High antinutrient levels and bruchid resistance of cowpea (Vigna unguiculata) seeds

Summary Seeds of some cowpea varieties, characterized by different degrees of storage pest resistance, were analyzed for inhibitors of the following enzymes: porcine amylase, Bacillus amylase, bovine chymotrypsin and trypsin. A broad variation was observed among samples for all tested inhibitors. Submitting the experimental data to the principal component analysis it has been observed that resistant lines are characterized by high levels of both antitryptic and antiamylasic activity. Moreover a high activity of a single inhibitor class is typical of the bruchid susceptible lines. Hence, breeding for high contents of these protein inhibitors could be an effective way of obtaining lines that are naturally resistant to storage pest attack. The limit of this breeding strategy seems to be the difficulty to reduce the antiamylasic activity before eating.     

Proteinase inhibitor polymorphism in the genus Vigna subgenus Ceratotropis and its biosystematic implications

The diversity of components for fourproteinase inhibitors found in species ofthe genus Vigna subgenus Ceratotropis are described. Trypsin,chymotrypsin, subtilisin and cysteineproteinase inhibitors were analyzedby isoelectric focusing followed by thegelatin replica method. Of these proteinaseinhibitors, trypsin inhibitors showedmost polymorphism both within and betweenspecies. Many trypsin inhibitor componentswere also active to chymotrypsin. Severalaccessions had very low levels or absenceof some inhibitors, such as very low levelsof trypsin inhibitor in two accessions ofthe V. tenuicaulis and absence ofchymotrypsin inhibitors in V.grandiflora and V. subramaniana.Proteinase inhibitor polymorphism broadlyagreed with the taxonomic system for thesubgenus Ceratotropis. Based oninhibitor variation species analyzed couldbe divided into three groups whichcorresponding to sections Aconitifoliae, Angulares and Ceratotropis. Some species have verylittle variation in trypsin inhibitorsdespite wide distribution, such as, V.radiata and V. reflexo-pilosa.Accessions of other species showedconsiderable intraspecific variation fortrypsin inhibitors, such as, V.grandiflora, V. aconitifolia andV. stipulacea. Proteinase inhibitorpolymorphism provides an indication of thespecies that may have contributed a genometo the tetraploid species, V. reflexo-pilosa.     

Interaction of insect digestive enzymes with plant protein inhibitors and host-parasite coevolution

Summary A complex of new approaches was used to study the insect-plant coevolution by using cereal pest digestive -amylases and proteinases and their proteinaceous inhibitors in cereals. During evolution, plants can weaken the destructive affects of insect hydrolases at the expense of inhibitors in various ways, including (1) increasing the inhibitor activity and heterogeneity, (2) increasing the complexity of an inhibitor set and (3) producing highly specific insect enzyme inhibitors and bifunctional amylase/proteinase inhibitors. Insects, in turn, can decrease the influence of inhibitors by (1) increasing digestive enzyme activities, (2) by modifying a set of related activities of various digestive hydrolases, (3) by decreasing enzyme sensitivity to inhibitors and (4) by destroying inhibitors in guts by proteinases.Abbreviations IEF isoelectric focusing - TI trypsin inhibitor - CI and C/SI chymotrypsin and chymotrypsin/subtilisin inhibitors - InsAI12 insect amylase inhibitor with molecular weight value of about 12 kDa     

Development of a molecular marker for a bruchid (Callosobruchus chinensis L.) resistance gene in mungbean

Bruchid, Callosobruchus spp. (Coleoptera: Bruchidae), is a serious pest during storage of seeds of mungbean (Vigna radiata (L.) Wilczek) and other Vigna species. A source of resistance to this pest has been identified in Vigna sublobata (Roxb.) Bairig. accession TC1966. Two hundred recombinant inbred lines at the F₁₂ generation have been developed for molecular mapping of bruchid resistance (Br) gene in TC1966. Through bulked segregant analysis (BSA), ten randomly amplified polymorphic DNA (RAPD) markers associated with the bruchid resistance gene were successfully identified. A total of four closely linked RAPDs were cloned and transformed into sequence characterized amplified region (SCAR) and cleaved amplified polymorphism (CAP) markers. Seven CAPs developed from the identified RAPD markers showed tighter linkage with the Br gene than the original RAPD. Through transformation of RAPDs into CAPs, codominant markers for bruchid resistance were successfully obtained. Homozygous genotypes of these PCR-based markers were estimated to contribute 85% of the variance for seed damage when the insect assay was performed under favorable growth conditions for bruchid.     

