Genetic dissection of bacterial speck disease resistance in tomato

The bacterial speck disease of tomato has been developed as a model system to elucidate the molecular basis of specificity in plant-bacterial interactions and to study signal transduction events involved in the expression of plant disease resistance. We have employed a mutagenic approach to define the steps involved in the expression of disease resistance to the bacterial pathogen, Pseudomonas syringae pv. tomato (Pst) Eleven disease susceptible mutants have been identified and characterized twith altered recognition of Pst strains that express the avirulence gene avrPto. Genetic analysis of these mutants has revealed that they fall into two complementation groups. Five of the mutants map at the Pto locus, while six map a new locus that we have termed Prf. Further characterization of these mutants has revealed that the mutants that map at Pto are still sensitive to the insecticide fenthion, while the prf mutants are altered in resistance and also are insensitive to fenthion. Genetic mapping has also determined that the Prf locus maps near Pto. We are currently employing a map-based cloning strategy to isolate the Prf locus.     

A genomic search for the gene conferring resistance to fusarium wilt in tomato

Fusarium wilt is an economically important disease of tomatoes, caused by the soil-born fungus Fusarium oxysporum f. sp. lycopersici. There are three host-specific races of this pathogen. The dominant tomato gene I-2 confers resistance to race 2. The I-2 fusarium resistance gene was mapped genetically to chromosome 11 of tomato, between the RFLP markers TG105 and TG36, 0.4 centiMorgan (cM) from TG105. A mean value of 43 kb for each cM was assigned in the vicinity of I-2. We have generated new RFLP markers in the region by chromosome walking from TG105 towards I-2 on lambda clones, and by subcloning a 350 kb long YAC clone (YAC 8) that contains TG105. These RFLP markers were mapped physically on YAC 8 by PFGE. The location of I-2 relative to these markers was genetically estimated using a recombinant inbred (RI) segregating population. The order of the markers according to the RI population is inconsistent with their order on the physical map. A cDNA clone, D14, that was isolated by YAC 8, turned out to be 53% similar to xanthine dehydrogenase from mammals and flies. Antibodies raised against a part of the protein encoded by D14 recognize cross reacting material of MW 80 kD, that is highly enriched in nodules of legumes, and seems to be induced by various environmental and pathogenic stress conditions.     

Molecular genetic characterisation of the Asc locus of tomato conferring resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici

The Alternaria stem canker disease of tomato is caused by the fungal pathogen Alternaria alternata f. sp. lycopersici and its host-selective AAL-toxins. Resistance to the pathogen and insensitivity to the toxins are conferred by the Asc locus on chromosome 3L. Sensitivity to AAL-toxins is a relative character; the toxins inhibit development of all tested tomato tissues but susceptible cultivars are much more sensitive than resistant cultivars. In addition to tomato, some other plant and animal species are sensitive to the toxins as well. The likely mode of action of AAL-toxins is interference with sphingolipid biosynthesis by specific inhibition of ceramide synthase activity. To molecularly isolate Asc, transposon tagging and positional cloning strategies are applied. As a first step, transposon insertions and restriction fragment length polymorphism (RFLP) markers are identified in proximity of the Asc locus. Subsequently, the transposons are used to inactivate Asc by insertion mutagenesis, and the RFLP markers are used to identify yeast artificial chromosomes (YACs) with tomato DNA inserts. Once an Asc-insertion mutant and/or a YAC encompassing Asc has been obtained, physical isolation and characterisation of Asc will be conceivable. Elucidation of the molecular role of Asc will illuminate the specificity of host recognition by Alternaria alternata f. sp. lycopersici.Abbreviations AAL-toxin Alternaria alternata lycopersici-toxin - A. a. lycopersici Alternaria alternata f. sp. lycopersici - Asc Alternaria stem canker - HST host-selective toxin     

RFLP characterization of Indian Musa germplasm for clonal identification and classification

