绿豆抗豆象遗传的初步研究

绿豆象(Callos0bruchun chinensis L.)是豇豆属豆类作物重要的仓库害虫.本研究通过抗豆象杂交育种后代VC1973A/TC1966 F1、F2和VC1973A/(VC1973A/TC1966 F2)BC1F1及TC1966/(VC1973A/TC1966 F2)BC1F1分离群体的遗传分析,发现绿豆抗豆象性状符合31的遗传分离规律,证明绿豆对豆象的抗性由1对显性单基因(Aa)控制,抗虫性为显性(A),感虫为隐性(a).     

Characterization of resistance to three bruchid species (Callosobruchus spp., Coleoptera,Bruchidae) in cultivated rice bean (Vigna umbellata)

Resistance of wild and cultivated rice bean (Vigna umbellata [Thunberg] Ohwi and Ohashi) to three bruchid species, Callosobruchus chinensis L., Callosobruchus maculatus F., and Callosobruchus analis F., was evaluated. All but three accessions of cultivated, and all wild rice bean accessions tested, exhibited complete resistance to all three bruchid species. Rice bean seeds with seed coat removed also showed complete resistance to the three bruchid species. Results indicate that physical attributes and/or chemical(s) present in the seed coat of rice bean are not the main factors responsible for resistance. Feeding tests were performed by using artificial beans prepared with varying proportions of rice bean (resistant) and azuki bean (susceptible) flour. Number of bruchid adults that emerged decreased, and larval developmental period (days) was extended, when artificial beans with an increasing proportion of rice bean flour were used. These tests revealed that a chemical compound(s) contained in the cotyledon of rice bean has an inhibitory effect on the growth of these bruchid species. The results also indicate that the chemical(s) in rice bean cotyledon is most effective against C. maculatus.     

栽培绿豆V2709抗豆象特性遗传分析

利用原产于印度的抗豆象栽培绿豆品种V2709与农艺性状优良的感豆象推广品种中绿1号(VC1973A)杂交,以杂交种F1、F2为试验材料,通过田间农艺性状调查、室内人工接虫鉴定及多元相关分析等,研究V2709中所包含的抗豆象特性的遗传背景。结果表明V2709的抗豆象特性由一对显性基因控制;通过相关性分析发现其抗虫性与植株高度呈极显著正相关,与主茎节数呈显著正相关,而与其他性状无明显相关性。     

Mapping of quantitative trait loci for a new source of resistance to bruchids in the wild species Vigna nepalensis Tateishi & Maxted (Vigna subgenus Ceratotropis)

Azuki bean breeders have long been interested in producing azuki bean [Vigna angularis (Willd.) Ohwi & Ohashi] varieties with bruchid resistance. A new bruchid (Callosobruchus spp.) resistance source was found in V. nepalensis Tateishi & Maxted, a species that is cross compatible with azuki bean. Quantitative trait loci (QTLs) analysis for resistance to C. chinensis (L.) and C. maculatus (F.) was conducted using F(2) (V. nepalensis x V. angularis) and BC(1)F(1) [(V. nepalensis x V. angularis) x V. angularis] populations derived from crosses between the bruchid resistant species V. nepalensis and bruchid susceptible species V. angularis. Resistance was measured using two traits, percentage of seeds damaged by bruchids and the time taken for adult bruchids to emerge from seeds. Based on the results from both populations seven QTLs were detected for bruchid resistance; five QTLs for resistance to C. chinensis and two QTLs for resistance to C. maculatus. The different locations found for some resistance QTL to the two bruchid species suggests different resistance mechanisms. QTLs on linkage group (LG) 1 and LG2 for bruchid resistance to C. chinensis co-localized with seed size QTLs suggesting that incremental increase in seed size accompanied susceptibility to C. chinensis. Based on linked markers the QTL on these two linkage groups appear to be the same as previously reported in other Asian Vigna. However, several other QTLs were newly detected including one on LG4 that appears unrelated to seed size. Transfer of these new sources of bruchid resistance from V. nepalensis to azuki bean will be aided by the progress being made in azuki genome mapping.     

