鹰嘴豆种质资源对Ascochyta rabiei的抗性评价及遗传多态性分析

以25 个鹰嘴豆品系为试验材料,通过叶面喷雾的方式进行Ascochyta rabiei菌悬液室内外人工接种,评价不同鹰嘴豆种质资源的抗病性;同时利用RAPD方法进行基因型鉴定,采用NTSYSpc 2.10t软件对分子标记结果进行遗传相似性的统计分析并建立各品系间的亲缘关系聚类图,探讨不同鹰嘴豆品系对A.rabiei抗性与遗传多态性间的关系。通过室内和田间鹰嘴豆抗A.rabiei鉴定结果综合分析表明：在25个鹰嘴豆供试品系中,“系选 03”和“216”品系均表现出稳定抗性特性;北园春品系表现出稳定中抗特性。通过RAPD多态性引物对这25 个供试品系进行PCR扩增,共获得129 个扩增条带,其中多态性条带共有67 条,多态性比例达51.94%,遗传相似系数为0.3731-0.9254。结合抗病性和遗传多态性,经方差分析表明,本研究所采用的鹰嘴豆品系对A.rabiei的抗性强弱与其遗传相似性之间无显著相关性。     

Biochemical and molecular biological studies on infection (Ascochyta rabiei)-induced thaumatin-like proteins from chickpea plants (Cicer arietinum L.)

A pathogenesis-related protein induced by infection with Ascochyta rabiei was purified from intercellular washing fluid of chickpea (Cicer arietinum L.) leaves. Amino-terminal sequencing identified the protein, named PR-5a, as a thaumatin-like protein. The isoelectric point was determined with 6.5 and the molecular mass is 16 kDa. Therefore, chickpea PR-5a is the first dicot member of a TLP subgroup containing small TLPs with a molecular weight between 15 and 18 kDa. PR-5a shows no antifungal activity towards A. rabiei. Screening of a chickpea cDNA library led to the isolation of a cDNA clone (p5a-241) for this protein. A second cDNA clone (ELR112) encoding a TLP was isolated using differential hybridisation of cDNA libraries obtained from elicited and water treated cell suspension cultures of chickpea. The deduced protein (PR-5b) has a molecular mass of 22 kDa. PR-5b is postulated to be located in the vacuole due to the presence of a respective N-terminal signal peptide and a carboxy-terminal extension. Southern blot analyses showed that ELR112 and p5a-241 represent single copy genes. During fungal infection of chickpea plants expression of both genes proceeds much faster in an A. rabiei resistant cultivar than in a susceptible one.     

Elicitor-induced metabolic changes in cell cultures of chickpea (Cicer arietinum L.) cultivars resistant and susceptible to Ascochyta rabiei

Cell-suspension cultures of two chickpea (Cicer arietinum L.) cultivars, resistant (ILC 3279) and susceptible (ILC 1929) to the fungus Ascochyta rabiei (Pass.) Lab., showed differential accumulation of the phytoalexins medicarpin and maackiain, and transient induction of related enzyme activities after application of an A. rabiei-derived elicitor. The chalcone-synthase (CHS) activity (EC 2.3.1.74) which is involved in the first part of phytoalexin biosynthesis exhibited a maximum 8–12 h after elicitation in the cells of both cultivars. Concomitant with the fivefold-higher phytoalexin accumulation, CHS activity increased twofold in the cells of the resistant cultivar. The maximum of the elicitor-induced CHS-mRNA activity was determined 4 h after onset of induction in the cultures of both cultivars, although in cells of cultivar ILC 3279 this mRNA activity was induced at a level twofold higher than that in cells of the susceptible race ILC 1929. Investigations of CHS isoenzymes by two-dimensional gel electrophoresis of immunoprecipitated in-vitro-translated protein indicated the presence of five proteins. In the cells of both cultivars only two of the isoenzymes were induced after elicitor treatment. Analysis of the total in-vitro-translated proteins by two-dimensional gel electrophoresis showed that the constitutively expressed patterns of mRNA activities in the cell cultures of the two cultivars were identical. After elicitation, considerably more translatable mRNAs were induced in the cells of cultivar ILC 3279. The few induced proteins, and their respective mRNA activities, which could be detected in the cells of the susceptible cultivar, all existed in the cells of the resistant cultivar, too. One highly induced protein (M_r 18 kDa) found in the cells of cultivar ILC 3279 reached its maximum mRNA activity 6 h after elicitor application. The amount of this protein was hardly increased in the cells of the susceptible cultivar. This protein appears to be excreted from the cells into the growth medium.Abbreviations CHS chalcone synthase - IEF isoelectric focussing - ILC international legume chickpea - PR-protein pathogenesis-related protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Financial support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. The authors thank Dr. K. Hahlbrock (Max-Planck-Institut für Züchtungsforschung, Köln, FRG) for provision of antisera and the International Centre for Agricultural Research in the Dry Areas (Aleppo, Syria) for plant material.     

