An improved test for evaluating the pathogenicity of isolates of Fusarium solani f.sp. pisi on pea

An improved in vitro test is described for determining the pathogenicity of Fusarium solani f.sp. pisi isolates on pea. This technique involves the use of polypropylene fibre Milcap plugs to suspend peas in boiling tubes containing spore suspensions in 0.1% water agar. Results were available after 14 days of incubation at 25°C. Four levels of pathogenicity were detected on pea cultivars Little Marvel and Dark Skinned Perfection using a total of eight isolates and strains of F. solani f.sp. pisi.     

A potential use of wild pea as a source of lower trypsin inhibitor activity

The aim of this work was to estimate the trypsin inhibitor activity (TIA) in seeds of cultivated pea varieties, wild varieties and selected crosses between varieties from the first group and wild pea varieties and to study the variation in genes coding trypsin inhibitors. Mean TIA in field pea varieties ranged from 3.12 TIU/mg of sample in field pea variety from Czech Republic to 12.90 TIU/mg of sample in field pea variety FP S4 of Serbian origin. Wild field pea varieties showed TIA between 0.98 TIU/mg of sample in Pisum elatius and 9.79 TIU/mg of sample in Pisum abyssinicum. Selected crosses between cultivated field pea varieties and Pisum elatius showed a decrease in TIA in comparison with a parent line that has higher TIA content. The PCR amplification resulted in variety-specific amplification. Varieties with low TIA activity showed amplification with At13/At5 primer pair, while varieties with higher TIA activity showed amplification with primer pairs At12/At5, At14/At5 and At14/At8. Thus, At13/At5 primer pair could be sufficient to distinguish most varieties. These markers can be applied during an early screening of the valuable materials for future breeding programs of pea cultivars with the low level of tripsin inhibitor.     

Effect of Alternaria solani culture filtrate on adventitious shoot regeneration in potato

Summary The effect of Alternaria solani culture filtrate on adventitious shoot regeneration from tuber discs was evaluated using five potato cultivars, which were selected based on their field reaction to Alternaria solani and which represented a range of disease reactions. The culture filtrate stimulated regeneration, a response that could prove to be very useful in the wider utilization of transformation and in vitro selection technology.Research conducted at the Scottish Crop Research Institute during a transfer of work of the senior author     

Chitosan as a Component of Pea-Fusarium solani Interactions

Chitosan, a polymer of β-1,4-linked glucosamine residues with a strong affinity for DNA, was implicated in the pea pod-Fusarium solani interaction as an elicitor of phytoalexin production, an inhibitor of fungal growth and a chemical which can protect pea tissue from infection by F. solani f. sp. pisi. Purified Fusarium fungal cell walls can elicit phytoalexin production in pea pod tissue. Enzymes from acetone powders of pea tissue release eliciting components from the F. solani f. sp. phaseoli cell walls. Hydrochloric acid-hydrolyzed F. solani cell walls are about 20% glucosamine. The actual chitosan content of F. solani cell walls is about 1%. However, chitosan assays and histochemical observations indicate that chitosan content of F. solani spores and adjacent pea cells increases following inoculation. Dormant F. solani spores also accumulate chitosan. Concentrations of nitrous acid-cleaved chitosan as low as 0.9 microgram per milliliter and 3 micrograms per milliliter elicit phytoalexin induction and inhibit germination of F. solani macroconidia, respectively. When chitosan is applied to pea pod tissue with or prior to F. solani f. sp. pisi, the tissue is protected from infection.     

Employing a Crude Toxin Preparation from Sarocladium oryzae as a Molecular Sieve to Select Sheath Rot-resistant Somaclones of Rice

This study was undertaken to explore the possibilities of using in vitro techniques for selection of sheath rot-resistant soma clones of rice cv Co43. Oryza nivara and O. longistaminata possessing sheath rot-resistance were included in the study, with a view to comparing the reaction of the susceptible somaclones of O. Sativa cv Co43 to a crude toxin preparation from culture filtrate of the pathogenic fungus, Sarocladium oryzae. A few somaclones of Co43, selecfed on the crude toxin preparation, regenerated into plantlets and their reaction to the toxin was reconfirmed up to R₁ generation.     

