Comparative analyses of cadmium and zinc uptake correlated with changes in natural resistance-associated macrophage protein (NRAMP) expression in Solanum nigrum L. and Brassica rapa

Environmental context Soils contaminated with metals can pose both environmental and human health risks. This study showed that a common crop vegetable grown in the presence of cadmium and zinc readily accumulated these metals, and thus could be a source of toxicity when eaten. The work highlights potential health risks from consuming crops grown on contaminated soils. Abstract Ingestion of plants grown in heavy metal contaminated soils can cause toxicity because of metal accumulation. We compared Cd and Zn levels in Brassica rapa, a widely grown crop vegetable, with that of the hyperaccumulator Solanum nigrum L. Solanum nigrum contained 4 times more Zn and 12 times more Cd than B. rapa, relative to dry mass. In S. nigrum Cd and Zn preferentially accumulated in the roots whereas in B. rapa Cd and Zn were concentrated more in the shoots than in the roots. The different distribution of Cd and Zn in B. rapa and S. nigrum suggests the presence of distinct metal uptake mechanisms. We correlated plant metal content with the expression of a conserved putative natural resistance-associated macrophage protein (NRAMP) metal transporter in both plants. Treatment of both plants with either Cd or Zn increased expression of the NRAMP, with expression levels being higher in the roots than in the shoots. These findings provide insights into the molecular mechanisms of heavy metal processing by S. nigrum L. and the crop vegetable B. rapa that could assist in application of these plants for phytoremediation. These investigations also highlight potential health risks associated with the consumption of crops grown on contaminated soils.

Races of Albugo candida causing white blister rust on brassica vegetables in Australia

Albugo Candida races of Australian isolates from B. oleracea var. italica (broccoli), B. rapa var. pekinensis (pak choi) and var. chinensis (Chinese cabbage), and Capsella bursa-pastoris (shepherd's purse) were identified on a set of Brassicaceae hosts. Isolates from broccoli were identified as A. candida race 9 (Ac 9), from pak choi and Chinese cabbage as the sub-race V of Ac 7 or as a mixed population of sub-races A and V of the same race. Isolates from Shepherd's purse were identified as Ac 4. Australian Ac 9 isolates caused white blister disease on broccoli, broccolini, cauliflower, Brussels sprout and other related Brassicaceae hosts including B. nigra (black mustard), B. napus (oilseed rape), and on B. rapa (turnip rape) 'Torch' (a differential host of Ac 7). All cultivars of cabbage (B. oleracea var. capitata) and one new broccoli 'Booster' inoculated with isolates from broccoli were immune to the isolate tested. This result indicates that the Australian A. candida varies from the European one that causes disease on cabbage as well as on other B. oleracea varieties and additionally on shepherd's purse and an American one that causes disease on cabbage. The genotype of B. nigra tested was susceptible to both Ac 9 and Ac 7. This result indicates that B. nigra can serve as a host for both races. This study provides the first record of white blister disease on B. napus ('Hobson' and 'Regent') in Australia.

Investigation of plasmodiophora brassicae (clubroot disease) in vegetable brassica using arabidopsis thaliana as a model system

Clubroot, caused by Plasmodiophora brassicae, is the most devastating soil-borne disease of vegetable brassicas. It occurs all over the world and is responsible for crop losses of up to 10% every year. In Australia, the disease is being managed effectively with chemicals and cultural practices, but ideally control can be improved in the long term by the introduction of resistant cultivars. The life cycle ofP. brassicae and mode of action of plant resistance has not been fully elucidated because of the technical difficulties of working with an obligate, soil-borne plant pathogen. However, Arabidopsis thaliana, which is a host ofP. brassicae, has great potential as a model system for studying the life cycle, the infection process and development of resistance. We have developed a sand-liquid-culture system for growing Arabidopsis that allows easy observation of all life stages and, most importantly, the primary plasmodial stages within the root hair. The method was first optimised for observations of the lifecycle of the pathogen in a susceptible Arabidopsis ecotype (Col-3) where all stages of the lifecycle have now been observed and characterised. Further screening of Arabidopsis ecotypes for disease resistance has utilised one of the most virulent Australian pathotypes of brassica (ECD number 16/19/31). To date, Arabidopsis ecotype Ta-0 has shown a level of tolerance to the disease even though the roots get infected. It has been reported earlier that resistance toP. brassicae in Arabidopsis is due to one or a small number of genes. To examine changes in gene expression during the early, critical stages of infection, RNA was extracted from the susceptible and resistant ecotypes at two time points, 4 days and 17 days after inoculation. Microarray analysis will be used to investigate genome wide changes in gene expression during infection but also to identify candidate genes that may confer resistance to Australian isolates of the pathogen.

Sperm cells of the pollen tubes of Brassica : ultrastructure and isolation

Sperm cells of pollen tubes grown both in vivo and in vitro form a male germ unit. Extensions from both sperm cells of each pollen tube are closely associated with the tube nucleus. A high yield (2.7 × 104. 20 mg−1 pollen grains germinated) of intact sperm cells was obtained following release by osmotic shock from pollen tubes grown in vitro. Structural integrity of isolated sperm was maintained by isolation at low temperature in an osmotically balanced medium. At 4° C many isolated sperm pairs were still enclosed within the pollentube inner plasma membrane. Sperm cells not enclosed within this membrane no longer remained connected as a pair. During isolation vesicles formed on the sperm cell surface from disruption of the fibrillar components bridging the periplasmic space. Both in the pollen tube and after isolation the sperm nucleus is in close association with at least one region of the sperm plasma membrane. Sperm isolated at room temperature showed the presence of nucleopores, and nuclei were euchromatic, instead of heterochromatic as in intact sperm in the pollen tube.