Amylase activity in triticale,X Triticosecale Wittmack

Summary Amylase activity in leaves and the seed exhibited variations among 12 cultivars of hexaploid triticales and was considerably higher than either octaploid triticale or hexaploid wheat. Amylase activity was influenced by the levels of nitrogen and the maturity stages of the kernel. The bran-free endosperm had much lower amylase activity than the whole meal. The measurement of dry matter, starch, total sugars and reducing sugars during the stages of kernal development indicated significant positive correlation between days after anthesis and amounts of starch and dry matter and negative correlation with total sugars, reducing sugars and amylase activity. However, a lower rate of starch accumulation and higher rate of amylase activity prior to kernel maturity was clearly evident.     

The effectiveness of evaluating wild species: searching for sources of resistance to bruchid beetles in the genus Vigna subgenus Ceratotropis

A species level germplasm collection representing 76% of known taxa in the genus Vigna subgenus Ceratotropis was evaluated for resistance to two species of bruchid beetles, Callosobruchus chinensis and C. maculatus. Seven taxa consisting of 29 accessions were found to be resistant to C. chinensis and 4 taxa consisting of 24 accessions were found to be resistant to C. maculatus. This compared with no resistant accessions being found in several hundred landrace accessions of mungbean, V. radiata var. radiata, in the same subgenus. Sometimes resistance was found in all accessions of a particular taxon, such as complete resistance to both C. chinensis and C. macualtus in V. umbellata. Other taxa showed intra taxon variation for resistance such as V. reflexo-pilosa andV. minima. The levels and patterns of resistance among taxa were diverse. The results suggest that various factors cause resistance to bruchid in the subgenus Ceratotropis. While the number of eggs laid on seeds generally reflected seed size, one small seeded cultivar of V. mungo var. mungo, black gram, had an unusually high number of eggs laid per seed. No correlation was found between seed size and levels of resistance. The species level germplasm collection, which reflects the core collection concept in trying to maximize genetic diversity in a limited number of accessions, has enabled a large number of potentially useful sources of resistance to bruchid beetles to be found efficiently. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytoplasmic male-sterility and source of pollen influence the expression of resistance to sorghum midge, Stenodiplosis sorghicola

Sorghum midge, Stenodiplosis (Contarinia) sorghicola (Coquillett), is an important pest of grain sorghum, and host plant resistance is an important aspect of control of this pest. This research investigated how cytoplasmic male-sterility and source of pollen influence the expression of resistance to sorghum midge. Sorghum midge emergence was significantly lower in panicles of midge-resistant and midge-susceptible cytoplasmic male-sterile lines when pollinated with AF 28 - a midge-resistant restorer line, than those pollinated with Swarna - a midge susceptible restorer line, indicating the presence of xenia effects. Maintainer lines (B-lines) of midge-resistant parents had significantly lower numbers of eggs and larvae than the B-lines of midge-susceptible parents. Male-sterile lines of the both midge-resistant and midge-susceptible lines were equally susceptible, indicating that resistance to sorghum midge is influenced by factors in the cytoplasm of the B-line. These findings will have an important bearing on the production of hybrids with resistance to insects. This revised version was published online in August 2006 with corrections to the Cover Date.     

Inheritance of resistance to Acanthoscelides obtectus in a wild common bean accession crossed to commercial bean cultivars

Summary The bean weevil Acanthoscelides obtectus, is an important storage pest of common beans in Latin America and Africa. A few wild bean accessions from Mexico have been identified as highly resistant to the weevil. One accession, G 12952, was crossed to two susceptible bean cultivars differing in seed size. Reciprocal F1 and F2 individual seed were evaluated for days to adult emergence (DAE) and emerged adult weight. Maternally inherited seed size affected resistance measurements only in the F1 reciprocal crosses, however, the overall resistance level of the F1 was more similar to that of the susceptible cultivars. The F2 showed a continuous, but skewed distribution from low to high DAE. Very few F2 individuals had the resistance level of G 12952. When the frequency distributions were divided into discrete categories based on parental response, resistance was found to be inherited as two recessive complementary genes. The F3 generation showed an overall lowering of resistance levels compared to their original F2 evaluations. However, none of the lines classified as resistant (50 DAE) in the F2, fell into the susceptible category in the F3, indicating that the resistant genotypes were relatively stable as expected with recessively inherited traits. Modifying genes from the commercial parents may be responsible for general lowering of resistance. Seed size was negatively correlated with adult weight but not with DAE. The unique resistance of the wild bean accessions is discussed in relation to its inheritance. The results and obstacles encountered in the A. obtectus breeding program at CIAT are described.     

