Temporal and Spatial Development of Cercospora Leaf Spot of Faba Bean Influenced by In Situ Inoculum.

The temporal and spatial dynamics of Cercospora leaf spot on susceptible and resistant lines of faba bean grown in or at defined distances from soil with residues infested by Cercospora zonata were examined in South Australia in 2005 and 2006. The disease was first observed on susceptible seedlings 49 days after sowing (DAS) in soil that had been sown with faba bean every 3 years since 1997 (positive soil zone for C. zonata) but was delayed by 1 week in adjacent soil (0 to 16 m away) with no history of cultivation of faba bean (negative soil zone). The incidence of diseased seedlings from 49 to 63 DAS showed a gradient from 4 to 16 m from the infested soil and was significantly greater for susceptible plants grown in the positive versus negative soil zones in field trials conducted in 2005 and 2006 (92 versus 30% in 2005, χ21 = 32.2, P < 0.001; 98 versus 55% in 2006, χ21 = 12.1, P < 0.001). The incidence of Cercospora leaf spot on the resistant line 1322/2 was significantly less (χ26 = 171.7; P < 0.001) than on the susceptible line 'Farah' at that time in both years, with fewer than 5% of the seedlings showing the disease. However, a gradient was shown at 70 to 84 DAS, where disease incidence was significantly greater on line 1322/2 in the positive soil zone than on plants in the negative soil zone in both years (62 and 18%, respectively, with χ21 = 27.9, P < 0.001 in the 2005 trial; and 47 and 6%, respectively, with χ21 = 33.3, P < 0.001 in the 2006 trial). At peak disease severity on Farah, Cercospora leaf spot mean leaf area diseased (%LAD) was severe (85 ± 4.3%) on leaves of the three nodes closest to the soil surface, and much less severe (1 ± 0.6%) in the upper canopy. Defoliation combined with %LAD was used to describe the loss of photosynthetic leaf area (%LPLA) in both cultivars, on both soil zones, in each year. Nonlinear regression analyses using a logistic model described disease development over time on susceptible plants grown in infested soil (e.g., for +12-m blocks within infested soil, y = 2.66 + 46.08/[1 + exp(-0.23 × [X - 40.92])] in 2005 and y = 0.49 + 5.02/[1 + exp(-0.14 × [X - 28.30])] in 2006, where X = DAS and y = %LPLA, with both regressions significant at P < 0.001), whereas an exponential model (e.g., for -12-m blocks from infested soil, y = 0.23 + 0.77 × 1.04X in 2005 and y = 0.44 + 0.56 × 1.04X in 2006, both at P < 0.001) best described disease gradients with increasing distance from the inoculum source. Paired t tests of %LPLA at 77 and 98 DAS showed significant differences in disease severity in the positive versus negative soil zones and a steep gradient in %LPLA from 0 to 4 m from the inoculum source. The role of infested faba bean residue in survival of C. zonata over time was also examined using a pot-bioassay and in situ field assay. When residues were removed from the soil surface or depleted rapidly by animal grazing, the amount of C. zonata inoculum in the soil was significantly less (P < 0.001) than for soil with residue remaining on the soil surface. C. zonata survived in soil and remained infective for at least 30 months after harvest of an infected faba bean crop.

Comparison of cultural growth and in planta quantification of Didymella pinodes, Phoma koolunga and Phoma medicaginis var. pinodella, causal agents of ascochyta blight on field pea (Pisum sativum).

