Rapid Screening Method of Cassava Cultivars for Resistance to Colletotrichum gloeosporioides f.sp. manihotis

An in vitro method for assessing cassava anthracnose disease (CAD) resistance was developed as a preliminary screen to a CAD-resistant breeding programme. Potato dextrose agar (PDA) media was amended by extracts from the stem cortex of 10 cassava cultivars (30001; 30572, 30211, 88/02549, 88/00695, 88/01336, 91/00344, 91/00313, 91/00684 and 91/00475), and assayed for efficacy of inhibition of the growth of Colletotrichum gloeosporioides f. sp. manihotis isolates (05FCN, 10FCN, 12FCN, and 18FCN). Morphological and physiological data indicated that there was a significant difference (P ≤ 0.05), in mycelial growth, spore germination and sporulation among the four isolates on PDA amended with cassava stem extracts. Extracts from cassava cultivars 30211, 91/00684 and 91/00313 showed higher inhibition of germ tube development, mycelial growth and sporulation of the fungal isolates, whereas cultivars 88/02549 and 88/01336 showed the least inhibition. The 10 cultivars were further tested in both greenhouse and field conditions, under disease pressure for two planting seasons, to corroborate resistance to the fungus as observed in vitro . Greenhouse and field trials with the 10 cassava cultivars showed a significant difference ( P  ≤ 0.05) in CAD resistance. Cultivars 88/02549 and 88/01336 were highly CAD-susceptible, as shown in the in vitro assays and confirmed in the greenhouse and field tests. The other eight cultivars were either resistant (30211, 91/00684), or moderately resistant (30572, 88/00695, 91/00475, 91/00344, 30001 and 91/00313) to CAD. The study shows that an in vitro screening assay of cassava for resistance to CAD could serve as a convenient preliminary screening technique to discriminate CAD-resistant from CAD-susceptible cassava cultivars. The in vitro screening method considerably reduces time and labour in comparison with the current screening techniques of cassava, which involve field planting, inoculation and evaluation.     

Screening of cassava and yam cultivars for resistance to anthracnose using toxic metabolites of colletotrichum species

Collectotrichum gloeosporioides f. sp. manihotis and C. gloeosporioides, causal agents of cassava (Manihot spp.) and yam (Dioscorea spp.) anthracnose diseases, respectively, produce toxic metabolites in culture that fluoresce at 254 nm and 366 nm, producing bands with Rf of 0.65 and 7.0, respectively. Symptoms induced on yam and cassava by the extracted metabolites were similar to those induced by the pathogens. Twenty-four clones of tropical D. rotundata (TDr), D. alata (TDa), D. esculenta (TDe), and D. cayenensis (TDc) were screened by applying toxic metabolites of C. gloeosporioides to their leaves and stems. Only TDr131, TDe 179 and TDc750 were resistant. Other clones were susceptible to varying degrees. Nineteen of the 45 clones of M. esculenta were resistant to varying degrees of toxic metabolites of C. gloeosporioides f. sp. manihotis. Results from in vitro screening of’ cassava and yam clones using toxic metabolites compared favourably with field screening based on natural epidemics. Using toxic metabolites appears to be a more effective technique for screeningfor disease resistance than conventional inoculation with plant. pathogens. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evaluation of selected geocarposphere bacteria for biological control of Aspergillus flavus in peanut

Selected bacterial strains isolated from the region of peanut pod development (geocarposphere) and two additional bacterial strains were screened as potential biological control agents against Aspergillus flavus invasion and subsequent aflatoxin contamination of peanut in laboratory, greenhouse, and field trials. All 17 geocarposphere strains tested delayed invasion of young roots and reduced colonization by the fungus in a root-radicle assay used as a rapid laboratory prescreen. In a greenhouse study, seven bacterial strains significantly reduced pod colonization by A. flavus compared to the control. In a field trial, conducted similarly to the greenhouse assay, pods sampled at mid-peg from plants seed-treated with suspensions of either 91A-539 or 91A-550 were not colonized by A. flavus, and the incidence of pods invaded from plants treated with either 91A-539 or 91A-599 was consistently lower than nonbacterized plants at each of five sampling dates. At harvest, 8 geocarposphere bacterial strains significantly lowered the percentage of pods colonized (> 51%) compared to the control. Levels of seed colonization ranged from 1.3% to 45% and did not appear related to aflatoxin concentrations in the kernels.     