Summary Nineteen single-copy clones isolated from a PstI genomic library (cv. Maiden Plantain), and eight Vigna chloroplast DNA clones were used to probe total genomic DNA digests of 57 genotypes of Musa from India. The 19 genomic clones detected a total of 107 polymorphisms among the 57 genotypes. Principal coordinates and phenetic analyses of these data placed cultivars and species into distinct groups that were in general agreement with a previously published RAPD-based classification of these same plant materials. The 107 polymorphisms were sufficient to differentiate each clone from every other clone. Heterologous Vigna chloroplast DNA probes were used to characterize the cytoplasm of Musa cultivars and species. PCO analysis of these RFLPs were detected both within and between the generally recognized genome groups, indicating multiple hybridization pathways in the origin of hybrid clones. Data presented demonstrate that RFLPs are sufficiently abundant to classify Musa germplasm and that genetic relationships among Musa cultivars, based upon RFLP data, are in general agreement with relationships determined by analysis of morphology and RAPDs.     

Studies on resistance to bacterial speck (Pseudomonas syringae pv. tomato) in tomato cv. Ontario 7710

Plants of 17 tomato cultivars and four wild Lycopersicon accessions were evaluated for their reaction to Pseudomonas syringae pv. tomato (Pst) in a greenhouse following a leaf‐spray inoculation. The genotypes exhibited a large amount of variation in response to Pst infection, with disease severity index (DSI) ratings from 0.2 to 3.9. The cultivar ‘Ontario 7710’ and two accessions of Lycopersicon hirsutum (LA 1773 and LA 1775) were the most resistant, with DSI values of 0.2, 0.4 and 0.6, respectively. Three varieties, M 1812, Kujawski and Warszawski, also showed a high level of tolerance. The most susceptible was ‘A 100’(DSI = 3.9). The inheritance of resistance to bacterial speck was investigated by disease tests in segregated populations obtained by hybridizing the tomato cv. Ontario 7710 with the susceptible variety ‘A 100′. Plants were rated for disease severity by inspecting each plant and were then evaluated according to phenotypic similarity to ‘Ontario 7710’ or ‘A 100’ in respect of the number and size of the spots. Genetic analysis in F1, F2 and backcross segregations indicated that resistance of'Ontario 7710’ to Pst is conferred by one incompletely dominant gene, Pto.     

Inheritance of resistance to angular leaf spot in Nicotiana tabacum L. cultivars burley 21 and kentucky 14

Summary The genetics of resistance to angular leaf spot caused by Pseudomonas syringae pv. tabaci in Nicotiana tabacum cultivars Burley 21 and Kentucky 14 was investigated by studying disease reactions to three isolates of parental, F₁, F₂ and backcross generations derived from crosses between the resistant cultivars and the susceptible cultivar Judy's Pride. Studies were conducted in the greenhouse and in field plant beds. Chi-square values were computed to determine whether the observed ratios for disease reactions deviated from expected Mendelian ratios for a single, dominant gene controlling resistance to angular leaf spot in tobacco. Based on the resistance of the F₁ and the backcross generation to the resistant parent (BC-R), a 3 resistant: 1 susceptible segregation ratio in the F₂, and a 1 resistant: 1 susceptible segregation ratio in the backcross generation to the susceptible parent (BC-S), it was concluded that resistance to three isolates of Pseudomonas syringae pv. tabaci is governed by a single, dominant gene.     

