Vital staining of plant cell suspension cultures: evaluation of the phytotoxic activity of the phytotoxins phomalide and destruxin B

 A cell suspension culture assay to determine the phytotoxicity of the fungal toxins phomalide, a host-selective toxin produced by the fungus Phoma lingam (Tode ex Fr.) Desm., perfect stage Leptosphaeria maculans (Desm.) Ces. et de Not., and destruxin B, the major host-selective toxin produced by the fungus Alternaria brassicae (Berk.) Sacc., was carried out with three Brassica spp. It was established that phomalide was significantly less phytotoxic to Cutlass (Brassica juncea), the cultivar resistant to L. maculans, than to Westar (B. napus), the cultivar susceptible to L. maculans, at concentrations ≤2×10^–5  M. Similar to phomalide, destruxin B, at concentrations ≤5×10^–5  M, decreased the viability of cells of the cultivar resistant to A. brassicae (Ochre, Sinapis alba) less than the viability of cells of the susceptible cultivar (Westar, B. napus). Considering the high selectivity of phomalide and its direct correlation with plant disease resistance, phomalide may have great potential application in breeding programs screening/selecting for blackleg resistance in brassicas. Received: 23 November 1999 / Revision received: 11 April 2000 / Accepted: 8 May 2000     

Brassica naponigra,a somatic hybrid resistant to Phoma lingam

Summary Brassica napus and B. nigra were combined via protoplast fusion into the novel hybrid Brassica naponigra. The heterokaryons were identified by fluorescent markers and selected by flow sorting. Thirty hybrid plants were confirmed by isozyme analysis to contain both B. nigra and B. napus chromosomes; of these, 20 plants had the sum of the parental chromosome numbers. A non-random segregation of the chloroplasts was found in the hybrids. Of 14 hybrid plants investigated, all had the B. napus type of chloroplast. The resistance to Phoma lingam found in the B. nigra cultivar used in the fusion experiments was expressed in 26 of the hybrid plants. The hybrids obtained in this study contain all of the three Brassica genomes (A, B and C) and have thus created unique possibilities for genetic exchanges between the genomes. Since most of the plants were fertile as well as resistant to P. lingam, they have been incorporated into conventional rapeseed breeding programs.     

A Detached Cotyledon Test for the Isolation of Mutants of Pyrenopeziza brassicae Defective in Pathogenicity Determinants

A detached cotyledon pathogenicity test was devised for the isolation of UV-induced mutants of the hemibiotrophie ascomycete pathogen of Brassica spp., Pyrenopeziza brassicae defective in pathogenicity determinants. At least 95 % of cotyledons of suitable susceptible cultivars of oilseed rape (Brassica napus L. ssp. oleifera cvs Shogun or Bolko) showed symptoms of disease (white spore pustules [conidiomata] on the surface of the cotyledon) within three weeks of inoculation of cotyledons with 10⁴ conidia of P. brassicae. The test allowed rapid screening of UV-induced mutants with a low frequency of false negatives. From 1,700 survivors of UV mutagenesis tested, 20 non-pathogenic mutants were obtained.     

Seedling and adult plant peaetions of Brassica napus-B. juncea recombinant lines towards A- and B-group isolates of Leptosphaeria maculans

The Brassica napus-B. juncea recombinant lines MX and MXS carrying a B. juncea major gene (JLml) in the genetic background of a spring- or a winter type B. napus cultivar, respectively, were tested for their resistance level to Leptosphaeria maculans under controlled conditions. Inoculation with three A-and four B-group individual isolates and with different mixtures of isolates realised within or between these groups was performed on cotyledons, leaves and stems. Cotyledons and leaves of the two recombinant lines were more resistant to A-group isolates than those of B. napus cultivars, except for one isolate recovered from the MX line. The recombinant lines were susceptible at cotyledon stage and resistant on leaves to B-group isolates, as were B. napus cultivars. On stems, severe cortical damage was usually produced on B. napus cultivars by some A-group isolates, whereas B-group isolates induced pith blackening on all genotypes. Stems of the MX line and the resistant donor species (B. juncea cv. Picra) were more resistant than those of the susceptible B. napus (cv. Westar) to the individual A-group isolates. Cultivar Picra was the most susceptible genotype to pith infection caused by the B-group isolates. The consequence of the host pathogen differential interactions on the durability of the monogenic resistance to L. maculans introduced from B. juncea into B. napus is discussed.     