Assessment of laboratory methods for evaluating cassava genotypes for resistance to root rot disease

Field evaluation of six cassava genotypes for resistance to root rot disease was compared with three rapid laboratory methods (whole root inoculation, root slice inoculation, and stem inoculation) for resistance screening. Both the field evaluation and the three laboratory methods separated the varieties into resistant and susceptible groups. Genotypes 30572 and 91/02324 were resistant while 92/0247, 92/0057 and TME-1 were susceptible. One genotype (30001) was not consistent in its reaction between field evaluation and laboratory assays. In the laboratory assays with three fungal pathogens, different pathogens varied in their levels of virulence on host genotypes. With the most virulent pathogen (Botryodiplodia theobromae), the majority of the genotypes reacted in the same way across trials with the root slice and whole root assays. Due to the good correlation between the whole root assay and the field results, we recommend this for the routine assessment of cassava resistance to root rot disease and for the analysis of virulence of pathogen isolates. However, because of the advantages in terms of economy of labour, space, time, quantity of root and inoculum required, the root slice assay could be used for the preliminary screening of large cassava accessions. The selected genotypes can then be further screened with the whole root inoculation method.     

In vitro mutation and selection of doubled-haploid Brassica napus lines with improved resistance to Sclerotinia sclerotiorum

This paper describes a new protocol to develop doubled-haploid (DH) Brassica napus lines with improved resistance to Sclerotinia sclerotiorum. In this protocol, haploid seedlings derived from microspore cultures of B. napus were used to produce haploid calli for in vitro mutation-selection. For routine screening, mutation was induced by EMS (ethylmethane sulfonate) or occurred spontaneously, and screening for resistant mutants occurred on media with added oxalic acid (OA) as a selection agent. In tests with selected lines, the optimal concentration of EMS for mutation was determined to be 0.15%, and the optimal concentration of OA for in vitro screening was 3 mmol/l (half lethal dose was 3.1 mmol/l) for the first cycle of screening. There was an accumulated effect of OA toxicity on calli over two cycles of screening, but the growth and capacity of the surviving calli for regenerating seedlings were not affected by OA. Of the 54 DH lines produced from the in vitro mutation-selection, two DH lines of resistant mutants, named M083 and M004, were selected following seedling and glasshouse tests. The resistance of M083 and M004 to S. sclerotiorum following tests with both mycelial inoculum and OA was greater than that of their donor lines and the resistant control Zhongyou 821. In both glasshouse and field disease nurseries, disease indices on M083 and M004 were less than 50% of those of the control. The time required for M083 and M004 to mature was 14 days and 10 days shorter, respectively, than that of their donor lines. Furthermore, M083 had more pods per inflorescence, a greater 1,000 seed weight and higher yield than its donor line. Random amplified polymorphic DNA characterisation showed that M083 had DNA band patterns that differed from its donor line.     

Transgenic plants of Vitis vinifera cv. Seyval blanc

Leaf discs of grapevine cv. Seyval blanc originating from in vitro cultures were transformed with Agrobacterium tumefaciens strain LBA 4404 harbouring the vector pGJ42 carrying genes for chitinase and RIP (ribosome-inactivating protein) in an attempt to improve fungal resistance. The gene for neomycin phosphotransferase II (nptII) was used as the selectable marker gene. The explants were cocultivated for 2 days with recombinant Agrobacteria and then submitted to selection on NN69 medium containing 100 mg/l kanamycin. Successful regeneration and conversion of transgenic plantlets were obtained. Stable integration of foreign DNA was confirmed by PCR and Southern blot analyses, and protein expression was detected by Western blot. The regenerated transgenic plants were adapted to the greenhouse and showed no evidence of phenotypical alterations. The foreign genes introduced into the transformed plants did not effect the expected improvement in fungal disease resistance under field conditions for the major pests Uncinula necator and Plasmopara viticola.     

