Cr Wilson

A polymerase chain reaction assay for cucumber mosaic virus in lupin seeds

Seed is the main source of infection of narrow-leafed lupin (Lupinus angustifolius) crops by cucumber mosaic virus (CMV). The ELISA procedure is currently used for large-scale, routine testing of lupin seed samples, but a more sensitive, reliable and labour-saving assay is needed which detects levels of seed infection as low as 0.1%. A Polymerase Chain Reaction (PCR) using ground dry seed samples was developed for this purpose. Primers based on concensus sequences of eight published CMV coat protein cDNAs (RNA3) of CMV subgroups 1 and 2 were used. The assay involved (1) a reverse transcription step for cDNA synthesis and (2) amplification of a specific fragment (482-501 bp depending on the strain) by PCR. Two methods of extracting virus from infected lupin material were used: (i) a rapid procedure which was effective for samples with higher levels of infection, e.g. infected leaves and 20.5% infected seed; (ii) a phenol-chloroform procedure, which led to greater sensitivity, enabling reliable detection of 0.1% seed infection. It detected CMV in 16 commercial seed samples (0.1-8% seed infection) belonging to seven cultivars from 12 different localities. Both methods were suitable for routine testing of the flour derived from grinding dry seed. On dissection of infected seeds, CMV was detected in the cotyledons and embryo and usually in or on the testa. The PCR assay detected virus from both CMV subgroups, but only subgroup 2 was found in lupin seed samples. The two CMV subgroups can be distinguished by digestion of amplified DNA with the restriction enzyme EcoRI; only CMV strains of subgroup 2 are digested to yield two fragments of size 330 and 170 bp.

Diseases of Pyrethrum in Tasmania: Challenges and Prospects for Management

Pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.) is a perennial plant and member of the Asteraceae that is endemic to the Dalmatian region of the former Yugoslavia (36). Pyrethrum is cultivated commercially solely for the production of six closely related esters called pyrethrins. The plant is tufted, slender, and herbaceous, growing to a height of approximately one meter (18). Leaves are alternate and pinnately lobed/narrowly lanceolate to oblong lanceolate. The daisy-like flowers are produced at the termini of stems and consist of a cluster of 40 to 100 bisexual, yellow disk florets encircled by a ring of 18 to 22 pistillate white ray florets atop a moderately convex to subglobose receptacle (Fig. 1; 100). Disk and ray florets both possess 3 to 10 ribbed achenes located between the floret and receptacle. Involucres generally range between 12 and 18 mm in diameter (17,18). Approximately 94% of the pyrethrins are produced within secretory ducts and oil glands of the achenes of the mature pyrethrum flower, with a minor percentage of oil glands and secretory ducts also found in leaves, stems, and roots (99). Pyrethrins can be separated into two groups of three ester compounds: pyrethrins I and II. The pyrethrin I fraction contains chrysanthemic acid products, including pyrethrin I, cinerin I, and jasmolin I. The pyrethrin II fraction is derived from pyrethric acid and made up of pyrethrin II, cinerin II, and jasmolin II (19,27). Compounds within both fractions contain insecticidal properties used in household and commercial pest control products. These compounds are referred to as “knockdown” and kill agents for many arthropods, yet are of low toxicity to mammals. Pyrethrins also have the advantage over other synthetic insecticides of being rapidly broken down upon exposure to light and air, are metabolized quickly, and can be used in the production of organic farm products. Thus, natural pyrethrins are generally considered to be nonpolluting (19,27). The major areas of pyrethrum production worldwide are located in East Africa (Kenya, Rwanda, and Tanzania), Tasmania (Australia), China, and Papua New Guinea (85,94). Production of pyrethrum in Kenya began in 1928, and despite some fluctuations in annual supply, Kenya is still one of the major suppliers to the world’s market. Pyrethrum cultivation in Kenya is centered in four production areas: the northern and southern Rift Valleys, Mount Kenya, and near Lake Victoria (94). Tasmania is the other major world producer and grows approximately 2,000 hectares. In Tasmania, pyrethrum is grown predominantly along the northwest coast of the island, between Deloraine (41° 31′ S; 146° 39′ E) and Table Cape (40° 56′ S; 145° 43′ E). The cultivation of pyrethrum differs markedly between Tasmania and the other production areas of the world. For example, in 2001 approximately 200,000 growers were involved in pyrethrum production

Spatiotemporal Description of Epidemics Caused by Phoma ligulicola in Tasmanian Pyrethrum Fields