Identification of candidate genes associated with resistance to bruchid (Callosobruchus maculatus) in cowpea

Cowpea is an important legume crop widely grown in sub‐Saharan Africa for food and feed. However, it is largely challenged by bruchid, a serious storage pest resulting in losses in quantity and quality of grains. Therefore, this research was designed to contribute to the breeding of cowpea resistance to bruchid through the identification of candidate genes associated with resistance to bruchid. A total of 217 mini‐core cowpea accessions were genotyped and phenotyped for their reactions to bruchid. To determine the genomic regions linked with bruchid resistance, 41,948 polymorphic SNP markers were used. Genome‐wide association study identified 11 SNPs linked to the average number of eggs, holes, insect emergence and development period and Dobie susceptibility index. Gene search via Phytozome identified six candidate genes (Vigun08g132300, Vigun08g158000, Vigun06g053700, Vigun02g131000, Vigun01g234900 and Vigun01g201900) associated with the resistance traits. These candidate genes could be incorporated into the farmers preferred but susceptible cowpea varieties to bruchid. The SNP markers associated with the resistance traits can be used in marker‐assisted breeding for accurate and rapid screening of cowpea resistant genotypes to bruchid.     

Insect-mediated seed-set evaluation of 21 soybean lines segregating for male sterility at 10 different loci

The cowpea trypsin inhibitor gene (CpTI) and neomycin phosphotransferase gene (nptII) were introduced into the embryonic callus cells of immature embryos of wheat elite line Shannong 995604 using Agrobacterium-mediated gene transfer. Independent plantlets were regenerated from kanamycin-resistant calli. PCR and real time PCR analysis, PCR-Southern and Southern blot hybridization indicated that there were three independently-dervied transgenic plants viz. transformed-I, II and III (T-I, T-II and T-III). The segregation of CpTI in the transgenic wheat progenies of T-Iand T-III were consistent with Mendelian inheritance. Resistance to the storage insect pest of wheat viz. the grain moth (Sitotroga cerealella Olivier) was improved significantly in seeds of the three transgenic wheat T₂ lines obtained from T₁ PCR-positive plants. The frequency of moth-eaten seed from T-I, T-IIand T-III was reduced 66.76%, 62.48% and 43.59% respectively. The investigation of agronomic traits of the three transgenic wheat T₁ PCR-positive plants revealed that the three transgenic lines had excellent agronomic traits. They provide good germplasm resource for wheat genetic improvement.     

Resistance to Spodoptera litura (Fabr.) in Helianthus species and backcross derived inbred lines from crosses involving diploid species

Spodoptera litura (Fabr.), the tobacco caterpillar, is a major defoliator on sunflower in the tropics. Genetic variability for resistance to S. litura is limited in the cultivar germplasm of sunflower. In the present investigation, 43 accessions of 17 wild Helianthus species of the annual and perennial habit groups were evaluated along with cultivated sunflower under field and no-choice conditions in the laboratory for resistance to this pest. Under field conditions, H. occidentalis and H. argophyllus were found to be immune with no leaf damage and few accessions of the species belonging to section Divaricati were found to be resistant. Laboratory bioassays against neonate, two and 4-day-old larvae confirmed resistance both in terms of high larval mortality and low larval weight gain in eight species viz., H. occidentalis, H. argophyllus, H. tuberosus, H. maximiliani, H. mollis, H. simulans, H. divaricatus and H. hirsutus. Intra-accessional variability was observed and accessions of few species showed varied reaction (resistant, partially resistant to susceptible) to the target pest. Field evaluation of 224 backcross derived inbred lines from five cross combinations involving diploid species under high natural pest incidence revealed low damage in plants derived from crosses involving H. argophyllus and H. petiolaris.     

Inheritance of resistance to spotted stem borer, Chilo partellus, in sorghum, Sorghum bicolor