Overexpression of defense response genes in transgenic wheat enhances resistance to Fusarium head blight

Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum and other Fusarium species, is a major disease problem for wheat production worldwide. To combat this problem, large-scale breeding efforts have been established. Although progress has been made through standard breeding approaches, the level of resistance attained is insufficient to withstand epidemic conditions. Genetic engineering provides an alternative approach to enhance the level of resistance. Many defense response genes are induced in wheat during F. graminearum infection and may play a role in reducing FHB. The objectives of this study were (1) to develop transgenic wheat overexpressing the defense response genes α-1-purothionin, thaumatin-like protein 1 (tlp-1), and β-1,3-glucanase; and (2) to test the resultant transgenic wheat lines against F. graminearum infection under greenhouse and field conditions. Using the wheat cultivar Bobwhite, we developed one, two, and four lines carrying the α-1-purothionin, tlp-1, and β-1,3-glucanase transgenes, respectively, that had statistically significant reductions in FHB severity in greenhouse evaluations. We tested these seven transgenic lines under field conditions for percent FHB disease severity, deoxynivalenol (DON) mycotoxin accumulation, and percent visually scabby kernels (VSK). Six of the seven lines differed from the nontransgenic parental Bobwhite line for at least one of the disease traits. A β-1,3-glucanase transgenic line had enhanced resistance, showing lower FHB severity, DON concentration, and percent VSK compared to Bobwhite. Taken together, the results showed that overexpression of defense response genes in wheat could enhance the FHB resistance in both greenhouse and field conditions.     

Research on the aflatoxin problem in groundnut at ICRISAT

Summary Aflatoxin contamination of groundnut is a serious problem in most groundnut producing countries and as such is given high research priority by the Groundnut Improvement Program of ICRISAT. Since 1979 we have concentrated on selecting cultivars resistant to seed invasion and colonization by toxigenicAspergillus flavus, and/or to aflatoxin production following invasion by the fungus. Resistance to invasion and colonization byA. flavus of rehydrated, mature seed has been found, and confirmed, in some cultivars. We have also screened several groundnut cultivars for seed resistance in the field, both under natural conditions and with the inoculum of the fungus added to the soil in the pod zone. Some cultivars with resistance to seed colonization also showed resistance to seed invasion byA. flavus. None of the cultivars tested has shown complete resistance to aflatoxin production but significant cultivar differences occurred in the amounts of aflatoxin produced in seeds inoculated with a toxigenic strain ofA. flavus.ICRISAT Journal Article No. JA-316     

Evaluation of Abiotic Stress Resistance in Mutated Populations of Cauliflower (Brassica oleracea var. botrytis)

An efficient in vitro screening method has been developed for cauliflower to create NEU and NMU induced mutant lines selected on hydroxyproline containing medium. Mutant lines and control plants were sub-cultured many times on maintenance medium and stored at 5°C for 2 years and then tested for salt and hydroxyproline resistance as in vitro and in vivo plants. In vitro shoot tips were also sub-cultured to media containing hydroxyproline and NaCl for 28 days and then assessed for their leaf proline content. Non-acclimated and acclimated in vivo plants were also assessed for resistance to freezing. Populations of control and selected lines were created by mass pollination and subsequently tested for their NaCl and frost resistance. Control plants had little or no NaCl or hydroxyproline resistance whilst selected plants showed varying degrees of resistance. In vitro and in vivo responses of selected lines were correlated. Leaf proline content was increased markedly in the mutant lines and the greatest proline contents occurred following NaCl stress with the most respondent line having 100-fold levels compared to the controls. Both non-acclimated and acclimated selected lines showed improved frost resistance over controls. Improvements in frost resistance were heritable but improvements in NaCl resistance were not. The results clearly demonstrated that NaCl, frost and hydroxyproline resistance were stable traits over repeated in vitro sub-cultures and prolonged low temperature storage. A complete range of mutants with single, double or triple resistance traits were produced. The level of resistance however was not necessarily correlated with the level of proline and some lines showed resistance without elevated proline. It is concluded that elevated proline is not essential for improved resistance to abiotic stress in cauliflower, but where it does occur it does improve resistance.     