In vitro Plant Formation from Stem Explants of Rape (Brassica napus cv. Zephyr)

Internodal segments from 6-weeks-old rape plants (Brassica napus L. cv. Zephyr) were induced to differentiate in vitro producing shoots or shoots and roots on synthetic nutrient medium under controlled conditions. Benzyladenine (BA) alone (5 × 10^?6 M) induced multiple shoot formation on all stem explants. Roots were induced on shoots when recultured on nutrient medium supplemented with auxins such as naphthalene-acetic acid (NAA) or indoleacetic acid (1AA) or when planted in vermiculite. Complete plant formation was obtained when NAA (2 × 1^?6, 5 × 10^?6 and 10^?5 M) was employed in conjunction with BA at 5 × 10^?6M. At higher concentrations (10^?5M) NAA retards the shoot development while 1AA suppresses it totally. Lower levels of auxins along with the cytokinin did not retard or inhibit shoot differentiation.     

Transfer of resistance against Phoma lingam to Brassica napus by asymmetric somatic hybridization combined with toxin selection

Summary Irradiated mesophyll protoplasts from nine different accessions of B. juncea, B. nigra and B. carinata, all resistant to Phoma lingam, were used as gene donors in fusion experiments with hypocotyl protoplasts isolated from B. napus as the recipient. A toxin, sirodesmin PL, was used to select those fusion products in which the resistant gene(s) was present. In the fusion experiments different gene donors, various irradiation dosages and toxin treatments were combined. Symmetric and asymmetric hybrid plants were obtained from the cell cultures with and without toxin selection. Isozymes were used to verify hybrid characters in the symmetric hybrids, whereas two DNA probes were used to identify donor-DNA in the asymmetric hybrids. Resistance to P. lingam was expressed in all symmetric hybrids, and in 19 of 24 toxin-selected asymmetric hybrids, while all the unselected asymmetric hybrids were susceptible.     

Influence of Cotyledon Excision and Sucrose on Root Formation in Pea Cuttings

Sucrose was supplied to stock plants of Pisum sativum L. cv. Alaska grown at different levels of irradiance. There was no significant effect on the rooting of the cuttings by sucrose supply to intact plants regardless of the irradiance. However, an increase in the number of roots per cutting was obtained at increasing concentrations of sucrose when the stock plants had been grown at 4 W m^?2 and their cotyledons had been removed two days before the cuttings were excised. Cotyledons were removed from stock plants at different times before the excision of cuttings with the intent to regulate the endogenous supply of carbohydrate. The number of roots per cutting was reduced by removal of the cotyledons and this reduction was correlated to the number of days the stock plants had grown without cotyledons as well as to the irradiance pre-treatment. A greater reduction occurred in cuttings from plants grown under 4 W m^?2 than from those grown under 38 W m^?2. The growth of the stock plants and the subsequent stem growth of the cuttings was determined by the irradiance to the stock plants and by the time of removal of the cotyledons. Exogenous supply of sucrose had no effect on the stem growth of the cuttings.     

Genetics of virulence in Leptosphaeria maculans (Desm.) Ces. et De Not., the cause of blackleg in rapeseed (Brassica napus L.)

The genetic basis of virulence of 24 isolates of L. maculans collected from various sites throughout south-eastern and south-western Australia were studied using five clone-lines of B. napus. The experimental design allowed the estimation of the environmental and genetic components of variance using a standard analysis of variance. Virulence of these isolates (as measured by the percentage of stem girdling, %G) on the clonelines NCII and Tap was found to be most likely controlled by a small number of genes; the broad-sense heritabilities were 79.7% and 67.5% for virulence on NCII and Tap, respectively. The significance of these results in relation to the potential of L. maculans in adapting to new resistant B. napus cultivars is discussed.     