Differential Induction of β-1,3 Glucanase in Resistant and Susceptible Varieties of Cicer arietinum L. Following Infection with Ascochyta rabiei

Infection of chickpea (Cicer arietinum L.) by Ascochyts rabiei resulted in marked induction of β-1, 3 glucanase activity. The induction of enzyme activity was two fold higher in resistant variety as compared to the susceptible variety. Slot blot analysis of total RNA using glucanase cDNA clone from tobacco as probe revealed that the induction of enzyme activity is at mRNA level.     

Histology of Disease Development in Resistant and Susceptible Cultivars of Chickpea (Cicer arietinum L.) Inoculated with Spores of Ascochyta rabiei

Abstract Histological studies were performed on a compatible and an incompatible interaction between chickpea ( Cicer arietinum L.) plants and the fungus Ascochyta rabiei (Pass.) Labr. The time course of infection, development on leaflets and stems of susceptible (ILC 1929) and resistant (ILC 3279) plants was monitored by light or scanning electron microscopy with the aim to compare histological changes as the basis for further work on biochemical changes in this plant-pathogen interaction.
Spores of A. rabiei began to germinate from 12 hpi on and developed a polar germ tube; fungal colonization, secretion of a mucilaginous exudate and appressoria formation (1–3 dpi) were identical on both cultivars. Leaves of susceptible plants were invaded by the fungus directly through the cuticle, the fungus then spread subepidermally followed by a rapid collapse of the leaf tissue (4–6 dpi). Development of leaf spots and fungal pycnidia could be observed 6–8 dpi. The resistant cultivarrapidly responded (24–48 hpi) to fungal infection and cells of the palisade parenchyma exhibited autofluorescence. In later stages of the infection (4–5 dpi) fluorescent areas developed to small necrotic spots all over the leaflet. These necrotic areas, were the result of cell death and a subsequent change in the leaf structure and were characterized by the accumulation of phenolic compounds. Leaves of the resistant cultivar were invaded by the fungus to less than 5%.     

In Vitro Selection Against Ascochyta Blight of Chickpea (Cicer arietinum L)

Callus cultures established on MS medium containing 2.0 mg l^-1 2, 4-D were inoculated on the regeneration medium supplemented with different concentrations (0.5, 1.0, 1.5, 2.0, 2.5 and 3%, v/v) of culture filtrate (CF) of Ascochyta rabiei infesting chickpea. Out of 486 callus pieces and 270 regenerants obtained from immature embryo derived callus screened, 50 callus lines and 74 regenerants were found resistant. Further, these resistant callus lines and regenerants were subjected to stability test by growing them on a medium containing 3% CF. Seventeen callus lines and 28 regenerants of the selected lines showed normal growth on the selection medium. The regenerated plants were tested in pots under artificial epiphytotic conditions where they showed normal growth behaviour and high degree of resistance.     

Assessment and comparison of AFLP and SSR based molecular genetic diversity in Indian isolates of Ascochyta rabiei, a causal agent of Ascochyta blight in chickpea (Cicer arietinum L.)