Antigibberellin-induced reduction of internode length favors in vitro flowering and seed-set in different pea genotypes

In vitro flowering protocols were developed for a limited number of early flowering pea (Pisum sativum L.) cultivars. This work was undertaken to understand the mechanisms regulating in vitro flowering and seed-set across a range of pea genotypes. Its final goal is to accelerate the generation cycle for faster breeding novel genotypes. We studied the effects of in vivo and in vitro applications of the antigibberellin Flurprimidol together with radiation of different spectral compositions on intact plants, plants with the meristem removed, or excised shoot tip explants. Based on our results, we present a simple and reliable system to reduce generation time in vitro across a range of pea genotypes, including mid and late flowering types. With this protocol, more than five generations per year can be obtained with mid to late flowering genotypes and over six generations per year for early to mid flowering genotypes.     

Screening for fire blight resistance in apple (Malus pumila) using excised leaf assays from in vitro and in vivo grown material

The apple cultivars Greensleeves and Novole and somaclones derived from Greensleeves were tested for resistance to fire blight (Erwinia amylovora) using excised leaf bioassays. Cv. Greensleeves was consistently susceptible to fire blight in in vitro, and in glasshouse and excised leaf bioassays whilst cv. Novole was resistant. Greensleeves somaclones, identified as having intermediate levels of fire blight resistance in glasshouse and in vitro screening tests, also showed intermediate levels of resistance in excised leaf bioassays. The length of leaves and the inoculum concentration employed affected the severity of symptoms observed. Excised leaf bioassays were unsuitable as tests for fire blight resistance using field grown material.     

Heritable variability induced by the in vitro culture and genetic improvement of cultivated plants

Abstract

Genetic variability is found among plants derived from in vitro cultures of somatic cells. A number of different factors, such as the pre-existing genetic variation developed in vivo during tissue differentiation, the variation induced during the in vitro culture and also the selection for specific genotypes during plant regeneration, are considered as possible causes of the phenomenon.

The nature of the genetic changes induced in somaclones (variation in chromosome number, gross and cryptic chromosomal rearrangements, transposition of genetic elements, gene amplification and somatic gene rearrangements) is also discussed.     

Physicochemical properties and in vitro digestibility of flour and starch from pea (Pisum sativum L.) cultivars

Flours and isolated starches from three different cultivars (1544-8, 1658-11 and 1760-8) of pea grown under identical environmental conditions were evaluated for their physicochemical properties and in vitro digestibility. The protein content, total starch content and apparent amylose content of pea flour ranged from 24.4 to 26.3%, 48.8 to 50.2%, and 13.9 to 16.7%, respectively. In pea starches, the 1760-8 showed higher apparent amylose content and total starch content than the other cultivars. Pea starch granules were irregularly shaped, ranging from oval to round with a smooth surface. All pea starches showed C-type X-ray diffraction pattern with relative crystallinity ranging between 23.7 and 24.7%. Pea starch had only a single endothermic transition (12.1-14.2 J/g) in the DSC thermogram, whereas pea flour showed two separate endothermic transitions corresponding to starch gelatinization (4.54-4.71 J/g) and disruption of the amylose-lipid complex (0.36-0.78 J/g). In pea cultivars, the 1760-8 had significantly higher setback and final viscosity than the other cultivars in both pea flour (672 and 1170 cP, respectively) and isolated starch (2901 and 4811 cP). The average branch chain length of pea starches ranged from 20.1 to 20.3. The 1760-8 displayed a larger proportion of short branch chains, DP (degree of polymerization) 6-12 (21.1%), and a smaller proportion of long branch chains, DP ≥ 37 (8.4%). The RDS, SDS and RS contents of pea flour ranged from 23.7 to 24.1%, 11.3 to 12.8%, and 13.2 to 14.8%, respectively. In pea starches, the 1760-8 showed a lower RDS content but higher SDS and RS contents. The expected glycemic index (eGI), based on the hydrolysis index, ranged from 36.9 to 37.7 and 69.8 to 70.7 for pea flour and isolated pea starch, respectively.     