The spotted stem borer, Chilo partellus, is one of the most important pests of sorghum, and host plant resistance is an important component for the management of this pest. Most of the sorghum hybrids currently under cultivation are based on cytoplasmic male-sterility (CMS). In order to develop a strategy for resistance to stem borer, we studied the traits associated with resistance, and their nature of gene action in F₁ hybrids derived from resistant, moderately resistant, and susceptible CMS and restorer lines. The hybrids based on stem borer-resistant, moderately resistant, or susceptible CMS and restorer lines were equally resistant or susceptible as the parents for leaf feeding [Damage rating (DR) 5.8 to 6.6 vs. 5.9 to 6.6], and had significant and decreasing trend in deadheart formation (resistant CMS × resistant restorer lines < moderately resistant CMS × moderately resistant restorer lines < susceptible CMS × susceptible restorer lines), respectively. Proportional contributions of restorer lines were greater than those of the CMS lines for leaf feeding, deadhearts, recovery and overall resistance, stalk length, nodes per plant, stem borer holes per plant, and peduncle tunneling. The general (GCA) and specific combining ability (SCA) estimates suggested that leaf feeding score, number of nodes, overall resistance score, panicle initiation, recovery score, and stalk length (dominance type of gene action) have been found to be associated with resistance to spotted stem borer, governed by additive type of gene action, their correlation and direct effects in the same direction, and explained 65.3% of the variation in deadhearts, and thus could be used as marker traits to select and breed for resistance to C. partellus in sorghum. The parents having significant SCA effects for two or more resistance traits for either or more parents have also been discussed for their use in the stem borer resistance breeding.     

Inheritance of seed resistance to bruchids in cultivated mungbean (Vigna radiata, L. Wilczek)

Bruchid beetles or seed weevils are the most devastating stored pests of grain legumes causing considerable loss to mungbean (Vigna radiata (L.) Wilczek). Breeding for bruchid resistance is a major goal in mungbean improvement. Few sources of resistance in cultivated genepool were identified and characterized, however, there has been no study on the genetic control of the resistance. In this study, we investigated the inheritance of seed resistance to Callosobruchus chinensis (L.) and C. maculatus (F.) in two landrace mungbean accessions, V2709BG and V2802BG. The F₁, F₂ and BC generations were developed from crosses between the resistant and susceptible accessions and evaluated for resistance to the insects. It was found that resistance to bruchids in seeds is controlled by maternal plant genotype. All F₁ plants derived from both direct and reciprocal crosses exhibited resistance to the bruchids. Segregation pattern of reaction to the beetles in the F₂ and backcross populations showed that the resistance is controlled by a major gene, with resistance is dominant at varying degrees of expressivity. Although the presence of modifiers was also observed. The gene is likely the same locus in both V2709BG and V2802BG. The resistant gene is considered very useful in breeding for seed resistance to bruchids in mungbean.     

Involvement of polyamines, diamine oxidase and polyamine oxidase in resistance of barley to Blumeria graminis f. sp. hordei

Polyamine levels and diamine and polyamine oxidase activities have been investigated in the first leaves of barley (Hordeum vulgare L.) in the absence of or following inoculation with conidia of powdery mildew (Blumeria graminis f. sp. hordei). Two cultivars with varying sensitivity to powdery mildew, viz., Chariot (resistant) and Golden Promise (susceptible) were used. The levels of putrescine, spermidine and spermine were found to be higher in the leaves of Chariot than in the leaves of Golden Promise and, with the exception of spermine, were generally higher in both cultivars after inoculation. In inoculated leaves of Chariot, levels of putrescine and spermidine peaked at 9 days and 12 days, respectively. In controls (uninoculated leaves), the activities of these enzymes, and putrescine and spermidine levels also increased but not to the same extent as in inoculated leaves. With Golden Promise, the levels of putrescine and spermidine in the inoculated leaves changed very little over sampling times but were always higher than in the controls. In this cultivar, there was little difference between inoculated leaves and the controls in diamine oxidase activity which reached a maximum value at 9 days post-inoculation. Activity of the bound form of diamine oxidase was low in both the cultivars. Polyamine oxidase was not detected at 3 days after inoculation in either cultivar but activity at fairly low levels was recorded at later times, usually reaching a maximum value at 9 days. The results suggest that polyamine metabolism and diamine oxidase activity in particular may be involved in the mechanism conferring resistance to barley powdery mildew in Chariot.     

Genetic control of Russian wheat aphid (Diuraphis noxia) resistance in wheat accession PI 47545

The Russian wheat aphid, Diuraphis noxia (Mordvilko), is a major pest of cereal crops in many areas of the world, causing serious reduction in grain yield in wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.). Incorporating genetic resistance to D. noxia into wheat cultivars is paramount to effectively reduce damage inflicted by this pest. Genetic resistance to D. noxia has been identified in wheat, barley and rye germplasm, and several resistance genes are available for use for cultivar improvement. In the United States of America, only a few Russian wheat aphid (RWA) resistant winter wheat cultivars are currently available, and these cultivars contain only one of the six known RWA resistance genes. The objective of this study was to determine the inheritance of RWA resistance in wheat accession PI 47545, using a screening method based on differences in the leaf morphology of resistant and susceptible types following insect challenge. PI 47545 was selected for study, since it displayed high levels of resistance in a white-grained wheat background, the predominant wheat class produced in the Pacific Northwest of the USA. Segregation analysis was conducted on an F₂ population developed by cross-hybridizing the susceptible soft white winter wheat cultivar ‘Daws’ to the resistant accession PI 47545. Russian wheat aphid screening data from this population indicated that the resistance in PI 47545 is controlled by a single, dominant gene (χ² = 1.72; p ≤ 0.189). This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic analysis and pyramiding of two gall midge resistance genes (Gm-2 and Gm-6t) in rice ( Oryza sativa L.)