Ascochyta blight (AB), caused by Ascochyta rabiei (Pass.) Labr. (anamorph), is the most damaging disease of chickpea (Cicer arietinum L.) and is a serious biotic stress constraint for chickpea production. To understand the molecular diversity in A. rabiei populations of India, a total of 64 isolates collected from AB-infected chickpea plants from different agroclimatic regions in the North Western Plain Zone (NWPZ) of India were analyzed with 11 AFLP (amplified fragment length polymorphism) and 20 SSR (simple sequence repeat) markers. A total of 9 polymorphic AFLP primer pairs provided a total of 317 fragments, of which 130 were polymorphic and showed an average PIC value 0.28. Of the SSR markers, 12 showed polymorphism and provided a total of 29 alleles with an average PIC value 0.35. To the best of our knowledge, this is the first report on a comparison of AFLP and SSR diversity estimates in A. rabiei populations. The dendrogram developed based on AFLP and SSR data separately, as well as on the combined marker dataset, grouped the majority of AB isolates as per geographic regions. Model based population structure analysis revealed four distinct populations with varying levels of ancestral admixtures among 64 isolates studied. Interestingly, several AFLP primer combinations and SSR markers showed the locus/allele specific to AB isolates of certain regions, e.g., Hisar, Sriganganagar, Gurdaspur, and Sundarnagar. Genetic variability present in AB isolates of the NWPZ of India suggests the continuous monitoring of changes in A. rabiei population to anticipate the breakdown of AB resistance in chickpea cultivars grown in India.     

Genetics of nodulation in Chickpea (Cicer arietinum L.)

Combining ability, components of genetic variance and graphic analysis revealed that nodulation in the cultivars of Chickpea (Cicer arietinum L.) under study, was predominantly under the control of non-additive gene action although substantial additive effect was also present. The crosses giving high specific combining ability effects also manifested highly significant positive heterosis. The parents F-61, Giza and Annegiri possessed mostly dominant alleles while Phule G-5, NEC-249 and N-31 possessed mostly recessive alleles having positive effect on nodule weight.     

Native β-glucuronidase activity in sugarbeet (Beta vulgaris)

β-Glucuronidase (EC 3.2.1.31) activity, initially thought absent from plants, has been found in a number of plant families. During an analysis of Agrobacterium -mediated transformation of sugarbeet ( Beta vulgaris L.), significant glucuronidase activity was observed in control (non-transformed) tissues when the fluorogenic substrates 4-methylumbelliferyl-β- d -glucuronic acid, resorufin glucuronic acid and 3-carboxyum-belliferyl-β- d -glucuronic acid were used to quantify β-glucuronidase activity under standard protocol conditions. Similarly, the colorigenic substrate p -nitrophenyl-β- d -glucuronide was hydrolyzed by this sugarbeet-derived glucuronidase. Biochemical and immunological data are presented to indicate significant differences between sugarbeet-derived glucuronidase and that from Escherichia coli (EC 3.2.1.31) encoded by gusA . These differences provide means of distinguishing between the two activities in extracts that contain a mixture of both. Use of X-glue, the substrate utilized in histochemical localizations of glucuronidase activity, gave no reaction product (i.e., indigo precipitate) at pH 7.0. However, at pH 3.0, 4.0 and 5.0 formation of the indigo precipitate was evident within 1 h at 37°C in sugarbeet callus and by 4 h in leaves and petioles. The specific activity of sugarbeet glucuronidase was observed to be strongly pH dependent, with an optimum near pH 4.0. The use of various β-glucuronidase assay techniques as applied to transformation of sugarbeet is discussed.     

Accumulation of phytoalexins and isoflavone glucosides in a resistant and a susceptible cultivar of Cicer arietinum during infection with Ascochyta rabiei

After infection with spores of a virulent strain of Ascochyta rabiei the chickpea (Cicer arietinum) cultivars ILC 1929 (susceptible) and ILC 3279 (resistant) were compared with regard to pterocarpan phytoalexin and isoflavone accumulation. Quantitative HPLC analyses of total extracts of aerial parts were used to measure the induced formation of the phytoalexins medicarpin and maackiain and the accumulation of the constitutive isoflavones biochanin A and formononetin together with their, 7-0-glucosides and their 7-0-glucoside-6″-0-malonates. The two cultivars showed no significant difference in the level of isoflavones and isoflavone conjugates. On the other hand, the resistant cultivar ILC 3279 rapidly accumulated large amounts of both, phytoalexins (20–26 nmole g^?1 fr.w.) whereas cultivar ILC 1929 only produced very small amounts (5 nmole g^?1 fr.w.) of medicarpin. The data are discussed with regard to isoflavonoid metabolism and the significance of induced and constitutive levels of phytoalexins and isoflavones in resistance of chickpea towards A. rabiei.     