Hypervariable 3′ UTR region of plant LTR-retrotransposons as a source of novel satellite repeats

The repetitive sequence PisTR-A has an unusual organization in the pea (Pisum sativum) genome, being present both as short dispersed repeats as well as long arrays of tandemly arranged satellite DNA. Cloning, sequencing and FISH analysis of both PisTR-A variants revealed that the former occurs in the genome embedded within the sequence of Ty3/gypsy-like Ogre elements, whereas the latter forms homogenized arrays of satellite repeats at several genomic loci. The Ogre elements carry the PisTR-A sequences in their 3′ untranslated region (UTR) separating the gag-pol region from the 3′ LTR. This region was found to be highly variable among pea Ogre elements, and includes a number of other tandem repeats along with or instead of PisTR-A. Bioinformatic analysis of LTR-retrotransposons mined from available plant genomic sequence data revealed that the frequent occurrence of variable tandem repeats within 3′ UTRs is a typical feature of the Tat lineage of plant retrotransposons. Comparison of these repeats to known plant satellite sequences uncovered two other instances of satellites with sequence similarity to a Tat-like retrotransposon 3′ UTR regions. These observations suggest that some retrotransposons may significantly contribute to satellite DNA evolution by generating a library of short repeat arrays that can subsequently be dispersed through the genome and eventually further amplified and homogenized into novel satellite repeats.     

Development and Study of SCAR Markers in Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

In order to develop more specific markers that characterize particular regions of the pea genome, the data on nucleotide sequences of RAPD fragments were used for choosing more extended primers, which may be helpful in amplifying a fragment corresponding to the particular DNA region. Of the 14 STS markers obtained from 14 polymorphic RAPD fragments, 12 were polymorphic, i.e., they are SCAR markers that can be used in genetic analysis. The transition from complex RAPD spectra to amplification of a particular SCAR marker substantially facilitates analysis of large samples for the presence or absence of the examined fragment. Inheritance of the developed SCAR markers was studied in F₁ and F₂. SCAR markers were used to identify various pea lines, cultivars, and mutants. It was established that the study of amplification of STS markers in various pea genotypes at varying temperatures of annealing and the comparison with amplification of the original RAPD fragments in the same genotypes provide an approach for analysis of RAPD polymorphism origin.     

Growth characteristics and ion contents of non-selected and salt-selected callus lines of highbush blueberry (Vacdnium corymbosum) cultivars Blue Crop and Denise Blue

Non-selected and sodium chloride selected callus lines of Vacdnium corymbosum L.cv Blue Crop and cv. Denise Blue were grown on media supplemented with 0–100 mM NaCl. For both cultivars, fresh weight and dry weight yields were greater in selected lines on all levels of NaCl. Selected lines of Blue Crop displayed better growth than selected lines of Denise Blue at most concentrations of NaCl. Internal Na⁺ and Cl^– concentrations in selected and non-selected lines of both cultivars increased as external concentration was raised. However, selected lines of Blue Crop and Denise Blue accumulated more Na⁺ and Cl^– than non-selected lines. Selected lines of both cultivars maintained higher levels of K⁺ than non-selected lines on all external NaCl levels. Selected lines of Blue Crop had higher levels of Na⁺ and Cl^– than that of Denise Blue. The results suggest Na⁺ and Cl^– accumulation could be a mechanism allowing better growth in selected lines at moderate salinity levels (50–75 mM NaCl).     