ABSTRACT Spatial and temporal patterns of foliar disease caused by Phoma ligulicola were quantified in naturally occurring epidemics in Tasmanian pyrethrum fields. Disease assessments (defoliation incidence, defoliation severity, incidence of stems with ray blight, and incidence of flowers with ray blight) were performed four times each year in 2002 and 2003. Spatial analyses based on distribution fitting, runs analysis, and spatial analysis by distance indices (SADIE) demonstrated aggregation in fields approaching their first harvest for all assessment times between September and December. In second-year harvest fields, however, the incidence of stems with ray blight was random for the first and last samplings, but aggregated between these times. Spatiotemporal analyses were conducted between the same disease intensity measures at subsequent assessment times with the association function of SADIE. In first-year harvest fields, the presence of steep spatial gradients was suggested, most likely from dispersal of conidia from foci within the field. The importance of exogenous inoculum sources, such as wind-dispersed ascospores, was suggested by the absence of significant association between defoliation intensity (incidence and severity) and incidence of stems with ray blight in second-year harvest fields. The logistic model provided the best temporal fit to the increase in defoliation severity in each of six first-year harvest fields in 2003. The logistic model also provided the best fit for the incidence of stems with ray blight and the incidence of flowers with ray blight in four of six and three of six fields, respectively, whereas the Gompertz model provided the best fit in the remaining fields. Fungicides applied prior to mid-October (early spring) significantly reduced the area under disease progress curve (P < 0.001) for defoliation severity, the incidence of stems with ray blight, and the incidence of flowers with ray blight for epidemics at all field locations. This study provides information concerning the epidemiology of foliar disease and ray blight epidemics in pyrethrum and offers insight on how to best manage these diseases.

Dietary Microbial Toxins and Type 1 Diabetes

Abstract: Toxins may promote type 1 diabetes by modifying or damaging the β cell causing release of autoantigens. Streptomyces is a common soil bacterium that produces many toxic compounds. Some Streptomyces can infect vegetables, raising the possibility of dietary exposure to toxins. We aimed to identify toxins that erode cellular proton gradients in extracts of Streptomyces and infested vegetables and to establish the effect of low doses of these toxins on pancreatic islets in mice. The vacuolar ATPase inhibitors, bafilomycin and concanamycin, and the ionophore, nigericin, were identified in extracts from 4 of 13 Streptomyces isolated from infested potatoes and in potatoes themselves. Injection of bafilomycin A1 into mice impaired glucose tolerance, reduced islet size, and decreased relative β cell mass. Thus, exposure to small quantities of bafilomycin in the diet may contribute to the cause of type 1 diabetes.

Stable and Extreme Resistance to Common Scab of Potato Obtained Through Somatic Cell Selection

Somatic cell selection with thaxtomin A as a positive selection agent was used to isolate variants of potato cv. Russet Burbank with strong to extreme resistance to common scab. Glasshouse and field trials identified 51 variants with significantly reduced disease incidence (frequency of infected tubers) and severity (tuber lesion coverage) compared with the parent cultivar. The most promising variants exhibited extreme disease resistance, rarely showing lesions, which were invariably superficial and shallower than those on the parent. Resistance traits were consistently expressed both in 10 glasshouse and two field trials at different locations, with varied inoculum and disease pressure. Disease-resistant variants differed in their response to thaxtomin A in tuber slice bioassays. Of 23 variants tested, 10 showed reduced thaxtomin A susceptibility, with the remaining 13 responding similar to that of the parent. Thus, toxin tolerance was not the only factor responsible for observed disease resistance; however, four of the five most disease-resistant variants had enhanced thaxtomin A tolerance, suggesting that this factor is important in the expression of strong disease resistance. Pathogenicity and toxin tolerance remained stable over a 6-year period, demonstrating that selected phenotypes were robust and genetic changes stable. The majority of disease-resistant variants had tuber yields equivalent to the parent cultivar in glasshouse trials. This suggests that selection for disease resistance was not associated with negative tuber attributes and that certain variants may have commercial merit, worthy of further agronomic testing.