Testing inoculation methods and sources of resistance to the halo blight bacterium (Pseudomonas syringae pv. phaseolicola) in Phaseolus vulgaris

Summary A comparative test of six inoculation methods was conducted using 2 halo blight race 2 virulent strains, Nebr. HB 16 and HB 21 (Pseudomonas syringae pv. phaseolicola), on five dry bean cultivars/lines (Phaseolus vulgaris L.) of known resistance and susceptibility. The water-soaking of leaves method caused the most severe reaction among the leaf inoculation methods, followed by the carborundum, spraying and multiple needle methods, respectively. The seed soaking method was considered too severe to be useful, since entries identified as resistant by the other methods, were susceptible with the former method. Great Northern Nebraska # 1 sel. 27 and PI 150414 had the highest level of leaf resistance, but the former developed systemic chlorosis with the stem stabbing method, but not the latter line. No systemic chlorosis was seen in either line with the other methods of inoculation. This suggests that there may be a different genetic mechanism conferring resistance/susceptibility to the toxin in these two lines when the stabbing method is used. No interaction occurred between method by genotype and isolate by method but significant interactions occurred between genotype by isolate and method by isolate by genotype. The leaf and pod reaction of forty cultivars/lines to the new halo blight Nebr. Charlevoix strain was also determined. Different combinations of degrees of resistance and susceptibility of leaves and pods were observed. GN Tara, GN Harris, and PI 150414 had the highest combination of leaf and pod resistance.Published as paper No. 7094, Journal Series, Nebraska Agricultural Experiment Station. Research was conducted under Project No. 20-036.     

A rapid screening technique to combine resistance to halo blight and bean common mosaic virus in Phaseolus vulgaris L.

Summary Seven bean lines (Phaseolus vulgaris L.) with differential resistance or susceptibility to race 2 of halo blight (Pseudomonas syringae pv. phaseolicola) and a necrosis-inducing isolate of bean common mosaic virus were inoculated with one or both pathogens in combination, to determine the feasibility of dual screening to identify resistance to both pathogens simultaneously. Dual screening yielded the same results as separate screenings. Neither pathogen affected the disease expression of the other. Simultaneously screening for resistance to both pathogens will shorten the recurrent screening-selection cycle of hybridization programs, and accelerate development of resistant cultivars.Abbreviations BCMV Bean Common Mosaic Virus - cvs Cultivars - HB Halo blight - Inoc. Pt. Inoculation point - NLL Necrotic local lesion - React Reaction - SVN Spreading veinal necrosis, System chloro-Systemic chlorosis - VN Vascular necrosis     

Molecular mapping of a new gene for resistance to rice blast (Pyricularia grisea Sacc.)

A single dominant blast resistance gene conferring resistance to a Korean rice blast isolate was identified in rice variety `Suweon 365'. We report the chromosomal localization and molecular mapping of this blast resistance gene designated as Pi-18, which confers resistance to Korean isolate `KI-313' of the blast pathogen. To know whether there is a relationship among genes conditioning resistance to location-specific isolates of the blast pathogen and thereby to identify linked markers to resistance gene for isolate KI-313 collected in Korea, RFLP markers previously reported to be linked to major blast resistance genes in different rice germplasm and other markers mapped to nearby regions were surveyed for polymorphism between a resistant (`Suweon 365') and a susceptible (`Chucheongbyeo') parent. Linkage associations of the RFLP markers with the resistance gene were verified using an F2 and F3 segregating population of known blast reaction. RFLP analysis showed that Pi-18 was located near the end of chromosome 11, linked to a single copy clone RZ536 at a distance of 5.4 centiMorgans (cM) and that this gene was different from Pi-1(t). An allelism test revealed that this gene was also different from Pi-k. Currently, a combination of RAPD and microsatellite primers is being employed to find additional markers in this region. Tightly linked DNA markers will facilitate selection for resistant genotypes in breeding programs and provide the basis for map based cloning of this new blast resistance gene. This revised version was published online in July 2006 with corrections to the Cover Date.     