Ethylene: Symptom, Not Signal for the Induction of Chitinase and beta-1,3-Glucanase in Pea Pods by Pathogens and Elicitors

Infection of immature pea pods with Fusarium solani f.sp. phaseoli (a non-pathogen of peas) or f.sp. pisi (a pea pathogen) resulted in induction of chitinase and β-1,3-glucanase. Within 30 hours, activities of the two enzymes increased 9-fold and 4-fold, respectively. Chitinase and β-1,3-glucanase were also induced by autoclaved spores of the two F. solani strains and by the known elicitors of phytoalexins in pea pods, cadmium ions, actinomycin D, and chitosan. Furthermore, exogenously applied ethylene caused an increase of chitinase and β-1,3-glucanase in uninfected pods. Fungal infection or treatment with elicitors strongly increased ethylene production by immature pea pods. Infected or elicitor-treated pea pods were incubated with aminoethoxyvinylglycine, a specific inhibitor of ethylene biosynthesis. This lowered stress ethylene production to or below the level of uninfected controls; however, chitinase and β-1,3-glucanase were still strongly induced. It is concluded that ethylene and fungal infection or elicitors are separate, independent signals for the induction of chitinase and β-1,3-glucanase.     

Bacterization of peanut withPseudomonas fluorescens for biological control ofRhizoctonia solani and for enhanced yield

Severity of stem-rot disease of peanut caused byRhizoctonia solani was reduced by 54.9 and 68% in plants of two cultivars treated in the greenhouse with antagonistic strains ofPseudomonas fluorescens. These strains were selected based on theirin vitro toxicity to mycelial growth and sclerotial germination ofR. solani. In field experiments, bacterization of peanuts withP. fluorescens resulted in taller plants (by 25.7%) and increased yields (by 59.0%).     

Antifungal Hydrolases in Pea Tissue : II. Inhibition of Fungal Growth by Combinations of Chitinase and beta-1,3-Glucanase

Chitinase and β-1,3-glucanase purified from pea pods acted synergistically in the degradation of fungal cell walls. The antifungal potential of the two enzymes was studied directly by adding protein preparations to paper discs placed on agar plates containing germinated fungal spores. Protein extracts from pea pods infected with Fusarium solani f.sp. phaseoli, which contained high activities of chitinase and β-1,3-glucanase, inhibited growth of 15 out of 18 fungi tested. Protein extracts from uninfected pea pods, which contained low activities of chitinase and β-1,3-glucanase, did not inhibit fungal growth. Purified chitinase and β-1,3-glucanase, tested individually, did not inhibit growth of most of the test fungi. Only Trichoderma viride was inhibited by chitinase alone, and only Fusarium solani f.sp. pisi was inhibited by β-1,3-glucanase alone. However, combinations of purified chitinase and β-1,3-glucanase inhibited all fungi tested as effectively as crude protein extracts containing the same enzyme activities. The pea pathogen, Fusarium solani f.sp. pisi, and the nonpathogen of peas, Fusarium solani f.sp. phaseoli, were similarly strongly inhibited by chitinase and β-1,3-glucanase, indicating that the differential pathogenicity of the two fungi is not due to differential sensitivity to the pea enzymes. Inhibition of fungal growth was caused by the lysis of the hyphal tips.     

Peroxidase, catalase, amine oxidase and acid phosphatase activities in Pisum sativum during infection with Fusarium oxysporum and F. solani

Two genotypes (cv. Smaragd and line DP1059) of Pisum sativum with different susceptibility to Fusarium oxysporum and F. solani and influence of pathogenesis on enzyme activities were studied. The increase of activity of studied enzymes was mostly observed in both roots and shoots during pathogenesis. Only activity of acid phosphatase decreased in the root and increased in shoots. The correlation between enzyme activity change and susceptibility of pea cultivars to F. oxysporum or F. solani was observed.     

Selection of turmeric callus tolerant to culture filtrate of <Emphasis Type="Italic">Pythium Graminicolum</Emphasis> and regeneration of plants

Root rot disease tolerant clones of turmeric variety Suguna of Curcuma longa L. were isolated using continuous in vitro selection technique against pure culture filtrate of Pythium graminicolum. Large amount of profuse, compact, creamish white callus was obtained from in vivo vegetative bud when cultured on LSBM fortified with 2,4-D (3 mg l⁻¹) after 45 days of culture. Callus was challenged with pure culture filtrate of P. graminicolum to isolate viable callus within 30 days of culture, which was further subjected to pure culture filtrate treatment. After three cycles of treatment, four cell lines which are tolerant to culture filtrate was isolated through continuous in vitro selection and subcultured on regeneration medium LSBM fortified with BAP (4 mg l⁻¹) along with the control non-selected callus to obtain complete plantlets through discontinuous in vitro selection technique. Plants regenerated from tolerant and non-selected calli were screened for disease tolerance by adopting in vitro sick plot technique. The data obtained from this experiment revealed a ratio of 225:49 tolerant: susceptible in vitro clones retrieved from tolerant callus. However, plants regenerated from the CL1a₁ and non-selected calli were susceptible under in vitro sick plot technique. The root rot disease tolerant clones were hardened and established in soil with 90% survival frequency.     