Host Genetic Analysis by Using Puccinia recondita tritici Induced Mutants for Increased Virulence

Monogenic lines derived by recombination from Buck Manantial wheat, a cultivar which has durable resistance, were used as hosts to detect Puccinia recondita tritici induced mutants for increased virulence. After treatments with ethyl methane sulphonate on clone 66 of P. recondita 9 types of mutants were obtained at approximate frequencies of 1 × 10^?4 and host lines were grouped in 6 classes, No increase virulence was obtained against B. Manantial after 2 cycles of treatments, but different combinations of virulences were observed on monogenic lines derived from it. Simultaneity of occurrence of some mutational events suggests complexity of virulence genes in the pathogen. At least 4 genes for incompatibility are present in B. Manantial when confronted with clone 66 and 4 to 7 mutational points are recognized in the pathogen. The specific relationships tending to equate the number of genes in both organisms would not be a general rule. Durable resistance can be explained by a combination of several specific disease reaction genes for which the pathogen population has not been able to accumulate all the corresponding alleles for virulence.     

Reduced rhizosphere colonization ability ofAgrobacterium tumefaciens chromosomal virulence (chv) mutants

Mutations in the chromosomal virulence (chv) region ofA. tumefaciens strain A723 reduce virulence, motility, and ability of the bacteria to bind to plant cells. We conducted experiments to assess the ability ofchv mutants to colonize the rhizosphere ofPisum sativum. The mutation had no effect on ability of bacteria to grow with a defined number of root cap cells as the sole carbon and nitrogen source. Ten days after inoculation, there were up to 103-fold more wild type thanchv mutant bacteria present in the rhizosphere of inoculated plants.     

Screening for Resistance to Blackleg Phoma lingam (Tode ex Fr.) Desm. within Brassicaceae

Ninety-six accessions within the Brassicaceae were inoculated with a wide range of isolates of Phoma lingam and screened for resistance. Initially, seedlings were sprayed with a spore suspension. The material with the highest level of resistance according to the seedling test was further inoculated with pycnospores in cotyledons, adult leaves and stem bases. All representatives with resistance at the cotyledon stage were found to be resistant at the adult, too. A large variability invirulence was found among the different isolates of the pathogen demostrating the importance of using a broad spectrum when screening for resistance. An evaluation based on the response to 17 different P. lingam isolates revealed that five accessions of B. juncea, two of B. nigra and two of B. carinata could be classified as resistant. Although all these representatives contain the B genome of Brassica, there were other accessions with the B genome that were sensitive.     

Interaction of an Escherichia coli mutator gene with a deoxyribonucleotide effector

Summary Strains of Escherichia coli carrying mutD5 display very high mutation rates (about 10⁴-fold above wild-type) when grown in rich medium, but relatively low mutation rates (about 10- to 50-fold above wild-type) in minimal medium. Thymidine, deoxycytidine, and deoxyuridine are all capable of stimulating mutation when added to minimal medium. Studies with mutants blocked in various steps of thymidine metabolism implicate a phosphorylated thymidine effector which mediates the mutagenic action of the added deoxyribonucleotides. In addition, an unidentified compound or compounds other than thymidine present in rich medium (L-broth) can also increase the mutation rate.     

A Mutant Brassica napus (Canola) Population for the Identification of New Genetic Diversity via TILLING and Next Generation Sequencing

We have generated a Brassica napus (canola) population of 3,158 EMS-mutagenised lines and used TILLING to demonstrate that the population has a high enough mutation density that it will be useful for identification of mutations in genes of interest in this important crop species. TILLING is a reverse genetics technique that has been successfully used in many plant and animal species. Classical TILLING involves the generation of a mutagenised population, followed by screening of DNA samples using a mismatch-specific endonuclease that cleaves only those PCR products that carry a mutation. Polyacrylamide gel detection is then used to visualise the mutations in any gene of interest. We have used this TILLING technique to identify 432 unique mutations in 26 different genes in B. napus (canola cv. DH12075). This reflects a mutation density ranging from 1/56 kb to 1/308 kb (depending on the locus) with an average of 1/109 kb. We have also successfully verified the utility of next generation sequencing technology as a powerful approach for the identification of rare mutations in a population of plants, even in polyploid species such as B. napus. Most of the mutants we have identified are publically available.     