Asian rice gall midge (Orseolia oryzae) is a major pest across much of south and southeast Asia. This pest is genetically diverse and many gall midge biotypes are known to exist in each country. During the last three decades, host plant resistance has proved to be the most effective mechanism of controlling the Asian rice gall midge. Seven genes conditioning resistance to gall midge larvae have been identified in rice (Oryza sativa) and are being used in cultivar improvement programs. However, some of these genes are rendered ineffective by new gall midge biotypes. Increased understanding of genetics, inheritance, allelic relationships and linkage is necessary to maximise the durability of major gene resistance by the pyramiding of these genes. The two genes, Gm-2 and Gm-6(t), are known to confer resistance against a number of biotypes in India and China, respectively. An F₃ population derived from a cross between Duokang #1 (donor of Gm-6(t)) and Phalguna (donor of Gm-2) was screened against Chinese gall midge biotype 4 at Guangdong, China, and Indian gall midge biotype 1 at Raipur, India. At each location, separately,a single gene governed resistance. The parallel segregation of 417 F₃progenies for both biotypes at two locations revealed that recombination had occurred between the two genes, establishing that the two genes are not allelic. However, the two genes Gm-2 and Gm-6(t), were found to be linked with a distance of ∼16.3 cM. A number of lines homozygous at one locus and segregating for the other locus were identified and selected. These lines were selfed to obtain lines homozygous for the favourable alleles at both loci (two locus pyramids). This is the first report on use of conventional host-pest interaction method for pyramiding two closely located Gm-resistance loci of dissimilar effects. The implications of deployment of these pyramids within and across country borders, with reference to the prevailing gall midge populations are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparison of sources of resistance to leaf aphids in lettuce (Lactuca sativa L.)

Summary Three lines of lettuce with resistance to Nasonovia ribisnigri, based on the dominant Nr-gene, and four lines selected for partial resistance to Myzus persicae were tested against three species of leaf aphid: N. ribisnigri, M. persicae and Macrosiphum euphorbiae. The effect of the Nr-gene was also studied in a segregating F2 population.In the material tested, resistance to N. ribisnigri was exclusively based on the Nr-gene, lines selected only for resistance to M. persicae showed no resistance to N. ribisnigri. The Nr-gene also induces partial resistance to M. persicae, but the level of this resistance is influenced by other genes, because the lines with Nr-gene differed significantly from each other for reproduction of M. persicae. The Nr-gene had no effect on the resistance of lettuce to M. euphorbiae.In lines with the Nr-gene, levels of resistance to M. persicae and to M. euphorbiae were correlated, suggesting that the resistance may be determined by the same genes. The Nr-line with highest resistance to M. persicae was comparable for this characteristic to the lines selected for resistance to M. persicae.The cultivars Taiwan and Ravel possess a resistance factor to M. euphorbiae that has no effect on M. persicae or N. ribisnigri. Lines selected for resistance to M. persicae also showed partial resistance to M. euphorbiae. Based on the present results no conclusions can be drawn whether this resistance is based on the same genes that provide resistance to M. persicae, or on a resistance factor comparable to that found in Taiwan and Ravel.     

Monosomic analysis of Russian wheat aphid (Diuraphis noxia) resistance in Triticum aestivum line PI137739

Summary The Russian wheat aphid, Diuraphis noxia (Mordvilko) (Homoptera: Aphididae), has become an important pest of wheat (Triticum aestivum L.) in the United States. The aphid causes a phytotoxemic reaction in wheat evidenced by local and systemic chlorosis and rolling of infested leaves. Developing resistance in wheat cultivars to D. noxia is an essential factor in controlling the damage caused by this pest. Several sources of genetic resistance to D. noxia have been identified in wheat germplasm. Monosomic analysis of the monogenic resistant T. aestivum accession PI137739 has shown that the gene (Dn1) for resistance is carried on chromosome 7D. It appears that chromosome 7B may carry a second resistance gene for D. noxia that might be a source of minor or complementary gene action for resistance.     

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.