Expression of a chitinase transgene in rose (Rosa hybrida L.) reduces development of blackspot disease (Diplocarpon rosae Wolf)

Blackspot, caused by the Ascomycete fungus Diplocarpon rosae, is the most widespread and pernicious disease of cultivated roses. While some species of rose possess resistance to D. rosae, none of the modern-day rose cultivars are fully resistant to the pathogen. In the current study, Biolistic gene delivery was used to introduce a rice gene, encoding a basic (Class I), chitinase into embryogenic callus of the blackspot-susceptible rose (Rosa hybrida L.) cv. Glad Tidings. The plasmid used for transformation carried the neomycin phosphotransferase (nptII) gene facilitating the selection and regeneration of transgenic plants on medium containing 250 mg/l kanamycin. Southern analysis confirmed integration of 2–6 copies of the chitinase gene into the rose genome; gene expression was confirmed by enzyme assay. Bioassays demonstrated that expression of the chitinase transgene reduced the severity of blackspot development by 13–43%. This degree of resistance to the pathogen correlated with the level of chitinase expression in the transgenic rose plants. The introduction of disease defence genes into rose provides a method of producing blackspot-resistant rose cultivars sought by breeders and growers.     

小钩丝壳属一新变种

本文报道了寄生在蔷薇科李属植物上的小钩丝壳属一新变种,即蔷薇小钩丝壳李变种Uncinuliella rosae Zheng & Chen var.pruni Z.Y.Zhao et Z.Q.Yuan,标本采自新疆霍城县野果林的樱桃李上。     

Characterisation of digestive protease in the rose sawfly,Arge rosae Linnaeus (Hymenoptera: Argidae)

Insect midgut proteases are excellent targets for insecticidal agents such as protease inhibitors. These inhibitors are used for producing transgenic plants, resistant to pests. For achieving this goal, it is necessary to find the nature of specific proteases and their properties for adopting possible pest management procedure. Therefore, characterisation of the enzymes in the gut of the rose sawfly, Arge rosae (Hymenoptera: Argidae), responsible for proteolysis, was performed using a range of synthetic substrates and specific inhibitors. The optimum conditions for general proteases and trypsin were achieved at pH 10. The highest activity for general proteases was obtained at a temperature of 45°C. The use of specific inhibitors and SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) provided evidence to suggest that most of the proteases belonged to the serine group because of high inhibitory effect of phenyl methane sulfonyl fluoride on total proteolytic activity. Also, inhibition assays and zymogram analysis showed that metalloproteases are present in A. rosae digestive system. These results indicated that A. rosae larvae mainly used serine proteases for protein digestion, with chymotrypsin as the dominant form. The kinetic parameters of trypsin-like proteases using N-benzoyl-dl-arg-p-nitroanilide as substrate indicated that the K _m and V _max values of trypsin in the gut of the fifth instar larvae were 730 ± 17.3 μM and 456 ± 13.85 nmol min^?1 mg^?1 protein, respectively.     

Microcystin-LR equivalent concentrations in fish tissue during a postbloom Microcystis exposure in Loskop Dam,South Africa

The effects of a decomposing cyanobacteria bloom on water quality and the accumulation of microcystin-LR equivalent toxin in fish at Loskop Dam were studied in May 2012. Enzyme-linked immunosorbent assay [ELISA] was used to confirm the presence of microcystin-LR equivalent in the water and to determine the microcystin (MCYST) concentration in the liver and muscle of fish. The lowest concentration of extracellular MCYST-LR equivalent was recorded in the lacustrine zone, where no cyanobacterial cells were observed, while the highest concentration (3.25 µg l^?1), 3.25 higher than World Health Organization standard, was observed in the riverine zone. Extremely high MCYST-LR equivalent concentrations of 1.72 µg MCYST-LReq kg^?1 in the liver and 0.19 µg kg^?1 in muscles of Labeo rosae, and 2.14 µg MCYST-LReq kg^?1 in the liver and 0.17 µg kg^?1 in muscles of Oreochromis mossambicus, indicate that the consumption of sufficient fish biomass might cause severe adverse effects in humans. Microscopic analyses of the stomach content of both fish species revealed low numbers of cyanobacterial Microcystis aeruginosa cells in comparison to other phytoplankton. The extracellular MCYST-LR equivalent of the decomposing bloom may have played a major role in the high levels observed in the livers of the two fish species. These findings are important for all downstream water users.     

Further investigations of the host range of the carrot fly, Psila rosae (F.)

Techniques were developed for testing umbelliferous species against naturally-occurring populations of carrot fly in the field. The most efficient technique involved exposure of plants to either first or second generations of the insect in the field, followed by caging of infested plots and trapping of emerged flies in water in yellow water dishes. In a series of field experiments between 1981 and 1989 inclusive, a total of 132 umbelliferous species and sub-species were tested against carrot fly. Seventy-eight of these proved to be new hosts, 27 were confirmed as hosts and 27 failed to support any carrot flies. Six non-umbelliferous plant species failed to be colonised by carrot fly. Sources of variability in investigations of the host range of insects are discussed.     