The interrelationship between the accumulation of lipids, protein and the level of acyl carrier protein during the development of Brassica napus L. pollen

Lipid accumulation during pollen and tapetal development was studied using cryostat sections of unfixed anthers from Brassica napus (rapeseed). Diamidino-2-henylindole (DAPI), a DNA fluorochrome, was used to stain the pollen nuclei in order to identify ten stages of pollen development in Brassica. Storage lipids (i.e. triacylglycerides) were stained using the fluorochrome Nile red. Pollen coat lipids are formed in tapetal plastids between the mid-vacuolate and early maturation pollen stages. The pollen coat components, including lipids and a proportion of the proteins, are derived from the remnants of the tapetum, after its rupture, during the second pollen mitosis. Quantitative microfluorometric analyses demonstrated four phases of lipid body accumulation or depletion in the developing pollen cytoplasm. The majority of storage lipids found in the cytoplasm of the mature pollen grain accumulated during the late vacuolate and early maturation stages when the pollen is bicellular. The level of acyl carrier protein, a protein integrally involved in lipid synthesis, was also found to be maximal in the developing pollen during the bicellular pollen stages of development. This coincided with the most active period of lipid accumulation. These data could indicate that the lipids of the pollen are synthesized in situ, by metabolic processes regulated by expression of genes in the haploid genome.

Bioanalytical evaluation of Lactobacillus delbrueckii subsp. lactis 313 cell-envelope proteinase extraction

Lactobacilli cell-envelope proteinases (CEPs) have demonstrated numerous biopharmaceutical applications in the development of new streams of blockbuster nutraceuticals; thus, the development of efficient and commercially viable methods for CEP extraction will promote their full-scale application. In this study, the sub-cellular location of CEPs in Lactobacillus delbrueckii subsp. lactis 313 (LDL 313) was identified and the effects of different extraction methods were investigated for their ability to efficiently release CEPs from LDL 313. Significantly high relative proteinase activity of~95% was detected in cell-wall fractions and ~5% activity was observed for osmotic fluids, implying that proteinases in LDL 313 are cell-wall bound. CEPs were released from cell-wall via incubation in calcium-free buffer, indicating the enzyme is liable to self-digestion and ionic misfolding. Of the different extraction methods investigated, the use of 5 M LiCl was the most suitable, under the conditions of experimentation, for releasing high levels of CEPs from LDL 313.

Evaluation of fixed sample-size plans for Plutella xylostella (Lepidoptera: Plutellidae) on broccoli crops in Australia

Fixed sample-size plans for monitoring Plutella xylostella (L.) (Lepidoptera: Plutellidae) on broccoli and other Brassica vegetable crops are popular in Australia for their simplicity and ease of application. But the sample sizes used are often small, ≈10–25 plants per crop, and it may be that they fail to provide sufficient information upon which to base pest control decisions. We tested the performance of seven fixed sample-size plans (10, 15, 20, 30, 35, 40, and 45 plants) by resampling a large data set on P. xylostella in commercial broccoli crops. For each sample size, enumerative and presence-absence plans were assessed. The precision of the plans was assessed in terms of the ratio of the standard error to the mean; and at least 45 and 35 samples were necessary for the enumerative and presence-absence plans, respectively, to attain the generally accepted benchmark of ≤0.3. Sample sizes of 10–20 were highly imprecise. We also assessed the consequences of classifications based on action thresholds (ATs) of 0.2 and 0.8 larvae per plant for the enumerative case, and 0.15 and 0.45 proportion of plants of infested for the presence-absence case. Operating characteristic curves and investigations of the frequency of correct decisions suggest improvements in the performance of plans with increased sample size. In both the enumerative and presence-absence cases, the proportion of incorrect decisions was much higher for the lower of the two ATs assessed, and type II errors (i.e., failure to suggest pest control upon the AT is exceeded) generally accounted for the majority of this error. Type II errors are the most significant from a producer’s standpoint. Further consideration is necessary to determine what is an acceptable type II error rate.

Studies on physiologic specialisation of albugo candida causing white blister on broccoli in Victoria

White blister caused by oomycete Albugo candida (Pers. Ex. Lev.) Kuntze, (AC), is an important disease affecting many cruciferous hosts, including vegetable brassicas. The outbreaks of white blister in broccoli and cauliflower crops (Brassica oleracea var. italica and var. botrytis) in Southern Australia in the last three years led to restrictions on movement of fresh produce and seedlings from the disease-affected areas. Current classification of AC races is based on physiologic specialisation of this pathogen. Race 9 has been identified to cause white blister on B. oleracea in the USA. We report on specialisation of AC causing disease in Victorian broccoli crops and the use of molecular tools for the separation of AC races. In a glasshouse, 12 Brassicaceae species/varieties replicated 6 times, were inoculated twice at the fully developed cotyledon stage with a distilled water suspension of zoosporangia (1x104 per ml) collected from a single broccoli leaf. Two weeks after inoculation the incidence of white blister on cotyledons and seedling leaves of cauliflower, broccoli, black mustard and Indian mustard was 79.7, 78.4, 73.7 and 6.9% respectively. Cabbage plants were symptomless indicating that further specialisation of the pathogen may have occurred in Australia. High disease incidence among black mustard plants shows that the Australian isolate differs from overseas AC race 9. The interaction of a number of B. oleracea varieties to a range of AC isolates from various hosts will be investigated. Degenerate primers are now being used to amplify actin and β-tubulin genes to identify race specific polymorphisms in AC isolates from three different hosts (wild radish, Chinese cabbage, and broccoli). Differing PCR amplification efficiencies from broccoli and wild radish isolates using degenerate actin primers indicates sequence differences in the two isolates. The fragments are now being cloned and sequenced for race comparison.