<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of safflower and the efficient recovery of transgenic plants via grafting

Background

Safflower (Carthamus tinctorius L.) is a difficult crop to genetically transform being susceptible to hyperhydration and poor in vitro root formation. In addition to traditional uses safflower has recently emerged as a broadacre platform for the production of transgenic products including modified oils and pharmaceutically active proteins. Despite commercial activities based on the genetic modification of safflower, there is no method available in the public domain describing the transformation of safflower that generates transformed T₁ progeny.

Results

An efficient and reproducible protocol has been developed with a transformation efficiency of 4.8% and 3.1% for S-317 (high oleic acid content) and WT (high linoleic acid content) genotypes respectively. An improved safflower transformation T-DNA vector was developed, including a secreted GFP to allow non-destructive assessment of transgenic shoots. Hyperhydration and necrosis of Agrobacterium-infected cotyledons was effectively controlled by using iota-carrageenan, L-cysteine and ascorbic acid. To overcome poor in vitro root formation for the first time a grafting method was developed for safflower in which ~50% of transgenic shoots develop into mature plants bearing viable transgenic T₁ seed. The integration and expression of secreted GFP and hygromycin genes were confirmed by PCR, Southern and Western blot analysis. Southern blot analysis in nine independent lines indicated that 1-7 transgenes were inserted per line and T₁ progeny displayed Mendelian inheritance.

Conclusions

This protocol demonstrates significant improvements in both the efficiency and ease of use over existing safflower transformation protocols. This is the first complete method of genetic transformation of safflower that generates stably-transformed plants and progeny, allowing this crop to benefit from modern molecular applications.

     

Phytotoxic polyketides produced by <Emphasis Type="Italic">Phomopsis foeniculi</Emphasis>, a strain isolated from diseased Bulgarian fennel

Recently, a new fungal disease caused by Diaporthe angelicae (anamorph Phomopsis foeniculi) has been found with increasingly frequency on fennel (Foeniculum vulgare) in Bulgaria. Using a bioassay-guided isolation and purification procedure, different metabolites were isolated from the fungal culture filtrates. They were identified by spectroscopic methods as nectriapyrone, a pentaketide monoterpenoid, and altersolanols A and J and macrosporin, three octaketides anthracenones. Leaf puncture bioassay was applied on detached tomato leaves to prove the phytotoxic activity of the fractions and of pure compounds. Nectriapyrone and altersolanols A and J showed a modulated phytotoxicity, while macrosporin was not toxic. Altersolanol A was the most active compound.     

PCR-SSCP analysis of <Emphasis Type="Italic">Fusarium</Emphasis> diversity in asparagus decline in Japan

The diversity of Fusarium populations in asparagus (Asparagus officinalis L.) decline fields in Japan was estimated by PCR-SSCP (single-stranded conformational polymorphism) analysis of the ITS2 regions of the nuclear rRNA genes. This method was used to rapidly and objectively identify pathogens associated with roots of plants showing symptoms of asparagus decline collected from fields in five regions across Japan. Over 651 fusarial isolates were obtained, and were easily differentiated into three principal species. Fusarium oxysporum f. sp. asparagi was most frequently isolated from the domestic five regions (68%), whereas Fusarium proliferatum (28.6%) was less frequent. Fusarium solani was found much rarely (2.5%). The frequency of isolation of Fusarium proliferatum increased gradually from the north to the south of Japan, though considerable differences were found between fields in each region, as well as regional differences among the Fusarium populations. Most of the fusarial isolates were highly pathogenic in vitro. These results reveal that Fusarium oxysporum f. sp. asparagi and Fusarium proliferatum are important biotic factors which lead to asparagus decline in Japan.     

<Emphasis Type="Italic">Cylindrocarpon</Emphasis> species associated with apple tree roots in South Africa and their quantification using real-time PCR

Cylindrocarpon species are known to be a component of the pathogen/pest complex that incites apple replant disease. In South Africa, no information is available on apple associated Cylindrocarpon species and their pathogenicity. Therefore, these aspects were investigated. Among the isolates recovered from apple roots in South Africa, four species (C. destructans, C. liriodendri, C. macrodidymum and C. pauciseptatum) were identified using β-tubulin gene sequencing and phylogenetic analysis. This is the first report of C. liriodendri, C. macrodidymum and C. pauciseptatum on apple trees. Cylindrocarpon macrodidymum was the most prevalent. Isolates within each of the four species were pathogenic towards apple seedlings, but varied in their virulence. With a single exception, all isolates were able to induce lesion development on seedling roots. Only 57% of the isolates, which represented all four species, were able to cause a significant reduction in seedling weight and/or height. The greatest seedling growth reductions were caused by two isolates of C. destructans, and one isolate each of C. liriodendri and C. macrodidymum. A quantitative real-time polymerase chain reaction (qPCR) method was developed for simultaneous detection of all four Cylindrocarpon species. qPCR analyses of Cylindrocarpon from the roots of inoculated seedlings showed that the amount of Cylindrocarpon DNA in roots was not correlated to seedling growth reductions (weight and height) or root rot. The qPCR method is, however, very useful for the rapid identification of apple associated Cylindrocarpon species in roots. The technique may also hold potential for being indicative of Cylindrocarpon disease potential if rhizosphere soil rather than roots are used.     