Down‐regulation of BnDA1, whose gene locus is associated with the seeds weight,improves the seeds weight and organ size in Brassica napus

Brassica napus L. is an important oil crop worldwide and is the main raw material for biofuel. Seed weight and seed size are the main contributors to seed yield. DA1 (DA means big in Chinese) is an ubiquitin receptor and negatively regulates seed size. Down‐regulation of AtDA1 in Arabidopsis leads to larger seeds and organs by increasing cell proliferation in integuments. In this study, BnDA1 was down‐regulated in B. napus by over expressed of AtDA1^R358K, which is a functional deficiency of DA1 with an arginine‐to‐lysine mutation at the 358th amino acid. The results showed that the biomass and size of the seeds, cotyledons, leaves, flowers and siliques of transgenic plants all increased significantly. In particular, the 1000 seed weight increased 21.23% and the seed yield per plant increased 13.22% in field condition. The transgenic plants had no negative traits related to yield. The candidate gene association analysis demonstrated that the BnDA1 locus was contributed to the seeds weight. Therefore, our study showed that regulation of DA1 in B. napus can increase the seed yield and biomass, and DA1 is a promising target for crop improvement.     

Three <Emphasis Type="Italic">nth</Emphasis> homologs are all required for efficient repair of spontaneous DNA damage in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Deinococcus radiodurans is a bacterium that can survive extreme DNA damage. To understand the role of endonuclease III (Nth) in oxidative repair and mutagenesis, we constructed nth single, double and triple mutants. The nth mutants showed no significant difference with wild type in both IR resistance and H₂O₂ resistance. We characterized these strains with regard to mutation rates and mutation spectrum using the rpoB/Rif^r system. The Rif^r frequency of mutant MK1 (△dr0289) was twofold higher than that of wild type. The triple mutant of nth (ME3)generated a mutation frequency 34.4-fold, and a mutation rate 13.8-fold higher than the wild type. All strains demonstrated specific mutational hotspots. Each single mutant had higher spontaneous mutation frequency than wild type at base substitution (G:C → A:T). The mutational response was further increased in the double and triple mutants. The higher mutation rate and mutational response in ME3 suggested that the three nth homologs had non-overlapped and overlapped substrate spectrum in endogenous oxidative DNA repair.     

Interaction of silicon and cadmium in <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Brassica napus</Emphasis>

The objective of this study was to determine the effect of silicon (Si) and cadmium (Cd) on root and shoot growth and Cd uptake in two hydroponically cultivated Brassica species (B. juncea (L.) Czern. cv. Vitasso and B. napus L. cv. Atlantic). Both species are potentially usable for phytoextraction. Inhibitory effects of Cd on root elongation were diminished by the impact of Si. Primary roots elongation in the presence of Cd + Si compared with Cd was stronger and the number of lateral roots was lower in B. juncea than in B. napus. Cd content per plant was higher in B. napus roots and shoots compared with B. juncea. Suberin lamellae were formed closer to the root apex in Cd + Si than in Cd treated plants and this effect was stronger in B. napus than in B. juncea. Accelerated maturation of endodermis was associated with reduced Cd uptake. Cd decreased the content of chlorophylls and carotenoids in both species, but Si addition positively influenced the content of photosynthetic pigments which was higher in B. napus than in B. juncea. Si enhanced more substantially translocation of Cd into the shoot of B. napus than of B. juncea. Based on our results B. napus seems to be more suitable for Cd phytoextraction than B. juncea because these plants produce more biomass and accumulate higher amount of Cd. The protective effect of Si on Cd treated Brassica plants could be attributed to more extensive development of suberin lamellae in endodermis.     

<Emphasis Type="Italic">MAM</Emphasis> gene silencing leads to the induction of C3 and reduction of C4 and C5 side-chain aliphatic glucosinolates in <Emphasis Type="Italic">Brassica napus</Emphasis>

