Response to Mycosphaerella pinodes in a germplasm collection of Pisum spp

Little resistance against Mycosphaerella pinodes is available in pea. In this work 78 accessions of Pisum were screened for resistance to M. pinodes. Fourteen accessions showed a good level of resistance in seedlings under controlled conditions, and in mature plants in the field. The highest levels of resistance were found in P. fuivum, followed by P. sativum ssp. eiatius and P. sativum ssp. syriacum. Resistance of five selected accessions was effective against different isolates of M. pinodes originating from different countries. Resistant accessions reported in this paper have been successfully hybridized with field pea cultivars.     

Diversity analysis and genotyping in Pisum with sequence tagged microsatellite site (STMS) primers

Pisum sativum specific sequence tagged microsatellite site primers were used to amplify genomic profiles from 15accessions of P. sativum L. that represented the genetic base of the Australian field pea-breeding program and five accessions of the wild related species P. fulvum. The STMS primers were used to assess genetic relationships among the Pisum accessions in two ways. Firstly, to produce RAPD-like multiple banding marker profiles using an adapted RAMS method, for intra- and interspecific diversity analysis. From the 14 flanking primer pairs assessed, 133 markers were obtained. Conservation and reproducibility of markers among individuals within accessions was demonstrated. The largest distance observed among P. sativumaccessions was 22% and among P.fulvum accessions was 40%, similar to that revealed with other PCR-based methods. The maximum distance between P.sativum and P. fulvum accessions was 46%. Phylogenetic clustering of P. sativum accessions, using the neighbour joining method and based on simple matching distances, was distinct and distant to P. fulvum. Secondly, PCR with a higher annealing temperature and fluorescent labeling identified simple and allelic loci markers useful for creating agenotype/fingerprint database for P. sativum cultivars. This is the first report to demonstrate the use of Pisum specific STMS sequences for both diversity analysis and genotype identification. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification and characterization of sources of resistance to Erysiphe pisi Syd. in Pisum spp.

A collection of 67 accessions of Pisum species originating from different countries was screened in a glasshouse test for resistance to Erysiphe pisi. All Pisum fulvum accessions were completely resistant. Incomplete resistance was identified in some accessions of P. sativum subsp. sativum var. arvense and P. sativum subsp. elatius and abyssinicum. Microscopy revealed several distinct cellular mechanisms governing resistance. In P. fulvum, it was mainly due to a high frequency of cell death that occurred both as a rapid response to attempted infection and a delayed response that followed colony establishment. Cell death following colony establishment was also key to the incomplete resistance in some accessions of P. sativum subsp. sativum var. arvense. In addition, impaired spore germination, and to a lesser extent appressorium formation, contributed to pre‐penetration resistance in some accessions. In some cases, resistance also retarded colony growth, possibly through effects on haustorial development or function in cells that survived the attack. Thus, these wild pea accessions offer diverse resistances that could be introduced to cultivated peas to increase the efficacy of powdery mildew resistance.     

The response of Pisum sativum L. germplasm to high concentrations of soil boron

Summary Tolerance to high levels of boron in the soil is an important aspect of the adaptation of crop varieties to southern Australian conditions. This paper reports investigations aimed at exploring the extent of genetic variation in Pisum sativum and at defining appropriate selection criteria for selection for boron tolerance in breeding programs.A collection of 617 accessions of Pisum was screened in controlled conditions and visually assessed for symptoms of boron toxicity. A high proportion of accessions were sensitive with only 3.5% being more tolerant than any of the Australian varieties. Relatively high proportions of tolerant and moderately tolerant accessions originated from Asia and South America.In a second experiment the responses of selected tolerant accessions were evaluated with respect to different parameters. The objectives were to confirm the performance of the putative boron tolerant accessions and identify appropriate parameters for selecting boron tolerant genotypes. In addition to the visual assessment of boron toxicity, measurements at the time of harvest included dry matter yield and concentration of boron in tissues. Symptom expression was highly correlated with dry matter yield and concentration of boron in tissues under high boron conditions and so could be used as a non-destructive selection criteria. A low degree of symptom expression by tolerant accessions could usually be attributed to low levels of boron in the vegetative tissues. The results of this study indicate that considerable genetic variation exists among exotic accessions of Pisum sativum and tolerance to boron could be transferred to sensitive varieties.     