RFLP variation and genetic relationships in cultivated cucumber

Summary Two sets of cucumber (Cucumis sativus L.) germplasm were used to determine the potential use of restriction fragment length polymorphisms (RFLPs) for estimating genetic relationships. Sixteen accessions [15 domesticated variety sativus and one feral variety hardwickii (PI 183967)] of diverse origin were used to assess RFLP variation in cucumber, and to determine if genetic relationships based on RFLPs were similar to those obtained by isozyme analysis. Additionally, 35 commercial lines or cultivars were surveyed to determine genetic relationships among and within common cucumber types (narrow genetic base). The 16 accessions were surveyed with 440 low copy clones from two libraries (Pst I partial genomic and cDNA) using two restriction enzymes. Data from a subset of 104 random (mapped and unmapped) and a set of 30 mapped RFLPs were used to estimate genetic relationships among the 16 cultigens. Variability was low among RFLPs (33% of all probes) and putative alleles ( 2.2 polymorphic fragments/probe). RFLP variation between sativus lines and hardwickii (21±4%) was greater than among sativus lines (12±2%). RFLPs among the 16 accessions revealed genetic relationships which agree with those obtained using isozymes. Genetic relationships estimated using mapped and unmapped RFLPs were similar. The 35 elite lines were surveyed using a set of 40 RFLPs from 3 libraries (Pst I and EcoR I partial genomic and cDNA) to evaluate the discriminatory value of RFLPs among and between commercial cucumber types. The RFLP-derived genetic relationships among this germplasm were in agreement with predictions based on fruit type and pedigree information. Thus, RFLPs are a useful addition to the morphological characters and isozyme loci currently used for taxonomic classification and plant variety protection of cucumber.     

Resistance to Pseudomonas solanacearum in the potato: I. Effects of sources of resistance and adaptation

Summary F1 hybrid true potato seed progenies with multiple sources of specific resistance to Pseudomonas solanacearum and adaptation were evaluated under tropical greenhouse conditions for resistance to a race 1 isolate of P. solanacearum. Results indicated that genes for adaptation are involved in conferring resistance to bacterial wilt. The effect of a particular resistant parent clone on the resistance level of its progeny depended on the resistance, adaptation or the combination of both characteristics of its mate. A heat tolerant parent gave a higher frequency of resistant offspring in combination with an ascertained source of resistance. Combining ability was an apparent feature of resistance to bacterial wilt and resistance was highest in the majority of the crosses that had a wide genetic background for both resistance and adaptation. The possible genetic nature of the resistance to bacterial wilt is discussed.     

Cytoplasmic genetic diversity in bananas and plantains

Summary Concerns over yield declines in bananas and plantains due to the spread of Black Sigatoka disease in Musa have drawn attention to the collection of Musa germplasm and its use in conventional and biotechnological improvement programs. This report demonstrates the use of chloroplast DNA (cpDNA) restriction fragment length polymorphisms (RFLPs) for differentiating cytoplasms of various Musa clones. DNA was extracted from lyophilized leaf blade tissue and digested with either Eco R1, Hind III, Bam H1 or Pst I. Southern blots onto nylon membranes were probed with radioactively labeled heterologous orchid and lettuce cpDNA fragments. Among the 14 Musa clones examined, a single balbisiana and four acuminata-type cytoplasms were differentiated. The ability to distinguish between cytoplasms and to place plants within a cytoplasmic grouping demonstrates the usefulness of RFLP technology in evaluating diversity and determining the ancestry of Musa clones.     

Identification of quantitative trait loci involved in resistance to <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">syringae</Emphasis> in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

Pseudomonas syringae is the main pathogen responsible for bacterial blight disease in pea and can cause yield losses of 70%. P. syringae pv. pisi is prevalent in most countries but the importance of P. syringae pv. syringae (Psy) is increasing. Several sources of resistance to Psy have been identified but genetics of the resistance is unknown. In this study the inheritance of resistance to Psy was studied in the pea recombinant inbred line population P665 × ‘Messire’. Results suggest a polygenic control of the resistance and two quantitative trait loci (QTL) associated with resistance, Psy1 and Psy2, were identified. The QTL explained individually 22.2 and 8.6% of the phenotypic variation, respectively. In addition 21 SSR markers were included in the P665 × ‘Messire’ map, of which six had not been mapped on the pea genome in previous studies.     