Degradation of the isoflavone biochanin a by isolates of Nectria haematococca (Fusarium solani)

Twelve isolates of Nectria haematococca, mating population VI (Fusarium solani) previously characterized for their virulence on pea plants and their ability to degrade the phytoalexin pisatin were assayed for the catabolism of the isoflavone biochanin A (5,7-dihydroxy-4′-methoxyisoflavone). Eleven isolates catabolized the isoflavone along the pathway: biochanin A → dihydrobiochanin A → 3-(p-methoxyphenyl)-6-hydroxy-γ-pyrone → p-methoxyphenylacetic acid → p-hydroxyphenylacetic acid → 3,4-dihydroxyphenylacetic acid.     

Chlorophyll fluorescence and lipid peroxidation changes in rice somaclonal lines subjected to salt stress

The aim of the present work was to explore physiological changes provoked by somaclonal variation in response to salinity. Two parental cultivars (La Candelaria and Yerua) and their derived somaclones were used as a source for breeding new rice lines with improved salt tolerance. We studied the effect of NaCl salt stress on chlorophyll fluorescence-related parameters, such as the maximum quantum yield of primary PSII photochemistry (F _v/F _m) and the performance index for energy conservation from photon absorbed by PSII antenna (PI_ABS). In addition malondialdehyde (MDA) content and leaf temperature (LT) responses were also measured. In somaclonal lines, F _v/F _m, PI_ABS, MDA and LT showed coefficients of variation of 13.7, 39.3, 25.5, and 3 %, respectively, for La Candelaria and 1.4, 17.6, 34.4 and 3 % for Yerua. However, the fragrant character did not differ in the aromatic somaclonal lines with respect to their parentals. Our results suggest that the F _v/F _m ratio would not be as good marker of PSII vitality as PI_ABS for salinized rice somaclones, unless they are highly susceptible to salinity. On other hand, the MDA content showed a strong negative correlation with the PI_ABS content in somaclones of both rice cultivars, suggesting that MDA levels could also be used as an oxidative damage index in rice somaclones.     

Selection for virus resistance in tomato exposed to tissue culture procedures

Protocols elaborated with the objective of achieving valuable material for selection procedure of variants with virusresistance traits in tomato genotypes are presented. Preliminary results are demonstrated in the domain of testing for variability in somaclones obtained through indirect adventitous organogenesis initiated on leaf explants of cultivated tomato (Lycopersicon esculentum Mill.). Somaclones were grown in greenhouse conditions and variation of their symptoms upon infection with tomato mosaic (ToMV) or cucumber mosaic (CMV) respectively was observed. Tests for resistance to the local isolates of the above cited viruses were performed using enzyme linked immunosorbent assay and back inoculation onto diagnostic plants. Screening data are presented. Desirable variants were selected from cultivars ‘Moneymaker’, ‘Potentat’ and ‘Rutgers’. Some of the ‘Moneymaker’ somaclones exhibited increased tolerance to cucumber mosaic virus, a few seemed to be even fully resistant though most were susceptible as donor plants. The most favourable somaclonal lines are actually further tested and monitored for changes in horticultural characteristics. The described procedure of searching for resistance trait in specific pathogen-free (SPF) plants regenerated from infected tissue looks promising and thus can serve as aid in attaining appropriate objectives of breeding programme. Additionaly experiments were initiated to obtain somaclones from cultivars ‘Beta’, ‘Krakus’ and Stevens Rodade hybrid via regeneration of isolated protoplasts. To this end the callus stage was obtained from all donors.