Life history of flight morph females of Callosobruchus maculatus F.: evidence of a reproductive diapause

Callosobruchus maculatus (F.) (Coleoptera : Bruchidae) is a tropical beetle that develops in the seeds of Vigna unguiculata. C. maculatus adults show an imaginal polymorphism with differences in morphological, behavioral and reproductive characteristics. Adults of the flight morph that emerge in cowpea storage systems were studied under natural climatic conditions. A large number of the flight morph females were in reproductive diapause and had a long imaginal life. These females did not synthesize vitellogenin, produced a specific diapause protein and possessed significant protein reserves. This suggests that the beetles survived in the tropical ecosystem for a long time and colonized the crops during the cowpea growing and flowering phases. Analysis of reproductive activity in females captured in the V. unguiculata crops indicates that they terminated their reproductive diapause and began to lay eggs as soon as the pods were formed. Few females of the flight morph were sexually active at the beginning of imaginal life. In this paper we discuss the adaptive significance of these two reproductive strategies in females of C. maculatus.     

Use of micro-CAT scans to understand cowpea seed resistance to Callosobruchus maculatus

The cowpea bruchid, Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae: Bruchini), is a worldwide pest of stored cowpea grain [ Vigna unguiculata (L.) Walp. (Leguminosae)], causing tens of millions of dollars' worth of damage annually. One method of managing this pest involves planting cultivars whose seeds are resistant to bruchids. Despite extensive research, we still do not know the mechanism by which TVu 2027, the original resistant cowpea line, resists C. maculatus . Using micro-CAT (computerized axial tomography) scan imaging, we visualized the feeding patterns of bruchids living and growing within cowpea seeds. We present evidence that an interior zone in TVu 2027 seeds is responsible for the high mortality and developmental delays experienced by avirulent larval bruchids. We observed that both virulent as well as avirulent bruchid strains have different feeding patterns in TVu 2027 seeds compared to susceptible seeds. It appears that the resistance factor is most concentrated in a zone in each cotyledon adjacent to the air space separating the two seed halves.     

Susceptibility of the bruchid Callosobruchus maculatus (Coleoptera: Bruchidae) and its parasitoid Dinarmus basalis (Hymenoptera: Pteromalidae) to three essential oils

The bruchid Callosobruchus maculatus (F.) causes major losses during the storage of seeds of Vigna unguiculata (Walp.) in West Africa. An endemic parasitoid, the pteromalid Dinarmus basalis (Rond.) reduces the increase in bruchid populations in stores and could be used for biological control. African farmers often introduce essential oils into granaries at harvest time. In Togo, essential oils were extracted from two Gramineae, Cymbopogon nardus (L.) and Cymbopogon schoenanthus (L.) and from a Lamiaceae, Ocimum basilicum (L.). The major components of these essential oils were citronellal in C. nardus, carene-2 and piperitone in C. schoenanthus and estragol in O. basilicum. Cymbopogon schoenanthus was the most toxic oil for C. maculatus adults. D. basalis adults were more susceptible to the three essential oils than the adults of their hosts C. maculatus. In the presence of cowpea seeds, the LC50s of the three essential oils were lower than in their absence, suggesting that the seeds may absorb a part of the volatiles. High doses of three essential oils slightly affected the survival of the fourth instar or the pupae of C. maculatus. This high survival was due to protection of larvae from volatiles by the surrounding seeds. The D. basalis were more affected by the oil volatiles than their hosts. Sub-lethal doses of essential oils reduced the duration of the adult life of both insect species and fecundity of the females. The differences in sensitivity of the host and its parasitoid could influence their population dynamics. The introduction of the essential oils into storage systems potentially could reduce density of parasitoid populations and increase seed losses.     

Talisia esculenta lectin and larval development of Callosobruchus maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae)

Bruchid larvae cause major losses in grain legume crops throughout the world. Some bruchid species, such as the cowpea weevil and the Mexican bean weevil, are pests that damage stored seeds. Plant lectins have been implicated as antibiosis factors against insects, particularly the cowpea weevil, Callosobruchus maculatus. Talisia esculenta lectin (TEL) was tested for anti-insect activity against C. maculatus and Zabrotes subfasciatus larvae. TEL produced ca. 90% mortality to these bruchids when incorporated in an artificial diet at a level of 2% (w/w). The LD(50) and ED(50) for TEL was ca. 1% (w/w) for both insects. TEL was not digested by midgut preparations of C. maculatus and Z. subfasciatus. The transformation of the genes coding for this lectin could be useful in the development of insect resistance in important agricultural crops.     