Overcoming hybridization barriers between pea and some of its wild relatives

Pea would benefit from the plasticity and adaptability of its cross-incompatible relatives Pisum fulvum and Lathyrus sativus L., and we have tested reciprocal sexual crossings by manually cross-pollinating plants of genotypes of these three species. Studies of in situ germination of pollen grains on stigmata showed that pollen tubes were generally unable to germinate or could not reach the ovary. A few putative hybrid pods were nevertheless harvested, with one grain per pod germinated in vitro, then micropropagated for flow cytometry, isoenzyme, molecular (ribosomal ITS PCR-RFLP) and genomic in situ hybridization (GISH) studies. One such grain was recovered from an inter-generic cross of P. sativum x L. sativus and four from an inter-specific P. sativum x P. fulvumcross. A strong cross-incompatibility was shown between pea and grass pea, where the putative hybrid turned out to be pea. Conversely, with the interspecific, P. sativum x P. fulvum cross, flow cytometry and isoenzymes with leaf tissues strongly suggested hybridity, while molecular approaches and GISH confirmed the production of inter-specific hybrids, and without the need for a wild type P. sativum accession as a bridging cross.     

Current status and future strategy in breeding pea to improve resistance to biotic and abiotic stresses

The economic importance and current progress made in studies of the host-parasite relationship and identification of sources of resistance and breeding strategies of some important biotic diseases of pea are reviewed in this paper. The root rot complex caused by Rhizoctonia solani, Fusarium solani, Aphanomyces euteiches, Pythium ultimum and Fusarium oxysporum f. sp. pisi, race 1 and 2 has been reported from all commercial pea growing areas of the world. Adequate sources of resistance have been identified and there has been impressive success in the control of the Fusarium wilt pathogen following the introduction of wilt-resistant cultivars. Leaf and stem diseases of pea caused by the Ascochyta complex, Peronospora viciae and Erysiphe pisi are prevalent in most temperate pea growing regions of the world. Several sources of resistance are available, some of which are surprisingly durable. The biochemical genetic parameters of phenolic content used for assaying resistance to Erysiphe pisi offers an alternative method of evaluating breeding material. Wild relatives of pea (Pisum fulvum and P. humile) are valuable additional sources of genetic variation and provide good sources of resistance to pests and diseases. In temperate rainfed pea growing areas of southern Australia, pea seed yield is more closely related to dry matter production than harvest index. Tall and leafy cultivars proved more productive than afila types.     

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.     

Pea weevil, Bruchus pisorum L. (Coleoptera: Bruchidae), resistance in Pisum sativum×Pisum fulvum interspecific crosses

The pea weevil, Bruchus pisorum (L.), is one of the most intractable pest problems of cultivated pea, Pisum sativum L., in the world. This study investigated the transfer of pea weevil resistance from two accessions (PI 595946, PI 343955) of wild pea, Pisum fulvum Sibth. & Sm., to interspecific populations derived from crossing these accessions with a weevil-susceptible pea cultivar ('Alaska 81'). Partial life tables characterized weevil stage-specific mortality and survivorship on parents and interspecific progeny in two glasshouse trials. Larval mortality rates on pods (F₃ plants) of several F_2:3 families were between 36.0% and 52.9%. These means were statistically similar to mean mortality rates on pods of resistant parents (45.4% and 46.2%), but significantly greater than mean rates on the susceptible parent (1.2% and 10.6%). Pod surface characteristics contributed to high neonate larval mortality on pods of resistant parents and interspecific progeny. Seed resistance was not broadly transferred to interspecific progeny [revealed by high weevil survivorship in seeds (means mostly >80%) and high seed damage ratings of 3–5 where ratings of 1–2 denote resistance (production of resistant seed averaged 4.2% to 22.8%)]. Estimates of total weevil mortality on pods and seeds of eight F_2:3 families were 50–70%. Thus, weevil resistance in the Pisum secondary gene pool can be transferred to interspecific progeny, thereby providing a potential avenue to develop weevil-resistant pea cultivars.     