Partial purification of cell wall β-galactosidases from Cicer arietinum epicotyls. Relationship with cell wall autolytic processes

The protein extracted from the cell wall of the epicotyls of Cicer arietinum L. cv. Castellana was separated by ion exchange chromatography in four different fractions with β-D-galactosidase (EC 3.2.1.23) activity. These were called βI, βII, βIII and βIV, according to their order of elution. βII was associated with a particularly high β-D-glucosidase (EC 3.2.1.21) activity. Gel filtration chromatography of each of the fractions gave further subdivision of fractions βI and βIII. Subfractions 1 βI, 1 βII and 1 βIV have glucosidase activity and subfractions 2 βI and 2 βIII have galactosidase activity.
The studies on the hydrolytic capacity of these fractions and its relationship with the autolytic process seem to show that subfraction 2 βIII is responsible for autolysis. The release of total and reducing sugars is very similar for autolysis and hydrolysis by 2 βIII. The sugars released are mainly galactose and, to a lesser extent arabinose and glucose. Galactose is released as a monosaccharide, while arabinose remains associated to a polysaccharide component together with glucose and small amounts of galactose.     

Agrobacterium mediated transformation of chickpea (Cicer arietinum L.) embryo axes

 Embryo axes of four accessions of chickpea (Cicer arietinum L.) were treated with Agrobacterium tumefaciens strains C58C1/GV2260 carrying the plasmid p35SGUSINT and EHA101 harbouring the plasmid pIBGUS. In both vectors the GUS gene is interrupted by an intron. After inoculation shoot formation was promoted on MS medium containing 0.5 mg/l BAP under a selection pressure of 100 mg/l kanamycin or 10 mg/l phosphinothricin, depending on the construct used for transformation. Expression of the chimeric GUS gene was confirmed by histochemical localization of GUS activity in regenerated shoots. Resistant shoots were grafted onto 5-day-old dark-grown seedlings, and mature plants could be recovered. T-DNA integration was confirmed by Southern analysis by random selection of putative transformants. The analysis of 4 plantlets of the T1 progeny revealed that none of them was GUS-positive, whereas the presence of the nptII gene could be detected by polymerase chain reaction. Received: 30 May 1997 / Revision received: 18 September 1997 / Accepted: 22 March 1999     

Elicitation and Suppression of Phytoalexin and Isoflavone Accumulation in Cotyledons of Cicer arietinum L. as Caused by Wounding and by Polymeric Components from the Fungus Ascochyta rabiei

Shortly after sowing cotyledons of chickpea (Cicer arietinum) start to accumulate the isoflavones biochanin A and formononetin together with their 7-0-glucosides and their 7-0-glucoside-6″-malonates. The additional accumulation of the pterocarpan phytoalexins medicarpin and maackiain can be induced by wounding of the cotyledons. Treatment of sliced cotyledons with a crude elicitor fraction obtained from the growth medium or the mycelium of the chickpea pathogenic fungus Ascochyta rabiei (Pass.) Lab. leads to a dramatic increase in the level of numerous aromatic compounds, especially of the isoflavone aglyca and the phytoalexins. Accumulation of isoflavone conjugates is not altered by elicitor treatment as shown by time course studies, and dose-response curves. A protein preparation (“suppressor”) isolated from the culture filtrate of the same fungus was shown to inhibit the accumulation of isoflavone aglyca, isoflavone conjugates and phytoalexins in the sliced cotyledons. The possible relevance of elicitor-suppressor counteraction with regard to the defence mechanisms of the host plant is discussed.     

Comparisons of Earlier- and Later-formed Pods of Chickpeas (Cicer arietinum L.)

In chickpeas (Cicer arietinum L.) flowering and pod developmentproceed acropetally. In plants grown under normal field conditionsat Hyderabad, in peninsular India, and at Hissar in north India,at successively apical nodes of the branches there was a declinein pod number per node, weight per pod, seed number per podand/or weight per seed. The percentage of nitrogen in the seedswas the same in earlier and later-formed pods at Hyderabad;at Hissar the later-formed seeds contained a higher percentage.Earlier- and later-formed flowers contained similar numbersof ovules. The decline in seed number and/or weight per seedin the later-formed pods of 28 out of 29 cultivars indicatedthat pod-filling was limited by the supply of assimilates orother nutrients. By contrast, in one exceptionally small-seededcultivar there was no decline in the number or weight of seedsin later-formed pods, indicating that yield was limited by ‘sink’size. Cicer arietinum L., chickpea, flowering, pod development, seed number, seed weight, nitrogen content     

Characterization of Vicilin Seed Storage Protein of Chickpea (Cicer arietinum L.)