Construction of bacterial artificial chromosome libraries and their application in developing PCR-based markers closely linked to a major locus conditioning bruchid resistance in mungbean (<Emphasis Type="Italic">Vigna radiata</Emphasis> L. Wilczek)

Bacterial artificial chromosome (BAC) libraries have been widely used in different aspects of genome research. In this paper we report the construction of the first mungbean (Vigna radiata L. Wilczek) BAC libraries. These BAC clones were obtained from two ligations and represent an estimated 3.5 genome equivalents. This correlated well with the screening of nine random single-copy restriction fragment length polymorphism probes, which detected on average three BACs each. These mungbean clones were successfully used in the development of two PCR-based markers linked closely with a major locus conditioning bruchid (Callosobruchus chinesis) resistance. These markers will be invaluable in facilitating the introgression of bruchid resistance into breeding programmes as well as the further characterisation of the resistance locus.     

Presence of the storage seed protein vicilin in internal organs of larval Callosobruchus maculatus (Coleoptera: Bruchidae)

Variant vicilins (7S storage globulins) of cowpea seeds (Vigna unguiculata) are considered as the main resistance factor present in some African genotypes against the bruchid Callosobruchus maculatus. It has been suggested that the toxic properties of vicilins may be related to their recognition and interaction with glycoproteins and other membrane constituents along the digestive tract of the insect. However, the possibility of a systemic effect has not yet been investigated. The objective of this work was to study the fate of 7S storage globulins of V. unguiculata in several organs of larvae of the cowpea weevil C. maculatus. Results demonstrated binding of vicilins to brush border membrane vesicles, suggesting the existence of specific receptors. Vicilins were detected in the haemolymph, in the midgut, and in internal organs, such as fat body and malpighian tubules. There is evidence of accumulation of vicilins in the fat body of both larvae and adults. The absorption of vicilins and their presence in insect tissues parallels classical sequestration of secondary compounds.     

Structure-based protein engineering for alpha-amylase inhibitory activity of plant defensin

The structure of a novel plant defensin isolated from the seeds of the mung bean, Vigna radiate, has been determined by (1)H nuclear magnetic resonance spectroscopy. The three-dimensional structure of VrD2, the V. radiate plant defensin 2 protein, comprises an alpha-helix and one triple-stranded anti-parallel beta-sheet stabilized by four disulfide bonds. This protein exhibits neither insecticidal activity nor alpha-amylase inhibitory activity in spite of showing a similar global fold to that of VrD1, an insecticidal plant defensin that has been suggested to function by inhibiting insect alpha-amylase. Our previous study proposed that loop L3 of plant defensins is important for this inhibition. Structural analyses and surface charge comparisons of VrD1 and VrD2 revealed that the charged residues of L3 correlate with the observed difference in inhibitory activities of these proteins. A VrD2 chimera that was produced by transferring the proposed functional loop of VrD1 onto the structurally equivalent loop of VrD2 supported this hypothesis. The VrD2 chimera, which differs by only five residues compared with VrD2, showed obvious activity against Tenebrio molitor alpha-amylase. These results clarify the mode of alpha-amylase inhibition of plant defensins and also represent a possible approach for engineering novel alpha-amylase inhibitors. Plant defensins are important constituents of the innate immune system of plants, and thus the application of protein engineering to this protein family may provide an efficient method for protecting against crop losses.     

绿豆抗豆象研究进展

绿豆象是豇豆属食用豆类作物重要的仓储害虫,严重影响其产量和品质。选育和利用抗虫品种是防治豆象最有效的途径。本文就绿豆抗豆象基因资源的发掘与利用及抗豆象特性的遗传机理与分离规律进行了总结与分析,对抗豆象的生化基础研究和分子标记进行了综述,探讨了抗豆象育种的重要性及尚需解决的问题,提出进行不同抗源的基因等位性鉴定、高效地利用抗源、培育多价抗豆象绿豆品种的重要性。     

Insecticidal action of Bauhinia monandra leaf lectin (BmoLL) against Anagasta kuehniella (Lepidoptera: Pyralidae), Zabrotes subfasciatus and Callosobruchus maculatus (Coleoptera: Bruchidae)