Inheritance of resistance to pea blight (Ascochyta pinodella) and induction of resistance in pea (Pisum sativum L.)

Summary Pea blight caused by Assochyta pinodella does considerable damage to the pea crop every year. To ascertain the inheritance of resistance to pea blight and incorporate resistance in the commercial cultivars, crosses were made between Kinnauri resistant to pea blight and four highly susceptible commercial pea cultivars — Bonneville, Lincoln, GC 141 and Sel. 18. Studies of the F₁'s, F₂'s, back crosses and F₃'s indicated that Kinnauri carries a dominant gene imparting resistance to pea blight.     

Genetical analysis of resistance to Mycosphaerella pinodes in pea seedlings

Summary In studies of the inheritance of resistance, pea seedlings of seven lines in which stems and leaves were both resistant to Mycosphaerella pinodes were crossed with a line in which they were both susceptible. With seven of the crosses resistance was dominant to susceptibility. When F₂ progenies of five crosses were inoculated on either stems or leaves independently, phenotypes segregated in a ratio of 3 resistant: 1 susceptible indicating that a single dominant gene controlled resistance. F₂ progenies of one other cross gave ratios with a better fit to 9 resistant: 7 susceptible indicating that two co-dominant genes controlled resistance. The F₂ progeny of another cross segregated in complex ratios indicating multigene resistance.When resistant lines JI 97 and JI 1089 were crossed with a susceptible line and leaves and stems of each F₂ plant were inoculated, resistance phenotypes segregated independently demonstrating that leaf and stem resistance were controlled by different genes. In two experiments where the F₂ progeny of the cross JI 97×JI 1089 were tested for stem and leaf resistance separately, both characters segregated in a ratio of 15 resistant:1 susceptible indicating that these two resistant lines contain two non-allelic genes for stem resistance (designated Rmp1 and Rmp2) and two for leaf resistance (designated Rmp3 and Rmp4). Evidence that the gene for leaf resistance in JI 1089 is located in linkage group 4 of Pisum sativum is presented.     

Identification of RAPD markers linked to the rust (<Emphasis Type="Italic">Uromyces fabae</Emphasis>) resistance gene in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>)

Summary Two RAPD markers linked to gene for resistance (assayed as pustule number cm⁻² leaf area) to rust [Uromyces fabae (Pers.) de Bary] in pea (Pisum sativum L.) were identified using a mapping population of 31 BC₁F₁ [HUVP 1 (HUVP 1 × FC 1] plants, FC 1 being the resistant parent. The analysis of genetics of rust resistance was based on the parents, F₁, F₂, BC₁F₁ and BC₁F₂ generations. Rust resistance in pea is of non-hypersensitive type; it appeared to be governed by a single partially dominant gene for which symbol Ruf is proposed. Further, this trait seems to be affected by some polygenes in addition to the proposed oligogene Ruf. A total of 614 decamer primers were used to survey the parental polymorphism with regard to DNA amplification by polymerase chain reaction. The primers that amplified polymorphic bands present in the resistant parent (FC 1) were used for bulked segregant analysis. Those markers that amplified consistently and differentially in the resistant and susceptible bulks were separately tested with the 31 BC₁F₁ individuals. Two RAPD makers, viz., SC10-82₃₆₀ (primer, GCCGTGAAGT), and SCRI-71₁₀₀₀ (primer, GTGGCGTAGT), flanking the rust resistance gene (Ruf) with a distance of 10.8 cM (0.097 rF and LOD of 5.05) and 24.5 cM (0.194 rF and a LOD of 2.72), respectively, were identified. These RAPD markers were not close enough to Ruf to allow a dependable maker-assisted selection for rust resistance. However, if the two makers flanking Ruf were used together, the effectiveness of MAS would be improved considerably.     