The causal agents of ascochyta blight on field pea in South Australia, Didymella pinodes, Phoma medicaginis var. pinodella and Phoma koolunga, are isolated from a single plant within a crop, suggesting competition for space and nutrients. Interactions among these pathogens were investigated. Diameters of colonies of D. pinodes and of P. medicaginis var. pinodella were significantly reduced on PDA amended with filtrate from broth cultures of P. koolunga as were diameters of colonies of D. pinodes on PDA amended with filtrate from P. medicaginis var. pinodella or D. pinodes. This effect was negated when cultures were transferred to unamended PDA, indicating filtrates were fungistatic instead of fungicidal. The diameter of P. koolunga colonies was not influenced by filtrate from any of the three species. When pathogens were co-inoculated in pairs onto leaves on field pea plants, the quantity of DNA of D. pinodes and of P. medicaginis var. pinodella was significantly reduced if co-inoculated with P. koolunga. The quantity of DNA of P. koolunga was not influenced by co-inoculation. When co-inoculated onto excised leaf disks on sterile water the mean lesion diameter due to D. pinodes and to P. medicaginis var. pinodella was significantly reduced if co-inoculated with P. koolunga isolate DAR78535. Lesions caused by D. pinodes were significantly reduced when inoculum was self-paired. Conversely the diameter of lesions caused by P. koolunga DAR78535 increased when self-paired or when co-inoculated with P. medicaginis var. pinodella. Unlike leaf disks on sterile water, co-inoculation had no influence on lesion size or quantity of pathogen DNA in leaf disks on water agar. Antagonism, including self-antagonism, was detected among these species, leading to reduction in lesion size and quantity of pathogen DNA. The slower growing species, P. koolunga, was not self-antagonistic, and in a few instances the effect of co-inoculation was additive or synergistic.

Distribution and Survival of Ascochyta Blight Pathogens in Field-Pea-Cropping Soils of Australia.

Phoma koolunga, Didymella pinodes, and P. medicaginis var. pinodella were detected in DNA extracted from soil following field pea crops across four states in the southeastern and western regions of Australia. P. koolunga was commonly detected in soil from South Australia but rarely in other states whereas D. pinodes plus P. medicaginis var. pinodella were widespread in all regions tested. The quantity of DNA of these pathogens detected in soils prior to growing field pea was positively correlated with ascochyta blight lesions on field pea subsequently grown in infested soil in a pot bioassay and also on field pea in naturally infected field trials. The quantity of DNA of the soilborne pathogens was greatest following a field pea crop and gradually decreased in the following 3 years. The DNA tests were used to quantify the DNA of the pathogens in field pea plants sampled from naturally infected field trials in South Australia over two seasons. The combined results of DNA tests and pathogen isolation from the plants indicated that P. koolunga and D. pinodes were equally responsible for the ascochyta blight symptoms in the diseased trials, while P. medicaginis var. pinodella had a minor role in the disease complex.

A new species of Phoma causes ascochyta blight symptoms on field peas (Pisum sativum) in South Australia.

Phoma koolunga sp. nov. is described, having been isolated from ascochyta blight lesions on field pea (Pisum sativum) in South Australia. The species is described morphologically and sequences of the internal transcribed spacer region compared with those of the accepted pathogens causing ascochyta blight of field peas. P. koolunga was distinct from Mycosphaerella pinodes (anamorph: Ascochyta pinodes), Phoma medicaginis var. pinodella and Ascochyta pisi. Under controlled conditions the symptoms on pea seedlings caused by P. koolunga were indistinguishable from those caused by M. pinodes, other than a 24 h delay in disease development. Isolates of P. koolunga differed in the severity of disease caused on pea seedlings.

Dynamics of introduced populations of Phragmidium violaceum and implications for biological control of European blackberry in Australia.

Phragmidium violaceum causes leaf rust on the European blackberry (Rubus fruticosus L. aggregate). Multiple strains of this pathogen have been introduced into southern Australia for the biological control of at least 15 taxa of European blackberry, a nonindigenous, invasive plant. In climates conducive to leaf rust, the intensity of disease varies within and among infestations of the genetically variable host. Genetic markers developed from the selective amplification of microsatellite polymorphic loci were used to assess the population genetic structure and reproductive biology of P. violaceum within and among four geographically isolated and diseased infestations of the European blackberry in Victoria, Australia. Despite the potential for long-distance aerial dispersal of urediniospores, there was significant genetic differentiation among all populations, which was not associated with geographic separation. An assessment of multilocus linkage disequilibrium revealed temporal and geographic variation in the occurrence of random mating among the four populations. The presence of sexual spore states and the results of genetic analyses indicated that recombination, and potentially random migration and genetic drift, played an important role in maintaining genotypic variation within populations. Recombination and genetic differentiation in P. violaceum, as well as the potential for metapopulation structure, suggest the need to release additional, genetically diverse strains of the biocontrol agent at numerous sites across the distribution of the Australian blackberry infestation for maximum establishment and persistence.

The influence of climate on foliar symptoms of eutypa dieback in grapevines.