Methylthioalkylmalate (MAM) synthases and their associated genes that have been extensively investigated in Arabidopsis control the side-chain elongation of methionine during the synthesis of aliphatic glucosinolates. A Brassica homolog of the Arabidopsis MAM genes was used in this study to analyze the role of MAM genes in B. napus through RNA interference (RNAi). The silencing of the MAM gene family in B. napus canola and B. napus rapeseed resulted in the reduction of aliphatic glucosinolates and total glucosinolate content. The results indicated that RNAi has potential for reducing glucosinolate content and improving meal quality in B. napus canola and rapeseed cultivars. Interestingly, MAM gene silencing in B. napus significantly induced the production of 2-propenyl glucosinolate, a 3-carbon side-chain glucosinolate commonly found in B. juncea mustard. Most transgenic plants displayed induction of 2-propenyl glucosinolate; however, the absolute content of this glucosinolate in transgenic B. napus canola was relatively low (less than 1.00 μmol g⁻¹ seed). In the high glucosinolate content progenies derived from the crosses of B. napus rapeseed and transgenic B. napus canola, MAM gene silencing strongly induced the production of 2-propenyl glucosinolate to high levels (up to 4.45 μmol g⁻¹ seed).     

A Comparison of the Disease Reactions of Stems and Detached Leaves of Soil and in vitro Grown Plants and Regenerants of Oilseed Rape to Leptosphaeria maculans and Protocols for Selection for Novel Disease Resistance

Protocols for selecting plant tissues of winter oilseed rape (Brassica napus subsp. oleifera) with resistance to Leptosphaeria maculans by either stem or leaf inoculation of both soil and in vitro grown plant material are described. The stem inoculation procedure gave good correlation (r = 0. 92) between the 50 day stem disease scores of eight out of nine cultivars of soil grown winter oilseed rape inoculated with isolate 41A4 of L. maculans and the N. A. B. esistance ratings or resistance data from field trials. The exception was the cultivar Liradonna. Inoculation of stems of five cultivars with isolates 41A4, 433 and 478 indicated a range of isolate virulence 478 > 41A4 > 433. This was the inverse of that observed in leaf inoculations. Application of the stem inoculation procedure to in vitro shoot cultures allowed differentiation of resistant and susceptible cultivars, including the cultivar Liradonna, after 20 days incubation at 20°C. The protocol was also applicable to plantlets regenerated from thin cell layer explants grown in vitro. Inoculations with isolate 433 allowed the differentiation of resistant, intermediately resistant and susceptible leaf material of soil grown plants, when leaf discs from young leaves were incubated on water agar supplemented with BAP (1 × 10^?5 M) at 25°C for 10 days. Intermediately resistant leaves were resistant after 10 days and susceptible after 15 days of incubation. Leaves of shoot cultures grown in vitro were more susceptible than the corresponding soil grown material. However, inoculation of old leaves with isolate 41A4 (an isolate of less virulence on leaves than 433) distinguished the cultivars after 15 days of incubation. These protocols allow the accurate assessment of resistance to L. maculans at the stem or leaf level and are of use in traditional as well as in vitro selection programmes.     

Effects of previous cropping and fungicide timing on the development of light leaf spot (Pyrenopeziza brassicae), seed yield and quality of winter oilseed rape (Brassica napus)

Light leaf spot, caused by Pyrenopeziza brassicae, was assessed regularly on double-low cultivars of winter oilseed rape during field experiments at Rothamsted in 1990-91 and 1991-92. Previous cropping and fungicide applications differed; seed yield and seed quality were measured at harvest. In each season, both the initial incidence of light leaf spot and the rate of disease increase were greater in oilseed rape crops sown after rape than those sown after cereals. The incidence of diseases caused by Phoma lingam or Alternaria spp. was also greater in second oilseed rape crops. In 1991-92 there was 42% less rainfall between September and March than in 1990-91, and much less light leaf spot developed. However, P. lingam and Alternaria spp. were more common. Only fungicide application schedules including an autumn spray decreased the incidence of light leaf spot on leaves, stems and pods, as indicated by decreased areas under the disease progress curves (AUDPC) and slower rates of disease increase. Summer sprays decreased incidence and severity of light leaf spot on pods only. In 1990-91, all fungicide treatments which included an autumn spray increased seed and oil yields of cv. Capricorn but only the treatment which included autumn, spring and summer sprays increased yields of cv. Falcon. No treatment increased the yields of cv. Capricorn or cv. Falcon in 1991-92. Fungicide applications decreased glucosinolate concentrations in the seed from a crop of cv. Cobra severely infected by P. brassicae in 1990-91, but did not increase yield.