Resistance in Lycopersicon species to black leaf mold caused by Pseudocercospora Fuligena

Over 540 accessions of wild Lycopersicon species or their crosses with L. esculentum were screened for resistance in a series of trials. Forty-six accessions were selected for the final screening trial based on lower disease ratings in previous trials. Of these, L. hirsutum had the greatest number of resistant accessions, followed by L. esculentum and L. peruvianum. Twenty accessions were quantified for their levels of resistance based on leaf area infected, area under disease progress curve (AUDPC), and the degree of sporulation. There was a significant positive correlation between the AUDPC calculated from 20 accessions evaluated under growth room and field conditions. Five L. hirsutum accessions had no sporulation associated with leaf lesions, whereas L. esculentum accessions had an average of 1.6×10⁴ conidia/cm² of leaf tissue. There was significant positive correlation between the AUDPC values and the number of conidia per cm² of leaf tissue.     

The use of RAPD technique for the identification and classification of Pisum sativum L. genotypes

Summary The genomic DNAs of 42 Pisum sativum genotypes, representing four wild and cultivated subspecies were used as templates in RAPD reactions. Amplification with eight decamer primers generated 149 polymorphic products. Genetic similarities of RAPD profiles were estimated via a coefficient of Jaccard and then the data were processed by cluster analysis (UPGMA). Each genotype was clearly identified and separated from the others. Our results show that RAPD technology is a rapid, precise and sensitive technique for identification of pea genotypes. However, the phylogenetic relationships within the Pisum sativum, which we tested by bootstrap analysis (Wagner parsimony), must be interpreted with caution.     

Cytological and Morphological Characterization of Pisum sativum and Pisum fulvum Tetraploids

Two colchicine-induced tetraploid lines, one belonging to Pisum sativum and the other to the wild species Pisum fulvum, were compared with their corresponding diploids and among themselves for chromosome number, meiotic behaviour and for pollen and pod fertility. For both lines, five generations were analyzed; the tetraploid level was maintained throughout the generations. In P. fulvum, aneuploids with 27, 29 and 30 chromosomes were observed, while in P. sativum aneuploids with 29 chromomes were found. Meiotic analysis evidenced a higher number of quadrivalents and a lower number of univalents in P. fulvum. This particular chromosomal behaviour may also have been responsible for determining the higher pollen and pod fertility in P. fulvum. Wild species of this genus seem to have better tolerance for ploidy variations.     

A method to select for reproductive frost tolerance in field pea (Pisum sativum L.)

This study describes an investigation to test whether genotypic differences for reproductive frost tolerance in field pea (Pisum sativum L.) can be measured in the field. The method involved individually tagging flowers or young pods at particular stages of development within 48 hr after a frost event and assessing pod survival and seed damage at maturity. Four field pea varieties were grown in 2011 in an experiment which measured the loss of pods following a specific frost event. This experiment also tested the impact of trellis and pathways sown with barley on the efficacy of the frost tolerance data. In 2012, an additional genotype was also tested and, in addition to pod loss, data were collected on seed damage in surviving pods. Results from both years showed significant genotypic differences. There was also a significant positive correlation between mean variety pod loss in 2011 and 2012 indicating reliability of this method across seasons.     

The effect of exogenous DNA on the nodulation of a non-nodulating line of Pisum sativum L.