Bruchid beetle larvae cause major losses in grain legume crops throughout the world. Some bruchid species, such as the cowpea weevil (Callosobruchus maculatus) and the Mexican bean weevil (Zabrotes subfasciatus), are pests that damage stored seeds. The Mediterranean flour moth (Anagasta kuehniella) is of major economic importance as a flour and grain feeder; it is often a severe pest in flour mills. Plant lectins have been implicated as antibiosis factors against insects. Bauhinia monandra leaf lectin (BmoLL) was tested for anti-insect activity against C. maculatus, Z. subfasciatus and A. kuehniella larvae. BmoLL produced ca. 50% mortality to Z. subfaciatus and C. maculatus when incorporated into an artificial diet at a level of 0.5% and 0.3% (w/w), respectively. BmoLL up to 1% did not significantly decrease the survival of A. kuehniella larvae, but produced a decrease of 40% in weight. Affinity chromatography showed that BmoLL bound to midgut proteins of the insect C. maculatus. 33 kDa subunit BmoLL was not digested by midgut preparations of these bruchids. BmoLL-fed C. maculatus larvae increased the digestion of potato starch by 25% compared with the control. The transformation of the genes coding for this lectin could be useful in the development of insect resistance in important agricultural crops.     

Vicilin variants and the resistance of cowpea (Vigna unguiculata) seeds to the cowpea weevil (Callosobruchus maculatus)

1. A globulin fraction prepared from the meal of Callosobruchus maculatus-resistant cowpea (Vigna wiguiculata) seeds was shown to be detrimental to this bruchid when incorporated in artificial seeds.2. The performance of C. maculatus was also shown to be strongly hindered by vicilins from resistant seeds when these storage proteins were incorporated in artificial seeds at the level of 2%.3. The purified vicilins from seeds of both resistant and susceptible cowpea varieties were shown to have the same SDS-PAGE pattern but different mobilities in non-denaturing polyacrylamide gel electrophoresis.4. These results and previous ones obtained by us (Silva and Xavier-Filho, 1991; Sales et al., 1992) strongly suggest that the resistance of cowpea seeds from the cultivar TVu 2027 and from others bred from it is associated with the presence of vicilin molecules which are refractory to digestion by bruchid midgut proteinases.     

Bruchid (Coleoptera: Bruchidae) ovicidal phenylbutanoid from Zingiber purpureum

The larvicidal activity of the dichloromethane extract of Zingiber purpureum Roscoe (Zingiberaceae) rhizome against the second instar of Aedes aegypti (L.) (Diptera: Culicidae) is shown to be due to 4-(3',4'-dimethoxyphenyl)buta-1,3-diene. The diene also showed ovicidal activity against the bruchid Callosobruchus maculatus (F.) (Coleoptera: Bruchidae). Most of the eggs laid by bruchids on treated cowpea seeds were transparent, and very few of them contained developing embryos. The few larvae produced from these embryos were unable to penetrate the seed coat and enter the seed. Similar effects were seen when adults were exposed to the compound and then placed on untreated cowpea seeds, suggesting that a new type of maternally mediated ovicidal effect was involved. Coated and impregnated granular formulations of the extract were evaluated for use in the control of bruchid infestation of stored cowpea seeds. Coated granules showed activity similar to that of the crude extract but were found to lose activity rapidly. Impregnated granules were found to be less active than the crude extract.     

Insecticidal activity of an alpha-amylase inhibitor-like protein resembling a putative precursor of alpha-amylase inhibitor in the common bean, Phaseolus vulgaris L.

alpha-Amylase inhibitor (alphaAI) in the common bean, Phaseolus vulgaris L., protects seeds from insect pests such as the cowpea weevil (Callosobruchus maculatus) and the azuki bean weevil (C. chinensis). Cultivars which lack alphaAI still show resistance to both bruchids. These cultivars have a glycoprotein that reacts with anti-alphaAI-1 antibodies. The glycoprotein with a molecular mass of 29 kDa (Gp29) was purified and the encoding gene was isolated. The primary structure of Gp29 is the same as alpha-amylase inhibitor-like protein (AIL) from which the encoding gene has already been isolated. AIL resembles a putative precursor of alphaAI, even though it does not form the active inhibitor. However, AIL has some inhibitory effect on the growth of C. maculatus but not C. chinensis. The presence of AIL alone is insufficient to explain the bruchid resistance of common bean cultivars lacking alpha-AI. Common bean seeds appear to contain several factors responsible for the bruchid resistance.     