ABSTRACT Foliar symptoms of Eutypa dieback, caused by Eutypa lata, in grapevines, cv. Shiraz, varied from year to year in a 6-year study conducted in South Australia and, although trends were similar for vineyards within geographical regions, differences were observed between regions. We attempted to elucidate the causes underlying this variation and hypothesized that it was influenced by climatic factors. A number of possible relationships were identified between climate and symptom expression: (i) increased symptom expression was related to increased winter rainfall 18 months earlier, (ii) decreased disease incidence and prevalence were related to increased temperature in spring, and (iii) a reduction in disease incidence was related to both very high and very low rainfall in October. Theories for these relationships are proposed and require further investigation. A conceptual model was developed which requires validation and has the potential to predict the incidence of foliar symptoms of Eutypa dieback. Information from this study could lead to an improved integrated pest management system to suppress foliar symptoms and sustain productivity of vines infected with E. lata.

The Influence of Grapevine Cultivar and Isolate of Eutypa lata on Wood and Foliar Symptoms.

Grapevine cultivar (Vitis vinifera) and isolate of Eutypa lata influence wood and foliar symptoms of Eutypa dieback. Foliar symptoms of Eutypa dieback developed within 8 months of inoculating young grapevines (cvs. Grenache, Cabernet Sauvignon, and Merlot) in a shadehouse. Isolates of E. lata from various wine regions in southern Australia varied in their ability to colonize inoculated grapevines and induce wood and foliar symptoms. Grapevine cultivars varied for wood and foliar symptom expression but not for mycelial colonization. However, the severity of foliar symptoms was not related to the rate of spread of the fungus in the grapevine. Furthermore, the staining of wood typically attributed to E. lata did not reflect the presence of the fungus because the fungus was detected up to 80 mm beyond the stain. A field trial with mature grapevines revealed significant differences in the rate of spread of wood staining due to E. lata among eight cultivars, with up to 50 mm/year detected in Cabernet Sauvignon and Shiraz grapevines. In the shadehouse, the maximum growth rate of E. lata was recorded to be 115 mm/year for Grenache rootlings. Information from this study may help to optimize management strategies for maintaining productivity of grapevines with Eutypa dieback, thus reducing the economic impact of the disease.

Sensor-augmented CSII therapy with predictive low-glucose suspend following total pancreatectomy.

Pancreatogenic diabetes is characterised by recurrent severe hypoglycaemia due to changes in both endocrine and exocrine functions. There are no guidelines to manage these individuals. Herein, we describe the post-operative management of two people who developed pancreatogenic diabetes following total pancreatectomy for neuroendocrine malignancy. In both individuals, diabetes was managed using sensor-augmented predictive low-glucose suspend continuous subcutaneous insulin infusion (CSII). We demonstrate the benefit of sensor-augmented CSII in averting hypoglycaemia whilst optimising glycaemic control. Expected rates of severe hypoglycaemia in individuals with pancreatogenic diabetes can be averted with the use of continuous glucose monitoring (CGM) technology, optimising quality of life and reducing the risk of diabetes-related complications. LEARNING POINTS: There are no clear guidelines to manage people with pancreatogenic diabetes.We describe the use of CGM with predictive low-glucose suspend continuous subcutaneous insulin infusion (CSII) in the management of two individuals post-pancreatectomy.Predictive low-glucose suspend technology can achieve excellent glycaemic control whilst avoiding recurrent and severe hypoglycaemia in people with pancreatogenic diabetes.Predictive low-glucose suspend CGM should be considered as an effective therapeutic option for the management of pancreatogenic diabetes.

Outcomes of twin pregnancies complicated by gestational diabetes: a meta-analysis of observational studies.