Vicilin, one of the major storage proteins of chickpea (Cicer arietinum L.) was purified and characterized during seed development. Vicilin was purified by zonal isoelectric precipitation followed by chromatography on DEAE-cellulose column. Vicilin on SDS-PAGE resolved into 5 major bands ranging in mol wt from 14 to 66 kD. More heterogenous pattern emerged on isoelectric focussing. This protein had high amount of amides and low amount of sulphur containing amino acids.     

Water Loss via the Glandular Trichomes of Chickpea (Cicer arietinum L.)

During late vegetative growth chickpea leaves and stems canbe covered with aqueous glandular droplets. If these dropletspersist at low humidities there may be substantial water lossvia the glandular trichomes Four solution culture experimentsin growth chambers tested for glandular water loss at differenthumidities. In the daytime, exudate persisted between relativehumidities of 55% and 95%, and the exudate water potential variedbetween - 2.0 M Pa and - 8.0 M Pa. Even by night, chickpea leaves,like wetted alfalfa leaves, were cooler than non-wetted alfalfaleaves or the ambient air. Daytime leaf temperatures were significantlyhigher in a mutant that produced fewer droplets than in itsnormal parent. It was concluded that water loss via the glandulartrichomes can be enough to lower leaf temperature by severaldegrees C within a wide range of atmospheric conditions. The exudate solutes, analysed to confirm the osmotic potentialmeasurements, were primarily malic, hydrochloric and oxalicacid. Without the strong acids a chickpea leaf, wet even ondry days, would be ripe for parasitic attack. Key words: Add exudate, leaf hairs, transpiration, leaf temperature     

Efficient transgenic plant regeneration throughAgrobacterium-mediated transformation of Chickpea (Cicer arietinum L.)

Three genotypes of chickpea ICCV-1, ICCV-6 and a Desi (local) variety were tested for plant regeneration through multiple shoot production. The embryo axis was removed from mature seeds, the root meristem and the shoot apex were discarded. These explants were cultured on medium containing MS macro salts, 4X MS micro salts, I35 vitamins, 3.0 mg/1 BAP, 0.004 mg/1 NAA, 3% (w/v) sucrose and incubated at 26⁰C. The explants were transformed withAgrobacterium tumefaciens strain LBA4404 with binary vector pBI121 containing theuidA andnptIl genes. Multiple shoots were repeatedly selected with kanamycin. The selected kanamycin resistant shoots were rooted on MS medium supplemented with 0.05 mg/1 113A. The presumptive transformants histochemically stained positive for GUS. Additionally, nptll assay confirmed the expression ofnptII in kanamycin resistant plants. Transgenic plants were transferred to soil and grown in the green house.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4dichlorophenoxy acetic acid - IAA Indole acetic acid - IBA Indole butaric acid - NAA Naphthalene acetic acid     

Inhibition of cell wall autolysis and auxin-induced elongation of Cicer arietinum epicotyls by β-galactosidase antibodies

Polyclonal antibodies were raised in response to βIII-galactosidase purified from cell wall of Cicer arietinum epicotyls. The antibody preparation generated, bound to βIII protein giving a major protein band in the zone corresponding to M_r 45 000, the molecular mass previously estimated for βIII-galactosidase. These antibodies clearly suppress autolytic reactions in isolated walls of Cicer arietinum epicotyl segments, while the preimmune serum had no effect on autolytic reaction. The results strongly support the idea that the autolytic degradation of the cell wall is carried out by the βIII-galactosidase.
The antibodies against β-galactosidase were also able to inhibit cell wall hydrolysis mediated by both total cell wall protein extracted by LiCl and cell wall hydrolysis mediated by βIII-galactosidase.
Since autolysis is thought to be related to the process of cell wall loosening, the effects of the antibodies against the autolytic enzyme was also tested on epicotyl growth. β-galactosidase antibodies consistently inhibited IAA-induced elongation.