Development of a Fungicide-Based Management Strategy for Foliar Disease Caused by Phoma ligulicola in Tasmanian Pyrethrum Fields

The efficacy of a range of fungicides were assessed under in vitro conditions in four field trials over three seasons for the management of ray blight of pyrethrum, caused by the fungus Phoma ligulicola in Tasmania. Fungicides belonging to the demethylation inhibitor group, including prochloraz, tebuconazole, difenoconazole, and cyproconazole, produced the most significant reduction in mycelial growth in vitro compared with unamended controls. Significant reductions in germination of conidia resulted from low concentrations of azoxystrobin. In the field, azox-ystrobin (as Amistar) at a rate of 300 g of product/ha significantly decreased disease intensity and increased pyrethrin yield (kg/ha) in all seasons. Applications of either azoxystrobin or difenoconazole (as Score) at a rate of 250 ml of product/ha in early spring were the most effective, whereas no significant benefit was demonstrated from applications in autumn. Over all seasons, azoxystrobin applications resulted in double the weight of flowers produced compared with nontreated areas, whereas increases in pyrethrin content within the flowers were season and location specific. Application of azoxystrobin and chlorothalonil (as Bravo 720) at a rate of 1.4 liters of product/ha resulted in a 45 to 48% increase in flowers, reduced defoliation, and increased stem diameter. The weight of individual flowers and flower maturity was not significantly affected by fungicide application. These data provide the basis for cost-effective management of this disease.

Confirmation of ray blight disease of pyrethrum in Australia.

Isolations from affected plant parts of pyrethrum exhibiting flower bud abortion and necrotic symptoms consistently yielded a Phoma sp., identified as the anamorph of Didpella ligulicola, the causal agent of ray blight of chrysanthemum. Pathogenicity of both mycelial and conidial inoculum of the isolated fungus to pyrethrum was proven. Infection of stem pieces occurred by direct penetration of the fungal hyphae with invasion being both inter- and intracellular. Infection was associated with red pigmentation and cytoplasmic crystalline inclusions of host tissues. This is the first confirmed record of this disease of pyrethrum in Australia.

Auxin-Induced Resistance to Common Scab Disease of Potato Linked to Inhibition of Thaxtomin A Toxicity

Production of the phytotoxin thaxtomin A by pathogenic Streptomyces spp. is essential for induction of common scab disease in potato. Prior studies have shown that foliar application of sublethal concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and other auxin or auxin-like compounds significantly reduced severity and occurrence of common scab in subsequently produced tubers. However, the means of disease suppression by these compounds was not known. We confirm the disease suppressive activity of 2,4-D. Detailed tuber physiological examination showed that lenticel numbers, lenticel external dimensions, and periderm thickness and structure, physiological features believed to be critical to Streptomyces scabiei infection, were not substantially changed by 2,4-D treatments, negating a possible mechanism for disease suppression through alteration of these structures. In contrast, our studies show accumulation of 2,4-D in tubers of treated plants occurs and is associated with an enhanced tolerance to thaxtomin A. Applying 2,4-D to cultures of S. scabiei did not significantly alter in vitro growth of the pathogen. Thaxtomin A production by the pathogen was inhibited by 2,4-D, but only at the highest rate tested (1.0 mM), which is at least 200-fold more than is found in 2,4-D treated tubers. These data suggest 2,4-D has no direct effect on the pathogen or its virulence. Confirmatory evidence from studies with Arabidopsis thaliana seedlings demonstrated that the auxins 2,4-D and IAA ameliorate thaxtomin A toxicity. The evidence presented whereby auxin treatment inhibits toxicity of thaxtomin A secreted by the pathogen suggests a novel indirect means of disease suppression.

Iris yellow spot virus found infecting onions in three Australian states

Iris yellow spot virus (IYSV) was detected for the first time in Australia, infecting onions in three and leeks in one state. Identification was confirmed using sap transmission to Nicotiana benthamiana, two IYSV-specific antisera in ELISA, RT-PCR with IYSV-specific primers, and sequence comparison with published IYSV sequences. Spring onion, onion seed and onion bulb crops were all infected, with spring onion being the most severely affected. The virus was also detected in nursery-grown onion and leek seedlings.

Pathogenicity of fungi commonly isolated from foliar disease in Tasmanian pyrethrum crops

The pathogenicity of Alternaria alternata, A. tenuissima, Stemphylium botryosum, Cladosporium cladosporioides and Ulocladium atrum commonly associated with diseased foliage of pyrethrum (Tanacetum cineariifolium) was characterised by inoculating glasshouse-grown pyrethrum plants in three replicated experiments. In the first experiment only one cultivar was used, whilst in the remaining two experiments the susceptibility of cultivars was evaluated. Inoculation with A. alternata, C. cladosporioides and U. atrum failed to induce disease in any cultivar. Inoculation with A. tenuissima resulted in the development of necrotic spots which coalesced to cover large areas of the leaves in all cultivars, whereas foliage of those inoculated with S. botryosum developed necrotic halos surrounded by a pink/brown margin. No significant differences were detected in disease severity following inoculation with A. tenuissima or S. botryosum between the four cultivars commonly grown in Tasmania. However, some significant differences were noted between cultivars in the number of lesions per leaf following inoculation with S. botryosum.