OBJECTIVE: Gestational diabetes mellitus (GDM) in singleton pregnancy is associated with large for gestational age neonates and adverse perinatal outcomes; however, the impact of GDM in twin pregnancy is unclear. Thus, the aim of this study is to assess the perinatal outcomes of twin pregnancies complicated by GDM by performing a meta-analysis of observational studies. STUDY DESIGN: Studies investigating GDM in twin pregnancy were identified through an online search of three databases: Medline, Embase and Web of Science. Selection criteria comprised full paper observational studies (retrospective or prospective) published in English that examined GDM in twin pregnancy compared with non-GDM twin pregnancy and reported on birth weight and/or adverse perinatal outcomes. Random-effects models with inverse-variance weighting were used to calculate standardized mean differences and unadjusted odds ratios. Sensitivity analyses were carried out to determine the impact of possible maternal confounders (body mass index and age) and GDM diagnostic criteria on perinatal outcomes. RESULTS: Thirteen observational studies were included. GDM twins were born at the same gestation as non-GDM twins, with marginally lower birth weight. There was no difference in the incidence of large or small for gestational age neonates. Although there was no correlation between GDM in twin pregnancy and respiratory distress, neonatal hypoglycemic or low Apgar score, GDM twins had a higher rate of neonatal intensive care unit admission (OR 1.49; 95% confidence interval: 1.10, 2.02; P<0.01). CONCLUSION: Identification and subsequent treatment of GDM in twin pregnancy demonstrates a similar risk of adverse perinatal outcomes compared with non-GDM twin pregnancies.

Australian research on ochratoxigenic fungi and ochratoxin A.

The presence of the mycotoxin, ochratoxin A (OTA), has been reported in Australian grape products. Comprehensive surveys of Australian wines have determined that the frequency and level of OTA contamination are low. Aspergillus carbonarius is the primary OTA-producing species associated with grapes in Australia, and all isolates tested to date produce OTA. Aspergillus niger is isolated more frequently from vineyards, however, few strains produce OTA. A. carbonarius and A. niger exist as saprophytes in the top layer of soil beneath vines, from where they are thought to be blown onto bunches. The level of A. carbonarius in soil may be reduced by temperatures above or below the optimum temperature for survival (25 degrees C), by high soil moisture content, and by modifications to tillage and mulching practices. A. carbonarius is an opportunistic pathogen of damaged berries. In the absence of damage, spores may exist on berry surfaces without causing visible rots. Aspergillus rots are associated with black Aspergillus species, primarily A. niger, A. carbonarius and A. aculeatus. The potential for such rots is increased with berry damage, inoculum coverage and berry maturity. Susceptibility to berry splitting is related, in part, to bunch structure, and may be variety-dependent or influenced by rainfall, irrigation and canopy management. Black Aspergillus spp. are closely associated with berries near the main stem of the bunch. During winemaking, around 80% of the OTA initially present in grapes is removed, primarily with the skins and pulp during pressing. Additional reductions occur with the removal of precipitated grape and yeast solids. Bentonite in white wine and yeast hulls in red wine were the most effective non-carbonaceous fining agents for the removal of OTA.

Absence of Oospores of Downy Mildew of Grape Caused by Plasmopara viticola as the Source of Primary Inoculum in Most Western Australian Vineyards.

Grapevine downy mildew, caused by the obligate, oomycete pathogen, Plasmopara viticola, was first recorded in Western Australia (W.A.) in 1998 (2) and has subsequently been observed in most viticultural regions of the state. Heterothallism in P. viticola was established by Wong et al. (3), whereby more than one mating type of the pathogen is required for sexual reproduction to occur. Oospores are considered to be the source of primary inoculum for this disease with further, secondary infection being advanced by asexual inoculum. However, recent research in European vineyards suggests that the majority of infection throughout the growing season arises via sexually derived (oosporic) inoculum (1). Since downy mildew is relatively new to W.A., few surveys have been conducted to study populations of the pathogen within the state. It is also noteworthy that the incidence of oospores in Australian vineyards has not been reported. The objective of this research was to assess the occurrence and type of inoculum of P. viticola in W.A. vineyards. A total of 1,266 P. viticola-infected leaf discs (LD) from eight wine-grape (775 LD), five table-grape (450 LD), and seven unknown (41 LD) cultivars grown in 16 vineyards in 10 geographically separate regions of W.A. were collected in the growing seasons of 2001-2003. These regions range from Chittering in the north to Albany in the south and received 700 to 1,200 mm annual rainfall, mostly in winter. Each LD was cleared in 1 M KOH at 60°C for 12 to 24 h and then was assessed for the presence of oospores with light microscopy. Leaves showing "mosaic"-type lesions (older infection) late in the season were collected where possible to ensure colony maturity and an increased likelihood of oospore formation. All LD from all regions were negative for the presence of oospores except for samples from a single vineyard (approximately 1,200 mm annual rainfall), where all 140 LD from six wine-grape cultivars contained oospores. The discovery that oospores were present in only one of 16 sampled vineyards provides a rare opportunity to study gene flow in field populations of the pathogen with time and to determine sources of primary inoculum where overwintering of P. viticola may not involve oospores. References: (1) S. McKirdy et al. Plant Dis. 83:301, 1999. (2) A. Rumbou et al. Eur. J. Plant Pathol. 110:379, 2004. (3) F. P. Wong et al. Plant Pathol. 50:427, 2001.