Summary The nodulation response of a non-nodulating field pea line (Pisum sativum L., PRL-H722) following treatment with DNA from an effectively nodulating cultivar (Trapper) has been investigated. DNA was introduced to the non-nodulating cultivar via imbibition and germination of seeds in DNA solutions under aseptic conditions. The DNA preparations used were >5×10⁶ Daltons, had a smooth melting profile showing 40% hyperchromicity with a Tm=70–72°C in 0.1×SSC⁰ buffer, and 260/280 nm ratio of >1.8. A significant correction (²(I d.f.) = 2032^**) of the mutant phenotype was observed in seeds treated with wild-type DNA. The nodulation response in treated plants was not observed in subsequent generations suggesting that the change observed did not involve integration of genetic information into the genome of germ line cells.NRCC no. 20986     

Sources of variation for sustainable field pea breeding

The pea crop (Pisum sativum L.) is a convenient source of plant protein for animal feeding. The objective of this research was to evaluate field pea breeding lines for agronomic performance and seed quality focussed to their use in a sustainable production. Thirty-five field pea breeding lines and six elite cultivars were evaluated for their agronomic value in field in Spain upon 20 traits related to flower, cycle, plant architecture, productivity and seed quality. The lines showed significant differences in all the quantitative traits evaluated and three of them, namely MB-0307, MB-0308, and MB-0319 were chosen to be evaluated, together with the advanced cultivar ZP-1233, in field and in growing chamber for agronomic performance, seed quality and ability for sustainable production. The four accessions displayed high seed protein content that had significant effect of cropping density with averages of 253.6 g kg⁻¹ under low cropping density of 60 plants m⁻² and 259.1 g kg⁻¹ under high cropping density of 90 plants m⁻², therefore, the low cropping density should be regarded as the most convenient. Average yield of the lines MB-0307, MB-0308 and MB-0319 and the cultivar ZP-1233 was fair (197.5 g m⁻²), probably due to the absence of fertilizers and irrigation, aiming for the sustainability of the crop. Intercropping with rye and herbicide application resulted in no differences on the seed yield; therefore, the ability of the breeding lines to grow without herbicide seems to be demonstrated, while the germination of the seeds at low temperature was very good. These results indicate that field pea could be a new protein crop in the North of Spain to satisfy the demand of plant protein for animal feed, based upon adapted breeding lines that combine the ability for growing under sustainable conditions with other desirable agronomic traits maintaining an adequate productivity.     

Analysis of tissue culture-derived variation in pea (Pisum sativum L.)-preliminary results

Summary The possibility of producing agronomically-useful somaclones via organogenesis and somatic embryogenesis from callus cultures of pea (Pisum sativum L.) was studied. Organogenic calli were induced from immature leaflets on MSB medium with NAA and BAP. Embryogenic calli were derived either from immature zygotic embryos (using 2,4-D) or from shoot apices (using picloram) of aseptically-germinated seedlings.The seed progenies (T₁ to T₃-generation) of primary regenerants were grown in field conditions and their phenotypic variation was evaluated and compared with control, non-tissue culture-derived plant material. In addition, electrophoretic analyses of selected isoenzyme systems and total proteins have been done. The results do not show dramatic changes in qualitative and quantitative traits. The evaluation of at least two future generations (T₄, T₅) is planned.Abbreviations BAP 6-benzylaminopurine - IBA indole-3-butyric acid - MSB medium (mineral salts after Murashige & Skoog, 1962, vitamins after Gamborg et al., 1968) - NAA -naphthalene-acetic acid, picloram-4-amino-3,5,6-trichloro picolinic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - ORG organogenesis - SE somatic embryogenesis     

Maintenance breeding and multiplication of pea and faba bean cultivars. Maintenance of protein peas (Pisum sativum) and field beans (Vicia faba)

Summary The maintenance of protein pea (Pisum sativum)and field bean (Vicia faba)cultivars is a part of the breedingscheme of these pulses at CEBECO-ZADEN. As soon as the cultivar is pure breeding a large quantity ofseed is stored under conditioned circumstances.     

Study of the variation in the somatic embryogenesis ability of some pea lines (Pisum sativum L. and Pisum arvense L.)

Summary Thirty lines of pea were tested in vitro to evaluate their ability to produce somatic embryos. Three distinct genotypic classes were detected (strong, medium and weak). The best responses were obtained in Pisum sativum. Abnormal somatic embryos and secondary embryogenesis seem to constitute the principal obstacle to the development of these structures.