Molecular identification of four different alpha-amylase inhibitors from baru (Dipteryx alata) seeds with activity toward insect enzymes

The endophytic bruchid pest Callosobruchus maculatus causes severe damage to storage cowpea seeds, leading to economical losses. For this reason the use of alpha-amylase inhibitors to interfere with the pest digestion process has been an interesting alternative to control bruchids. With this aim, alpha-amylase inhibitors from baru seeds (Dipteryx alata) were isolated by affinity chromatographic procedures, causing enhanced inhibition of C. maculatus and Anthonomus grandis alpha-amylases. To attempt further purification, this fraction was applied onto a reversed-phase HPLC column, generating four peaks with remarkable inhibition toward C. maculatus alpha-amylases. SDS-PAGE and MALDI-ToF analysis identified major proteins of approximately 5.0, 11.0, 20.0 and 55 kDa that showed alpha-amylase inhibition. Results of in vivo bioassays using artificial seeds containing 1.0% (w/w) of baru crude extract revealed 40% cowpea weevil larvae mortality. These results provide evidence that several alpha-amylase inhibitors classes, with biotechnological potential, can be isolated from a single plant species.     

Effect of host quality of Callosobruchus maculatus (Coleoptera: Bruchidae) on performance of the egg parasitoid Uscana lariophaga (Hymenoptera: Trichogrammatidae)

Development and reproductive success of the solitary egg parasitoid Uscana lariophaga Steffan were examined after development in eggs of the bruchid storage pest Callosobruchus maculatus Fabricius reared at either low or high densities on cowpea seeds and laid at day 1 and 4 of maternal life. Both bruchid larval competition and maternal age negatively affected egg size, but the latter more than the former. Uscana lariophaga reared in small hosts developed slower, were smaller and produced fewer eggs compared to parasitoids reared in large hosts. Fecundity of the parasitoid was heavily influenced by host egg size. This was reflected in the values for the intrinsic rate of increase of U. lariophaga, which differed for wasps that developed in host eggs laid by bruchid females of different age. Wasps allocated marginally more female offspring to larger hosts.     

Effects of search experience in a resource-heterogeneous environment on the oviposition decisions of the seed beetle, Callosobruchus maculatus (F.)

Abstract.  1. This study investigates how female seed beetles, Callosobruchus maculatus , distribute their eggs on various-sized seeds when the size of seed was varied during the egg-laying period.
2. Beetles were allowed to lay eggs on one of three arrays of 64 adzuki beans ( Vigna angularis ). Each array contained four size classes of seed, ranging from small (5.0–5.5 mm diameter) to large (6.5–7.0 mm), but differed in how they were distributed within the environment. In the most heterogeneous condition (the 64-patch design), the four sizes were interspersed, while in the least heterogeneous condition (the four-patch design) they were grouped into four separate blocks. Thus, a beetle exploring the 64-patch design would frequently encounter all four seed sizes, whereas a beetle exploring the four-patch design would only rarely encounter a change in bean size.
3. Beetles experiencing greater seed size heterogeneity were more likely to lay eggs on larger seeds, whereas those in the blocked condition were more likely to oviposit on small seeds. Beetle responses to seed size heterogeneity suggest that the degree of preference for large seeds depends on a female's recent experience.
4. Female beetles exhibited size discrimination throughout their egg-laying process; however, there was a trade-off between seed size and egg discrimination (i.e. avoiding those seeds already containing developing eggs) in response to the change in fitness gained from either laying on larger seeds or lower egg-load seeds during the egg-laying process.
5. Our model provides the first evidence that evolving seed size discrimination ability is adaptive for the seed beetle with egg-discrimination ability.     

亚蔬中心绿豆品种(系)抗白粉病评价

采用苗期人工接种鉴定法,在大棚种植条件下对12个亚蔬中心(AVRDC)绿豆品种白粉病抗性进行了鉴定评价。结果显示,VC1560C、V4785和VC2768A三个品种高抗(HR)白粉病,VC6173-14、V1132为中抗(MR)白粉病品种。其它品种对白粉病表现高度感病。在田间种植条件下对亚蔬中心16个抗豆象回交9代品系(BC9)进行了成株期白粉病抗性鉴定。与对照感病品种 VC1973、VC1178A 相比,VC6459-3-6-37和 VC6458-6-3-16对白粉病具有一定抗性,但白粉病感染程度仍很严重,其它14个 BC9品系均对白粉病表现高度感病。