Resistance to bacterial blight (Pseudomonas syringae pv. pisi) in Spanish pea (Pisum sativum) landraces

Pea bacterial blight (Pseudomonas syringae pv. pisi) has long been known to be present in pea growing areas of Spain and to cause serious crop losses, although there is no published record of its occurrence. A collection of 16 isolates from a winter pea trial in Valladolid in 1991 which were shown in this study to be P.s. pv. pisi races 4 and 6 would appear to be the first published record of the disease in Spain. This occurrence of races 4 and 6 is the same as reported for winter-sown peas in the South of France. Ten Pisum sativum landraces from different geographical areas of Spain and considered to be representative of the traditional pea crop, were tested for resistance to seven races of P.s. pv. pisi. Seedlings of each landrace were stem inoculated with the type strain of each race in a glasshouse. Resistance exhibited by the different landraces mainly conformed to those previously described in pea cultivars indicating various combinations of the main resistance genes: R3, R2+4, R3+4 and R2+3+4. R3 was the most frequent R gene, being present in all landraces. R4 was present in four and R2 in three of the landraces tested. Variation for resistance within landraces was limited except for landrace accessions ZP-0102, ZP-0109 and ZP-0137 which also showed variation for morphological traits. The resistance responses of landrace ZP-0109 were difficult to interpret, but suggested a genetic mixture with some evidence of less well documented R genes, R5 and/or R6, and possibly some unknown resistance to race 6. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic mapping of quantitative resistance to race 5 of Pseudomonas syringae pv. phaseolicola in common bean

Halo-blight is an important worldwide bacterial disease of common bean (Phaseolus vulgaris L.) caused by Pseudomonas syringae pv. phaseolicola. Nine races of the pathogen and five race-specific resistance genes have been previously described. However, a quantitative response to this pathogen has also been described. The objective of this study was to identify halo-blight resistance loci linked to molecular markers that could be used in resistance breeding. Chromosomal regions related to race 5 halo-blight resistance were localized on a genetic map of RAPD and AFLP molecular markers and constructed by the analysis of a “Jules” × “Canela” F₂ progeny. “Jules” shows quantitative resistance to halo-blight and “Canela” is a very appreciated but susceptible Spanish bean landrace. Two QTL for resistance to halo-blight were mapped in two linkage groups. There were four large groups, with 14–22 molecular markers each, five with 4–8 markers each, and three with 2 or 3 markers each.     

Genetic analysis of forage grasses based on heterologous RFLP markers detected by rice cDNAs

Genetic polymorphism within and between three species of forage grasses, perennial ryegrass (Lolium perenne L), meadow fescue (Festuca pratensis Huds.) and tall fescue (Festuca arundinacea Schreb.), was analyzed using restriction fragment length polymorphism (RFLP) markers detected by rice cDNA probes developed at the Rice Genome Research Programme of Japan (RGP). One hundred and ninety‐seven rice cDNA clones were used for hybridization to genomic DNA of forage grasses. Many of the rice cDNA clones produced no visible band or only a smear with no discrete bands. Twenty‐three clones showed high efficiency cross‐hybridization to the genomic DNA of forage grasses. Genetic variation was evaluated for five varieties and one population of forage grasses using 12 polymorphic rice cDNA RFLP probes. Genetic variability within varieties as measured by Rogers’ genetic distance was considerably lower for the F. pratensis variety ‘Tomosakae’ than for the L. perenne and F. arundinacea varieties. To determine the genetic diversity between varieties of different species, cluster analysis was performed using data from the 12 RFLP probes. The two accessions of Lolium perenne were clustered more closely together than the three varieties of F. arundinacea. Two Japanese varieties of F. arundinacea were grouped in the same cluster. The variety‐specific RFLP markers were seen among six accessions of L. perenne, F. pratensis and F. arundinacea. Such variety‐specific RFLP markers would provide very useful tools for breeding programmes such as the intergeneric hybridization of Lolium and Festuca genera.     

