf assay was developed to assess disease response of Lupinus albus to phomopsis leaf blight caused by Diaporthe toxica. A spore concentration of 1 × 107 spores mL−1 using spray inoculation gave the greatest discrimination between genotypes. The effect of leaf position was studied using the first five fully expanded leaves above the cotyledons. Leaf position did not alter the disease response for genotypes with a resistant reaction, but older leaves were more susceptible in less-resistant genotypes. There was good agreement between disease response using detached leaves and leaves inoculated in planta, indicating the utility of the detached leaf assay. In this study, 110 Lupinus albus cultivars were evaluated for resistance to phomopsis leaf blight caused by Diaporthe toxica in two detached leaf experiments. In one experiment, 95 genotypes were assessed for phomopsis leaf blight resistance and phomopsis stem blight resistance after the leaves used in this study were removed. There was...

Historically, in Australia, Phomopsis stem blight in Lupinus albus crops is rare. However, in 2004 an outbreak of this disease occurred in southern New South Wales affecting stems, pods and seeds of the cultivar Kiev-Mutant. This virulent outbreak represents a potential threat to the Australian lupin industry. The current research was therefore initiated to optimise disease screening protocols for evaluation of the disease. Screening for resistance to Phomopsis stem blight is important because stubble is a potential high source of inoculum in no-tillage systems, and grazing of Phomopsis infected stubble can cause lupinosis in stock animals. A single spore isolate of the fungal pathogen Diaporthe toxica collected from the 2004 outbreak was used to assess the levels of disease resistance in stem tissue in L. albus cultivars in some experiments and in other experiments field-infected stubble was used as inoculum. Resistance of current L. albus cultivars, breeding lines, and landraces to D. toxica was assessed in both glasshouse and field experiments in 2007 and 2008. The results showed that resistance existed in some cultivars and several germplasm accessions. Environmental effects and possible differences in pathogen race structure lead to some poor correlations of resistance ratings between different experiments (ranging from r = −0.489 to 0.800, depending on the experiment and tissue assessed). In field experiments consistent expression of the disease was dependent on rainfall. Screening in an irrigated disease nursery improved the methodology. Nevertheless, results suggest that it will be possible to develop new L. albus varieties that are resistant to Phomopsis stem blight should the disease become widespread in south-eastern Australia.

Phenotyping assays in plant pathology using detached plant parts are multi-phase experimental processes. Such assays involve growing plants in field or controlled-environment trials (Phase 1) and then subjecting a sample removed from each plant to disease assessment, usually under laboratory conditions (Phase 2). Each phase may be subject to non-genetic sources of variation. To be able to separate these sources of variation in both phases from genetic sources of variation requires a multi-phase experiment with an appropriate experimental design and statistical analysis. To achieve this, a separate randomization is required for each phase, with additional replication in Phase 2. In this article, Phomopsis leaf and pod blight (caused by Diaporthe toxica) of Lupinus albus was used as a case study to apply a multi-phase experimental approach to identify genetic resistance to this pathogen, and demonstrate the principles of sound experimental design and analysis in detached plant part assays. In seven experiments, 250 breeding lines, cultivars, landraces, and recombinant in-bred lines from a mapping population of L. albus were screened using detached, inoculated leaves, and/or pods. The experimental, non-genetic variance in Phase 2 varied in magnitude compared to the Phase 1 experimental, non-genetic variance. The reliability of prediction for resistance to Phomopsis pod blight was high (mean of 0.70 in seven experiments), while reliability of prediction for leaf assays was lower (mean 0.35–0.51 depending on the scoring method used).

A novel method for the determination of phomopsin A (1) in lupin flour, pea flour, and bean flour as well as whole lupin plants was established based on stable isotope dilution assay (SIDA) LC-MS/MS using (15)N6-1 as an isotopically labeled internal standard. Artificially infected samples were used to develop an optimized extraction procedure and sample pretreatment. The limits of detection were 0.5-1 μg/kg for all matrices. The limits of quantitation were 2-4 μg/kg. The method was used to analyze flour samples generated from selected legume seeds and lupin plant samples that had been inoculated with Diaporthe toxica and two further fungal strains. Finally, growing lupin plants infected with D. toxica were investigated to simulate a naturally in-field mycotoxicosis. Toxin levels of up to 10.1 μg/kg of 1 were found in the pods and 7.2 μg/kg in the stems and leaves.