EMS and UV irradiation induce unstable resistance against CAA fungicides in <Emphasis Type="Italic">Bremia lactucae</Emphasis>

Wild type (WT) field isolates of Bremia lactucae failed to germinate in vitro or infect lettuce leaves in the presence of CAA (carboxylic acid amide) fungicides. Minimal inhibitory concentrations (MIC) for mandipropamid, dimethomorph, benthiavalicarb and iprovalicarb were 0.005, 0.5, 0.5 and 5 μg ml⁻¹, respectively. Mutagenesis experiments showed that spores exposed to EMS (ethyl methane sulphonate) or UV irradiation (254 nm) could infect lettuce leaves in the presence of up to 100 μg ml⁻¹ CAA. The proportion of infected leaves relative to the number of spores inoculated (infection frequency) was inversely related to the concentration of CAA used, ranging between 0 and 160 per 1 × 10⁶ spores. Resistant mutants (RM) lost their resistance within 1–14 reproduction cycles on CAA-treated plants. Crosses were made between RMxWT isolates and RMxRM isolates with an attempt to obtain stable homozygous resistant off-springs. Such crosses yielded few resistant but unstable progeny isolates. Mutagenic treatments given to hybrid isolates also failed to produce stable resistance. Previous gene sequencing data showed that stable resistance to CAAs is based on a single SNP in the cellulose synthase 3 (CesA3) gene of Plasmopara viticola. Therefore, we sequenced a 582 bp DNA fragment of Ces3A of WT, RM and hybrid isolates of B.lactucae. No mutation in this gene fragment was found. We conclude that mutagenic agents like EMS or UV may induce resistance to CAA in Bremia lactucae but this resistance is not stable and not linked to mutations in CesA3 gene.     

Morphological and molecular analysis of genetic variability within isolates of <Emphasis Type="Italic">Corynespora cassiicola</Emphasis> from different hosts

Twenty-two isolates of Corynespora cassiicola obtained from cucumber, papaya, eggplant, tomato, bean, Vigna, sesame and Hevea rubber (Hevea brasiliensis) were analysed by morphological features, the differences of the ribosomal DNA internal transcribed spacer (rDNA-ITS) region sequence and the inter simple sequence repeat (ISSR) technique. Variability of morphological features was observed among the isolates. Pathogenicity tests showed that isolates from different hosts attacked Hevea rubber. Sequences of two outgroup taxa, C. proliferata and C. citricola, were downloaded from GenBank. The phylogenetic trees were constructed by using the rDNA-ITS region sequences from 24 Corynespora spp. isolates. In this analysis, the 24 sequences grouped into two clusters (A and B). Cluster A consists of sequences from all isolates of C. cassiicola; whereas cluster B consists of the two outgroup taxa, C. proliferata and C. citricola. However, the ITS region is conservative, and is not fit for studying differences among isolates. A total of 114 DNA fragments was amplified with 16 ISSR primers, among which 102 were polymorphic (89.5%). A dendrogram was created by the unweighted pair-group method with arithmetic averaging (UPGMA) analysis, and 22 isolates grouped into three clusters (C, D and E). Cluster C is composed of all of the Hevea rubber isolates, whereas cluster D is composed of nine isolates: four from papaya, five from cucumber, eggplant, bean, vigna and sesame. Cluster E is composed of two isolates from cucumber and tomato. These analyses showed that the genetic diversity was very rich among the tested isolates. There are no correlations between the morphological characteristics or rDNA-ITS region sequences of the 22 isolates and their host or geographical origin, but there is a link between ISSR clusters and their host origins. ISSR markers appear to be useful for intra-species population study in C. cassiicola.     

Screening of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> isolates from vineyards in Israel for resistance to fungicides

Plots in two vineyards in the Golan Heights, Israel were treated with six botryticides during three growing seasons with 3 applications per season. Applications of fenhexamid, pyrimethanil and cyprodinil + fludioxonil were effective, resulting in 52–65% and 53–63% mean reduction in grey mould incidence and severity, respectively. Carbendazim, fluazinam and iprodione were ineffective or slightly effective. Five hundred and sixteen B. cinerea isolates were collected from infected berries or trapped from the air in the vineyards, and profiles of sensitivity to benomyl, fenhexamid, fluazinam, fludioxonil, iprodione and pyrimethanil were established for each of the isolates based on a mycelial growth test. Seventy-four percent of the isolates were sensitive to the six tested fungicides, and the other 26% of the isolates were classified into 10 phenotypes characterized by resistance to one or more fungicides. Resistant isolates showed fitness parameters similar or reduced in comparison to sensitive isolates. Resistance to benzimidazoles and to dicarboximides was the most frequent (up to 25%) and apparently pre-existed in the populations tested. Increased frequency of benzimidazole resistance, but not dicarboximide resistance, was observed following the 3 years of applications of the fungicides. High level resistance to pyrimethanil was present at a frequency of about 2% in both vineyards in the first 2 years of the sampling survey and reached 10% in the third year at Site 2. A few isolates were resistant to fenhexamid or fludioxonil (0.8 or 0.2%, respectively). No strong resistance to fluazinam was detected, although numerous, less sensitive isolates, presumably possessing multi-drug resistance traits, were recovered at higher frequency from the plots treated with fluazinam than from the untreated plots.     