Assessment of somaclonal variability in hop (Humulus lupulus L.) in vitro meristem cultures and clones by molecular methods

In vitro meristem tissue cultures are used for production of virus-free rootstocks of hop (Humulus lupulus L.). Because use of plant tissue cultures is associated with occurrence of somaclonal variability, we assessed somaclonal variability in hop meristem in vitro cultures before and after thermotherapy by different molecular methods (RFLP, RAPD, STS, ISSR and AFLP) and compared it with existing clonal variability of Osvald's clones 31, 72 and 114. No molecular differences were observed between mother plants and in vitro mericlones by RFLP and STS analyses. Amplified molecular differences were found in RAPD and ISSR products of one from five in vitromericlones cvs. Eroica (E5) and Southern Brewer (SB2), respectively. Similarities with mother plants were 0.965 and 0.913 (JSC), respectively. Specific amplified polymorphic products were found for every mericlone and mother plant in AFLP reactions and variability of DNA sequence ranged from 0.824 to 0.993 (JSC). This variability was very similar to determined intra-clonal variability within Osvald's clones 31, 72 and 114 by AFLP analysis. Inter-clonal variability of DNA sequence was exactly higher than intra-clonal variability of DNA sequence in these clones. The molecular differences between Osvald's clone 72 normal and meristem derived were not verifiable. Thermotherapy increased frequency of molecular changes, since amplified differences were found in 14 from 20 in vitro mericlones of cv. Eroica, in 6 from 11 in vitro mericlones of cv. Yeoman and in 15 from 23 in vitro mericlones of cv. Southern Brewer by RAPD and ISSR analyses.     

Mitochondrial DNA variation in finger millet (Eleusine coracana L. Gaertn)

Summary This study was conducted to classify 26 lines of finger millet from Africa and India into cytotype groups based on the Southern blot hybridization patterns obtained with maize and sorghum mitochondrial cloned gene probes. Five restriction endonuclease enzymes were used for single digestions on total cellular DNA, giving a total of 20 enzyme/probe combinations. There was a low level of polymorphism, with identical RFLP banding patterns in 23 of the 26 lines. However, mtDNA clone atp9 hybridized to a 3.6 Kb Xba1 fragment in ecotype SDFM-1143 from Malawi; but did not hybridize to a 3.0 Kb fragment present in all other ecotypes. Two Zimbabwean lines, SDFM 63 and SDFM 77, had an extra, low intensity 6.5 Kb Xba1 fragment hybridized by mtDNA clone cox1. This data enabled classification of the lines into 3 cytopype groups.     

Interactions between plant and aphid genotypes in resistance of lettuce to Myzus persicae and Macrosiphum euphorbiae

Summary Six lettuce lines, representing two types of resistance to the green peach aphid, Myzus persicae, and a control line with high susceptibility to M. persicae were tested for resistance to six different clones of Myzus persicae and two clones of the potato aphid, Macrosiphum euphorbiae.The clones of M. persicae showed very different levels of aggressiveness on lettuce: two had a high level of reproduction, two had an intermediate level and two were poorly adapted to lettuce as a host. Differences between lettuce lines in aphid reproduction increased with increasing aggressiveness of the aphid clone, which means that aggressive clones are most effective for selection purposes. No evidence was found for clone-specific plant genotype reactions, meaning that lines resistant to one clone will also be resistant to other clones of M. persicae, allthough not neccessarily at the same level. The lettuce lines selected for partial resistance to the aggressive clone WMp1 were completely or almost completely resistant to less aggresive clones.No differences in level of reproduction were found between the two clones of M. euphorbiae and no relation was observed between resistance to M. persicae and M. euphorbiae, indicating the species-specific character of resistance to leaf aphids in lettuce.