The genetics of resistance to Phomopsis stem blight caused by Diaporthe toxica Will., Highet, Gams & Sivasith. in narrow-leafed lupin (Lupinus angustifolius L.) was studied in crosses between resistant cv. Merrit, very resistant breeding line 75A:258 and susceptible cv. Unicrop. A non-destructive glasshouse infection test was developed to assess resistance in the F1, F2, selected F2-derived F3 (F2:3) families, and in selfed parent plants. The F1 of Unicrop × 75A:258 (and reciprocal cross) was very resistant, and the F2 segregated in a ratio of 3:1 (resistant: susceptible), which suggested the presence of a single dominant allele for resistance in 75A:258. In Merrit × Unicrop (and reciprocal), the F1 was moderately resistant, and the F2 segregated in a ratio of 3:1 (resistant: susceptible). Thus Merrit appeared to carry an incompletely dominant resistance allele for resistance. The F1 of Merrit × 75A:258 (and reciprocal) was very resistant and the F2 segregated in a ratio of 15:1 (resistant: susceptible), which supported the existence of independently segregating resistance alleles for resistance in 75A:258 and Merrit. Alleles at loci for early flowering (Ku) and speckled seeds (for which we propose the symbol Spk) segregated normally and independently of the resistance alleles. Resistant F2 plants gave rise to uniformly resistant or segregating F2:3 families, whereas susceptible F2 plants gave rise only to susceptible F2:3 families. However, the variation in resistance in the F2 and some F2:3 families of crosses involving 75A:258, from moderately to extremely resistant, was greater than that expected by chance or environmental variation. We propose the symbols Phr1 to describe the dominant resistance allele in 75A:258, and Phr2 for the incompletely dominant resistance allele in Merrit. Phr1 appears to be epistatic to Phr2, and expression of Phr1 may be altered by independently segregating modifier allele(s).

Immunological and biochemical assays were developed for screening for resistance to Diaporthe toxica in individual plants of narrow-leafed lupins (Lupinus angustifolius). The former was an enzyme-linked immunosorbent assay (ELISA) for measuring phomopsin mycotoxins and the latter gave an estimation of glucoseamine in infected stem pieces. Stems of L. angustifolius seedlings were inoculated with conidia from D. toxica cultures and, as expected with this latent disease, remained symptomless for 21 days after inoculation. At this time, phomopsins were measured in excised stems that had been incubated for 6 or 8 days to allow mycelial growth from latent infection structures, thereby increasing the phomopsins to detectable levels in individual plants. The estimation of glucoseamine was carried out on the same stems that had been assayed for phomopsins. The method was based on the alkaline deacetylation of chitin to chitosan, the glucoseamine residues of which are de-aminated with nitrous acid, yielding an aldehyde which is determined colorimetrically. At six days after excision, both tests clearly distinguished the very resistant, resistant, intermediate and susceptible lines and they may be useful in large-scale resistance screening in lupin breeding programmes. The ELISA of phomopsins is easier to use and would be particularly useful in the elimination of susceptible plants and those plants expressing intermediate levels of resistance during early generations of the breeding programme.

In Phomopsis leptostromiformis two varieties, leptostromiformis and occidentalis have been known. The present investigation confirmed that only P. l. var. occidentalis produced its teleomorph in vitro . For this fungus, isolated in Western Australia, the name Diaporthe woodii is recognized. Observations of the fungal development on infected lupin stubble in the field resulted in the discovery of the teleomorph of the Phomopsis state earlier recognized as var. leptostromiformis , which causes the lupinosis problem in grazing animals. The teleomorphs of the two taxa are sufficiently different to regard them as two distinct species. A new species, D. toxica , is described for the teleomorph of the toxigenic Phomopsis .

Phomopsis leptostromiformis, and its teleomorph Diaporthe toxica, is a lupin pathogen that causes stem blight in young lupins and, as a saprophyte, has been detected on dead lupine material. Under favourable conditions, the fungus produces phomopsins (PHOs), a family of macrocyclic hexapeptide mycotoxins capable of binding tubulin through the tripeptide side chain. The toxic effects appear largely confined to the liver. In particular, the ingestion of PHO contaminated lupin stubble has been linked to lupinosis, a debilitating disease of sheep (the most sensitive animal) characterised by disorientation, blindness, lethargy, and eventually death. The chemical structures of PHO A, B and D have been identified. Analytical methods to determine PHOs are mainly enzyme-linked immunosorbent assays or chromatographic separations, in combination with ultraviolet and mass spectrometric detection. The data about the PHOs occurrence are limited to Australia, restricted to lupin seed. Only one survey has been carried out on lupin seeds and flours from the Swiss market. Not many strategies have been developed to limit lupin seed contamination. Efforts devoted to control lupinosis in Australia focused on the development of cultivars resistant to Phomopsis infection. There are few examples in literature of decontamination or detoxification of PHOs; moreover, they have been shown to be resistant to extensive processing, including cooking. Australia and New Zealand are the only countries that included PHOs in their mycotoxin regulations, with a limit of 5 µg/kg in lupin seeds and derived products. Phomopsins are poorly studied mycotoxins and risk assessment on PHOs has not been done at the European level. The collection of all available scientific data was requested by EFSA in a specific project and partners involved considered it of general interest preparing this review to highlight the limited available information, which indicate that the assessment of potential risk related to PHOs is currently not feasible.