Improving sooty blotch and flyspeck severity estimation on apple fruit with the aid of standard area diagrams

Sooty blotch and flyspeck is caused by numerous species of fungi that colonize the surface of apple fruit and thereby lower its market value. Although this disease poses a substantial threat to apple growers’ profitability in some regions, reliable and cost-effective methods for epidemiological and disease control studies have not been validated, nor are they widely available. We modified a standard area diagram to aid sooty blotch and flyspeck severity assessments and quantified its impact on accuracy and precision of visual estimates. Samples of ‘Fuji’ and ‘Mutsu’ fruit were photographed both from the top and laterally. Severity was assessed from a sub-sample of 160 images using image analysis software. Validation of the diagram was performed by eight raters who independently assessed severity in two series of selected images representing the lateral view and the top view, initially unaided and subsequently with the aid of the scale. Severity estimates ranged from 0.4% to 98% (most fruit had <10% severity). Accuracy and precision of the estimates were significantly improved when using the diagrammatic scale; concordance correlation coefficient values increased from 0.81 to 0.95. A strong tendency to underestimate severity for the mid-range to high levels was minimized when using the aid, which also improved reproducibility of the estimates among raters. In addition to strengthening evidence that a standard area diagram can be used reliably in sooty blotch and flyspeck studies, we expanded its application to disease assessment in the peduncle region, which enhances the usefulness of the method for evaluating efficacy of management practices.     

Phytoplasmas infecting sour cherry and lilac represent two distinct lineages having close evolutionary affinities with clover phyllody phytoplasma

Phytoplasmas infecting sour cherry and lilac in Lithuania were found to represent two lineages related to clover phyllody phytoplasma (CPh), a subgroup 16SrI-(R/S)C (formerly 16SrI-C) strain exhibiting rRNA interoperon sequence heterogeneity. 16S rDNAs amplified from the cherry bunchy leaf (ChBL) and lilac little leaf (LcLL) phytoplasmas were identical or nearly identical to those of operon rrnA and operon rrnB, respectively, of CPh. There was no evidence of 16S rRNA interoperon sequence heterogeneity in either LcLL or ChBL phytoplasma. Based on collective RFLP patterns of 16S rDNA, ChBL was classified in subgroup 16SrI-R, and LcLL was classified in new subgroup 16SrI-S. The ribosomal protein (rp) gene sequences from LcLL phytoplasma were identical to those of CPh, and strain LcLL was classified in rp subgroup rpI-C. By contrast, rp gene sequences from ChBL phytoplasma differed from those of subgroup rpI-C; based on RFLP patterns of rp gene sequences, ChBL was classified in new rp subgroup rpI-O. Single nucleotide polymorphisms (SNPs), designated here by a new SNP convention, marked members of rp subgroup rpI-C, and distinguished LcLL and CPh from ChBL and other non-rpI-C phytoplasmas in group 16SrI. The results raise questions concerning phytoplasma biodiversity assessment based on rRNA genes alone and encourage the supplemental use of a single copy gene in phytoplasma identification and classification, while drawing attention to a possible role of horizontal gene transfer in the evolutionary history of these lineages.     

Haplotypes of “<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter solanacearum” suggest long-standing separation

Three haplotypes of the recently discovered bacterium species “Candidatus Liberibacter solanacearum” are described and related to geographic ranges. The first two are associated with Zebra Chip/Psyllid Yellows of potatoes and other solanaceous plants, vectored by the tomato/potato psyllid Bactericera cockerelli in North and Central America and New Zealand. The third is associated with diseased carrots in Finland and vectored by the carrot psyllid Trioza apicalis. The haplotypes are described by SNPs on the 16s rRNA, 16s/23s ISR and 50s rplJ and rplL ribosomal protein genes. These SNPs are inherited as a package across the three genes. Haplotype “a” has been found primarily from Honduras and Guatemala through western Mexico to Arizona and California, and in New Zealand. Haplotype “b” is currently known from eastern Mexico and northwards through Texas to south central Washington. These haplotypes show some range overlap in Texas, Kansas and Nebraska. The haplotypes are not yet known to elicit biological differences in the plant or insect hosts. These apparently stable haplotypes suggest separate bacterial populations of long standing.