Biological control of alligatorweed (Alternanthera philoxeroides) with a Fusarium sp.

Alligatorweed (Alternanthera philoxeroidesG.) has become a serious weed in different crops in China. A fungal pathogen was found in Chongqing and Sichuan Provinces and was identified as a species in the Fusarium genus. The fungus produced macroconidia and chlamydospores abundantly on potato sucrose agar (PSA) plates. The bestconidial production and germination and colonygrowth of Fusarium sp. were at 23–31°C and pH 6.7–7.0. Light period and flooding did not affect fungal growth and conidium formation. The herbicides, glyphosate and paraquat, inhibited the fungal development in vitro. The fungus did not affect seed germination and seedling growth of paddy rice, wheat, maize, oilseed rape and broad bean inlaboratory or greenhouse trials. Inoculum density and wetness duration influenced the efficiency of Fusarium sp. to control alligatorweed; a concentration of 1.0 × 10⁵ spores^–1 ml and 12 h of high humidity duration after inoculation produced goodinfections on the weed at 23°C in the laboratory. When the fungus was applied to alligatorweed grown in greenhouse and in the field, good biocontrol efficiency was obtained: the plants started to wilt after four to five (greenhouse) or six days (field), and were killed 9–10 (greenhouse) or 13–14 (field) days after spraying the fungal inoculum. This was similar to the control efficiency resulting from glyphosate treatment. Therefore, this Fusarium sp. appeared to be a good candidatefor further studies and a promising biocontrol agent to manage alligatorweed in some terrestrial and aquatic crops.     

Transfer and Expression of an Artificial Storage Protein (ASP1) Gene in Cassava (Manihot Esculenta Crantz)

In order to increase the nutritional quality of cassava storage roots, which contain up to 85% starch of their dry weight, but are deficient in protein, a synthetic ASP1 gene encoding a storage protein rich in essential amino acids (80%) was introduced into embryogenic suspensions of cassava via Agrobacterium-mediated gene transfer. Transgenic plants were regenerated from suspension lines derived from hygromycin-resistant friable embryogenic callus lines. Molecular analysis showed the stable integration of asp1 in cassava genome and its expression at RNA level in transformed suspension lines. PCR and Southern analyses proved the transgenic nature of the regenerated plant lines. The expression of asp1 at RNA level was demonstrated by RT-PCR. The ASP1 tetramer could be detected in leaves as well as in primary roots of cultured transgenic plants by western blots. These results indicate that the nutritional improvement of cassava storage roots may be achieved by constitutive expression of asp1 in transgenic plants.     

Half sib selection for resistance to powdery mildew (Erysiphe graminis DC. f.sp. secalis Marchal) in rye populations

Summary Three rye populations originating from different cultivars were subjected to half sib progeny selection for three cycles to improve their resistance to powdery mildew (Erysiphe graminis DC. f.sp. secalis Marchal). Selection was carried out in the greenhouse (measurement of spore production at the three-leaf stage), in the field (scoring of mildew infections at ear emergence) or at both locations. All selections resulted in significant responses, the level of which varied depending on the genetic base of mildew resistance in the populations. Greenhouse selection was most effective when the frequency of dominant genes could be increased, whereas field selection was less effective. Estimates for heritability in the narrow sense ranged from 0.42–0.94 indicating that mildew resistance can be increased effectively. Inbred lines should be selected in base populations with a high level of quantitative resistance and/or a high frequency of dominant genes. For an effective resistance strategy in hybrid breeding such dominant genes are most valuable, and can be selected in young growth stages. In Strain CG the increase of dominant resistance genes by early selection resulted in a significant improvement of kernel yield.     