An immunoassay for the detection of phomopsin was used to detect levels of the mycotoxin in epidermal peels from resistant and susceptible lupin cultivars asymptomatically infected with Diaporthe toxica. Quantifiable levels (> 6,25μg/kg) of phomopsin were detected in susceptible lupin cultivars, but not in very resistant breeding lines or in the control. These differences reflect the differences in resistance observed in microscopical assays and mature plants in the field.

Phomopsis stem blight (PSB) caused by Diaporthe toxica is a major disease in narrow-leafed lupin ( Lupinus angustifolius L.). The F(2) progeny and the parental plants from a cross between a breeding line 75A:258 (containing a single dominant resistance gene Phr1 against the disease) and a commercial cultivar Unicrop (susceptible to the disease) were used for development of molecular markers linked to the disease resistance gene. Two pairs of co-dominant DNA polymorphisms were detected using the microsatellite-anchored fragment length polymorphism (MFLP) technique. Both pairs of polymorphisms were isolated from the MFLP gels, re-amplified by PCR, sequenced, and converted into co-dominant, sequence-specific and PCR-based markers. Linkage analysis by MAPMAKER suggested that one marker (Ph258M2) was 5.7 centiMorgans (cM) from Phr1, and the other marker (Ph258M1) was 2.1 cM from Ph258M2 but further away from Phr1. These markers are suitable for marker-assisted selection (MAS) in lupin breeding.

The lupin industry in Australia is based largely on Lupinus angustifolius, the narrow-leafed lupin. This species was developed as a new crop in Australia by J S Gladstones in the 1960s from semi-domesticated L. angustifolius introduced from Europe. The genes for low alkaloid content (iuc) and soft seeds (moll) were available in the Swedish forage variety Borre. Spontaneous mutants with white flowers and seeds (leuc) and non-shattering pods (le and ta) were found in the bitter landrace New Zealand Blue. These were recombined with iuc and moll to release the first commercial sweet varieties Uniwhite (1967) and Uniharvest (1969). However, it was not until early flowering (spontaneous mutant Ku) was found in Borre and recombined to produce the variety Unicrop (1973) that the narrow-leafed lupin became a viable crop for the main cropping regions of southern Australia. Breeding since that time has focussed on developing disease resistance, and concurrently yield and quality. Resistance to grey leaf spot (Stemphylium vesicarium) in Marri (1976) and Illyarrie (1979) and resistance to Phomopsis stem blight (Diaporthe toxica) in Gungurru (1988) and Merrit (1991) were major breakthroughs in the development of the crop. The role of wild lupins in these genetic advances was paramount. Genetic advances in yield of narrow-leafed lupins from Unicrop (1973) to Merrit (1991) occurred at an average rate of approximately 2% per year. Breeding and testing of lupins in Australia is funded partly by industry funds through a nationally co-ordinated project. All new varieties must contain less than 0.02% alkaloids on average. In addition to L. angustifolius, L. albus cv. Kiev Mutant has been very successful on more fertile loamy soils. A low alkaloid and early flowering variety of L. luteus, cv. Wodjil, was released in 1997 and is adapted to very acid sands in low rainfall areas. Breeders are also domesticating new lupin species such as L. atlanticus and L. pilosus for more alkaline and loamy soils. The greatest challenge facing breeders at present is the urgent need for anthracnose resistance.

Lupinus albus has been grown commercially in Australia since the 1970s and has long been regarded as resistant to phomopsis caused by Diaporthe toxica. Isolates capable of infecting L. albus were collected following an outbreak of phomopsis in 2004 in southern New South Wales. Glasshouse screening of L. albus cultivars with the pathogen revealed that genotypes differ in their response to D. toxica with some current cultivars having useful resistances. Several breeding lines and old cultivars were susceptible in either vegetative or reproductive tissue or both. The commonly grown commercial cultivar Kiev Mutant was moderately susceptible in all tissues assessed. A principle component analysis gave weak to moderate correlation (r = 0.14 to r = 0.54, depending on the two-way comparison) between vegetative and reproductive tissue, which is suggestive that resistance in different tissues may be under distinct genetic control. The germination of infected seed from the outbreak in 2004 was severely compromised, and much of the seed harboured the pathogen. A survey of 171 commercial seed samples from 2004 to 2006 showed that phomopsis discoloured seed was present in only 20 samples, all of them confined to southern New South Wales. While the pathogen presents a risk to L. albus cropping zones in southern New South Wales, it is not widely spread at this stage. However, vigilance is required to prevent infected seed from being sown so that the disease can be kept in check.