Comparison of the sensitivity of four Delia species to host and non-host plant compounds

Abstract. The behavioural facilitation hypothesis, tested in the present study, suggests that evolution of host-plant shifts by phytophagous insects is based on the preadaptation of insects to the chemistry of potentially novel plant hosts. Thus, closely-related insects should have similar sensitivities to compounds that are shared by different host plants. The chemoreception is investigated for four phytophagous flies, Delia radicum, Delia floralis, Delia antiqua and Delia platura (Diptera, Calyptratae: Anthomyiidae), belonging to the same genus but developing mainly on different plant families, with particular secondary plant compound profiles. In addition, the carrot fly, Psila rosae, an acalyptrate Diptera, is included as an unrelated species that is associated with completely different host plants. For the comparison, the known oviposition stimulants of the cabbage root fly (glucobrassicin, sinalbin, sinigrin and a thia-triaza-fluorene compound; CIF-1) present on the cabbage leaf surface were chosen. Responses from prothoracic tarsal sensilla are recorded to contact stimulation in a dose-dependent manner. Among the different flies tested, only D. radicum responds to all the compounds. By contrast, D. floralis is only sensitive to CIF-1, and not specifically on the C₅ sensillum, a finding that is in conflict with previously published results. This discrepancy is possibly an indication of the variability among flies originating from different cultures or habitats. With the exception of sinigrin at high concentration, the various compounds tested do not stimulate D. antiqua or D. platura. However, the carrot fly appears to be completely insensitive to sinigrin even at the highest tested concentration of 10⁻¹ M. The responses of the contact-chemoreceptor neurones to the selected compounds therefore provide little evidence of common sensitivities that would explain host shift in Delia species and specialization at the physiological level. The wide divergence within closely-related species and rearing cultures appears to indicate that the sensitivity and distribution of sensory receptor neurones is very variable on an evolutionary scale.     

Efficacy of triazoles and strobilurins in controlling black spot disease of roses caused by Diplocarpon rosae

Black spot disease is an important disease of roses with worldwide occurrence. It is caused by Diplocarpon rosae , an ascomycetous fungus and its control relies on fungicides. The effects of strobilurins and triazoles on D. rosae development within the host and on disease symptoms have not been well studied. Strobilurins completely inhibited germination of conidia when applied protectively 1 week before inoculation or on the same day with the inoculum. They were, however, not effective in eradicating the disease when applied after the fungus was established in the host. Triazoles reduced the germination rate of the conidia when applied protectively and they inhibited disease symptom development when applied after the fungus was established in the host but before symptom expression. Application of triazoles after symptom development suppressed further development of the disease, but in the case of treatment with myclobutanil yellowing and defoliation still occurred. Post-infection application of triazoles led to the apparent breakdown of subcuticular mycelia, intercellular mycelia, and hyphae in the epidermal cells, while the effects of strobilurins were limited to the subcuticular mycelia. Triazoles were more effective than strobilurins because they are more systemic.     

Trade‐offs in oviposition choice? Food‐dependent performance and defence against predators of a herbivorous sawfly

The sawfly Athalia rosae L. (Hymenoptera: Tenthredinidae) is a feeding specialist on plant species of the Brassicaceae, which are characterised by secondary metabolites, called glucosinolates. The larvae can take up the respective glucosinolates of their hosts and concentrate them in their haemolymph to protect themselves against predators. Oviposition preferences of naïve females were tested for three species, Sinapis alba L., Brassica nigra (L.) Koch, and Barbarea stricta Andrz., and were related to larval performance patterns. Larvae were reared on either one of these plants and it was investigated how host‐plant quality influences both the developmental times and growth of larvae (bottom‐up) and the defence efficiency against predators (top‐down). Innately, almost all adult females avoided B. stricta for oviposition and clearly preferred B. nigra over S. alba. On average, larvae developed best on B. nigra. Female larvae reached similar final body masses on all host‐plant species, but males reared on S. alba were slightly lighter. The developmental time of larvae reared on B. stricta was significantly longer than on the other two plants. However, larvae reared on B. stricta were best protected against the predatory wasp Polistes dominulus Christ (Hymenoptera: Vespidae). The wasps rejected these larvae most often, while they attacked larvae reared on S. alba most frequently. Thus, larvae feeding on B. stricta theoretically run a higher risk of predation due to a prolonged developmental time, but in practice they are better protected against predators. Overall, oviposition preferences of A. rosae seem to be more influenced by bottom‐up effects on larval performance than by top‐down effects.     

Exploitation of the resistance to carrot fly in the wild carrot species Daucus capillifolius

Under field conditions a wild Daucus species from Libya, D. capillifolius, supported less than one tenth as many carrot flies (Psila rosae) as the susceptible carrot cultivar Danvers Half Long 126. Breeding lines developed from crosses between D. capillifolius and three different carrot types were grown in a series of field experiments at Wellesbourne between 1980 and 1989. Each year selections were made for agronomic quality and/or for increased resistance to carrot fly. The programme produced lines which for size, shape and colour represented most of the commercially-important carrot types. Some of these lines were also significantly more resistant to carrot fly than selections from the partially-resistant cv. Sytan. However, the best lines were not as resistant as the wild parent. The highest quality resistant lines were sold to seed companies for variety production.     

Identification of molecular markers linked to Rdr1, a gene conferring resistance to blackspot in roses

Blackspot resistance in the tetraploid rose genotype 91/100–5 had been characterised previously as a single dominant gene in duplex configuration. In the present study a tetraploid progeny (95/3) segregating for the presence of the blackspot resistance gene Rdr1 were screened with 868 RAPD and 114 AFLP primers/primer combinations. Seven AFLP markers were found to be linked to Rdr1 at distances between 1.1 and 7.6 cM. The most closely linked AFLP marker was cloned and converted into a SCAR marker that could be screened in a larger population than the original AFLP and was linked at a distance of 0.76 cM. The cloned fragment was used as an RFLP probe to locate the marker on a chromosome map of diploid roses. This is the first report of markers linked to a resistance gene in roses, and the possibilities of using them for a marker-assisted selection for blackspot resistance as well as for map-based cloning approaches are discussed. Received: 23 December 1999 / Accepted: 25 March 2000