The effect of fungicides on vesicular-arbuscular mycorrhizal symbiosis: I. The effects on vesicular-arbuscular mycorrhizal fungi and plant growth.

This paper describes the effects of the fungicides Benlate, Aliette and Ridomil on plant growth and on mycorrhizal development in onion plants. An attempt was made to distinguish effects on plants from those on the fungus by making comparisons between mycorrhizal plants in the absence of added phosphorus and non-mycorrhizal equivalent size plants. Vital staining techniques were used to analyse the effects of the fungicides on the living fungus both within the root and in the soil. Benlate had no effect on shoot dry weight or root length of onion plants whereas a reduction in plant growth was observed following the application of Aliette or Ridomil, in comparison to control plants. Benlate had negative effects on the numbers of living internal hyphae, arbuscules, fungal-plant interface and living external hyphae in the soil. Aliette had no effect on the number of living intercellular hyphae and arbuscules. However, it markedly reduced the root length and the length of infected root per plant. These combined effects led to a reduction in the area of the interface between fungus and plant. The length of external hyphae per gram of soil was reduced following the application of Aliette, though not as severely as with Benlate. Ridomil had more complex effects on vesicular-arbuscular mycorrhizal symbiosis; treated plants showed a reduction in plant growth and also a reduction in all fungal parameters, namely the number of living intercellular hyphae, arbuscules and fungal-plant interface, the length of infected root and the development of external hyphae in the soil.

The effect of fungicides on vesicular-arbuscular mycorrhizal symbiosis: II. The effects on area of interface and efficiency of P uptake and transfer to plant.

Two experiments were conducted under controlled environmental conditions to determine the effects of the three fungicides, Benlate®. Aliette® and Ridomil®, on efficiency of P uptake from the soil and transfer across the living plant-fungal interface- of onion plants (Allium cepa L.) associated with Glomus sp. 'City Beach' (WUM 16), P applied to the soil did not apparently increase the rate of transfer (flux) of P to the plant via the fungal partner of the mytorrhiza. Benlate reduced P inflow and transfer across the interface in one of the experiments. The rate of P uptake per m living external hyphae was not affected but, as development of living external hyphae in the soil was reduced, the contribution of the fungus to P uptake was small. Aliette reduced growth of both shoots and roots, but apparently increased the accumulation of P in the tissues compared with controls. Ridomil reduced P inflow per m of root and P uptake per m living external hyphae, hut had no effect on the rate of P transfer across the interface. This led to a reduction in the overall contribution of the fungus to P nutrition.

Diaporthe perjuncta Does Not Cause Phomopsis Cane and Leaf Spot Disease of Grapevine in Australia.

In Australia, Diaporthe perjuncta (formerly known as Phomopsis taxon 1) and Phomopsis viticola (Phomopsis taxon 2) have been associated with Phomopsis cane and leaf spot of grapevine. Although P. viticola causes distinct leaf spots, as well as lesions on shoots and canes, the pathogenicity of D. perjuncta is relatively unknown. The pathogenicity of D. perjuncta and P. viticola was studied in relation to symptom expression and bud loss. Only P. viticola caused brown-black, longitudinal, necrotic lesions on stem tissue and leaf spots characteristic of the disease, whereas both D. perjuncta and P. viticola induced bleaching of dormant canes. Inoculation of dormant buds with D. perjuncta did not cause bud death. D. perjuncta and P. viticola were reisolated from inoculated tissue and into pure culture. D. perjuncta colonized the epidermis and cortex of the grapevine shoot but not the vascular tissue. D. perjuncta appears to be an endophyte, rather than a pathogen of grapevine.

Aspergillus species producing ochratoxin A: isolation from vineyard soils and infection of Semillon bunches in Australia.