Resistance to Diaporthe toxica in 12 lines of narrow-leafed lupin (Lupinus angustifolius) was assessed by a competitive enzyme-linked immunosorbent assay (ELISA). Seedlings were inoculated with the pathogen and after 21 days resistance was assessed by counting latent infection structures in epidermal tissue under the microscope. Infected stem pieces were then incubated for 6 days in moist chambers before treatment with antisera to D. toxica. There was a high correlation between the ELISA reaction and the frequency of latent infection structures on the 12 lines (r = 0.92, P< 0.001). Visual confirmation of the ELISA result was obtained by further incubating the excised stems for up to 12 days and assessing the degree of fungal development. There was a high correlation between the ELISA result after 6 days incubation and visual ratings of fungal development after 12 days incubation (r = 0.89, P

Resistance to latent infection by Diaporthe toxica was examined in resistant and susceptible cultivars and breeding lines of Lupinus angustifolius (narrow-leafed lupin or blue lupine). Conidial germination (91 ± 1% at 3 days after inoculation) or penetration of the cuticle (14 ± 1% at 3 days and 24 ± 1% at 5 days) by D. toxica were not affected by host resistance. After 5 days, the relative size and number of subcuticular coralloid infection hyphae differed on resistant and susceptible hosts. On susceptible hosts, the majority of coralloid hyphae were large (>100 μm long at 21 days). Resistant hosts had the same density of coralloid hyphae (approximately 300 hyphae per cm 2 ), but the majority were small (

This study examines the effect of calcium (Ca) supply on infection of narrow-leafed lupins (Lupinus angustifolius) by Diaporthe toxica the cause of lupinosis in animals. Susceptible cv. Yandee and resistant breeding lines CE2:435 and 75A:258 were grown in solution culture at varying levels of Ca supply. Epidermal Ca concentration increased with increased Ca supply in both resistant and susceptible narrow-leafed lupins. There was an inverse relationship between the concentration of epidermal Ca and the frequency of infection by the fungus.

Inoculation of a susceptible cultivar of lupin (Lupinus angustifolius cultivar Yandee) with pycnidioconidia of Diaporthe toxica was carried out in the glasshouse to evaluate the effects of simulated dew period, inoculum concentration, temperature of incubation and host tissue maturity on infection using the assessment of the latent infection structures as the assay method. Optimum infection occurred after a dew period of 72 h. Infection was highest at inoculum levels between 1 x l05 and 1 x l07 and at 20�C. One-week-old stem tissues were most susceptible to infection with very few infections evident on 10 week old tissues.

The events leading to latent infection of narrow-leafed lupins (Lupinus angustifolius) by Phomopsis leptostromiformis were examined by light microscopy. One-centimeter stem segments of 28-day-old plants of cultivar Yandee were inoculated with a suspension of 1×10 6 conidia ml -1 and kept under high humidity for 48 hr

Latent infection and tissue colonization by Diaporthe toxica was examined by light, scanning, and transmission electron microscopy in stems, leaves, and petioles of resistant and susceptible narrow-leafed lupins (Lupinus angustifolius). Resistance was observed during the latent phase of the disease as an incompatible reaction between the host and pathogen that appeared to occur after penetration of the cuticle. Conidia were attached firmly to the cuticle by an extracellular substance presumably exuded from the conidia. Conidia penetrated the cuticle directly via an infection peg and formed subcuticular coralloid hyphae. The frequency of subcuticular coralloid hyphae was similar on stems, leaves, and petioles of each line. At 14 days after inoculation, resistant plants had a high frequency of small coralloid hyphae (10-80 µm length). The epidermal cells beneath these small coralloid hyphae appeared necrotic and collapsed with accumulation of polyphenolics and electron-dense substances and a loss of internal organisation in the cytoplasm. Necrosis was occasionally observed in small coralloid hyphae as well. Susceptible plants had a high frequency of large coralloid hyphae (80-400 µm length) in which intrahyphal hyphae were observed, and host epidermal cells beneath large coralloid hyphae appeared normal. Colonization of tissues below the cuticle began immediately after excision of stems from susceptible plants, but was delayed in resistant plants. At 8 days after excision, hyphae had invaded all stem tissues and initiated the formation of pycnidia in susceptible plants, but few hyphae were observed in stems of resistant plants.Key words: Diaporthe toxica, coralloid hyphae, Lupinus angustifolius, resistance.

Two metabolites of P. leptostromijormis (phomopsins A and B) have been isolated as a crystalline mixture from a culture of the fungus on lupin seed. The mixture has been shown to be capable of inducing lupinosis in sheep and in young rats. Key steps in the isolation were the transfer of the phomopsins from crude aqueous solution to tetrahydrofuran and chromatography on macroreticular polystyrene resin. The bioassays used in monitoring fractions were based on inhibition of cell cultures and the mitosis-arresting effect of the metabolites on liver cells in vivo.