AIMS: The incidence of toxigenicity among Australian isolates of Aspergillus niger and Aspergillus carbonarius was assessed. Aspergillus rot and concomitant production of ochratoxin A (OA) in bunches inoculated with A. carbonarius were also investigated. METHODS AND RESULTS: Aspergillus niger and A. carbonarius were isolated from vineyard soils. Aspergillus niger was more widespread than A. carbonarius, and two restriction fragment length polymorphism types of A. niger, N and T, were present. Three of 113 A. niger isolates and all 33 A. carbonarius isolates produced OA. Aspergillus carbonarius was inoculated onto Semillon bunches with and without damage in the month before harvest. Damaged berries at greater than 12.3 (o) Bx were particularly susceptible to Aspergillus rot and production of OA, which was concentrated in severely mouldy berries. CONCLUSIONS: OA in Australian grapes results mainly from infection of berries by A. carbonarius. It is concentrated in discoloured, shrivelled berries. The potential for Aspergillus rot and OA production appears to commence after veraison and increase with berry damage and ripeness. SIGNIFICANCE AND IMPACT OF THE STUDY: Minimizing damage to grapes between veraison and harvest significantly reduces Aspergillus rot and OA formation. Monitoring the extent of Aspergillus rot in bunches infected with toxigenic Aspergillus spp. may give some indication of OA contamination.

Intraspecific variation of Rhizoctonia solani AG 3 isolates recovered from potato fields in Central Iran and South Australia.

Pectic zymogram, RFLP and PCR analyses were used to characterize Rhizoctonia solani AG 3 isolates collected from diseased potatoes in South Australia. The pectic zymogram data were compared with those obtained for isolates collected from central Iran. Analyses of bands corresponding to pectin esterase and polygalacturonase revealed three zymogram subgroups (ZG) in AG 3. In addition to the previously reported ZG7 (here renamed ZG7-1), two new zymogram subgroups, ZG7-2 and ZG7-3, were identified. Of the 446 isolates tested, 50% of the South Australian and 46% of the Iranian isolates were ZG7-1. The majority of the isolates originating from stem and root cankers were ZG7-1, whereas most of the isolates designated ZG7-2 and ZG7-3 originated from tuber-borne sclerotia. Pathogenicity tests revealed that ZG7-1 generally produced fewer sclerotia and more severe cankers of underground parts of the potato plants than the other two ZGs. Two random DNA clones, one originating from an AG 3 isolate and the other from an AG 4 isolate, were used as probes for RFLP analyses of Australian isolates. The AG 3 probe, previously identified to be specific to this group, detected a high level of genetic diversity, with 11 genotypes identified amongst 50 isolates analysed. The low-copy AG 4 probe resolved three genotypes amongst 24 isolates. For 23 isolates analysed with both markers, the combined data distinguished a total of six genotypes and similarity analysis resolved the isolates into two main groups with 50% homology. PCR, using primers for the plant intron splice junction region (R1), also revealed variation. No obvious relationship among pectic zymogram groups, RFLP and PCR genotypes was observed.

Ochratoxin A-producing Aspergilli in Vietnamese green coffee beans.

AIMS: To determine the incidence and severity of infection by ochratoxin A (OA)-producing fungi in Vietnamese green coffee beans. METHODS AND RESULTS: Aspergillus carbonarius, A. niger and yellow Aspergilli (A. ochraceus and related species in section Circumdati) were isolated by direct plating of surface-disinfected Robusta (65 samples) and Arabica (11 samples) coffee beans from southern and central Vietnam. Significantly, more Robusta than Arabica beans were infected by fungi. Aspergillus niger infected 89% of Robusta beans, whereas A. carbonarius and yellow Aspergilli each infected 12-14% of beans. OA was not produced by A. niger (98 isolates) or A. ochraceus (77 isolates), but was detected in 110 of 113 isolates of A. carbonarius, 10 isolates of A. westerdijkiae and one isolate of A. steynii. The maximum OA observed in samples severely infected with toxigenic species was 1.8 microg kg(-1); however, no relationship between extent of infection and OA contamination was observed. CONCLUSIONS: Aspergillus niger is the dominant species infecting Vietnamese coffee beans, yet A. carbonarius is the likely source of OA contamination. SIGNIFICANCE AND IMPACT OF STUDY: Vietnamese green coffee beans were more severely infected with fungi than the levels reported for beans from other parts of the world, yet OA contamination appears to be infrequent.