木薯UGT41基因对细菌性枯萎病的抗性研究北大核心CSCD

糖基转移酶广泛存在于植物中,其中UDP依赖型糖基转移酶(UDP-glycosyltransferases,UGTs)基因家族是糖基转移酶中的一大类。该研究以华南124木薯品种(Manihot esculenta cv.SC124)为材料,利用RT-PCR技术克隆木薯MeUGT41基因,以病毒诱导干扰木薯MeUGT41基因的表达量,并对基因干扰植株进行细菌性枯萎病抗性评价,为研究MeUGT41基因在木薯抵抗细菌性枯萎病的抗病机理奠定基础。结果表明:(1)地毯草黄单胞菌(Xamthomonas axonopodis pv.Manihotis,Xam)可显著诱导木薯MeUGT41基因表达。(2)成功构建MeUGT41的病毒诱导基因沉默(VIGS)载体,将干扰载体转化至木薯叶片进行MeUGT41基因沉默,荧光定量PCR检测结果显示,木薯叶片中MeUGT41基因的表达量显著下降。(3)Xam侵染实验表明,干扰抑制MeUGT41基因表达可显著降低木薯植株叶片对Xam病菌侵染的抵抗能力。研究认为,木薯叶片中MeUGT41基因具有抵抗Xam病菌侵染的能力。     

Analysis of somatic hybrids between two sterile dihaploid Solanum tuberosum L. breeding lines. Restoration of fertility and complementation of G. pallida Pa2 and Pa3 resistance

Fourteen somatic hybrids generated by electrofusion of mesophyll protoplasts from a non-flowering dihaploid S. tuberosum clone, DHAK-11, and a male-sterile dihaploid clone S. tuberosum, DHAK-33, were grown in the greenhouse and subjected to morphological assessments and tests for fertility and resistance to the white potato cyst nematode Globodera pallida pathotypes Pa2 and Pa3. The ploidy level of the hybrids ranged from 38 to 63 chromosomes. All hybrids developed flowers with violet petals except for one, hy-56, that possessed red petals. The colour of the tuber skin was purple in all hybrids except in hy-56 where the tuber skin was red. All of the hybrids were female fertile and generated viable seeds. Near-tetraploid hybrids produced the highest number of seeds per fruit and these seeds had a normal size. Hybrids with 58 or more chromosomes produced smaller seeds and less seeds per fruit. The germination frequency of the seeds was not influenced by the chromosome number of the hybrids. Pollen viability was determined and the male fertility of three hybrids was tested. Pollination with these three hybrids gave rise to fruit development, but only one produced viable seeds. The hybrids were tested for resistance to G. pallida pathotypes Pa2 and Pa3. A high level of resistance to Pa3, inherited from one parental clone, DHAK-11, and a high level of resistance to Pa2, inherited from the other parental clone, DHAK-33, was combined in four hybrids. These results demonstrate, that protoplast fusion is an efficient method for restoring the fertility of somatic hybrids generated from sterile parent clones, and is a powerful procedure for the complementation of multigenetic disease resistance traits in potato breeding lines.     

Scaling up tests on virulence of the cassava green mite fungal pathogen <Emphasis Type="Italic">Neozygites tanajoae</Emphasis> (Entomophthorales: Neozygitaceae) under controlled conditions: first observations at the population level

Virulence of entomopathogens is often measured at the individual level using a single host individual or a group of host individuals. To what extent these virulence assessments reflect the impact of an entomopathogen on their host in the field remains largely untested, however. A methodology was developed to induce epizootics of the cassava green mite fungal pathogen Neozygites tanajoae under controlled conditions to evaluate population-level virulence of two (one Beninese and one Brazilian) isolates of the entomopathogen—which had shown similar individual-level virulence but different field impacts. In unrepeated separate experiments we inoculated mite-infested potted cassava plants with either 50 or 25 live mites (high and low inoculum) previously exposed to spores of N. tanajoae and monitored the development of fungal infections for each isolate under the same conditions. Both isolates caused mite infections and an associated decline in host mite populations relative to the control (without fungus) in all experiments, but prevalence of the fungus varied with isolate and increased with inoculum density. Peak infection levels were 90% for the Beninese isolate and 36% for the Brazilian isolate at high inoculum density, and respectively 17% and 25% at low inoculum density. We also measured dispersal from inoculated plants and found that spore dispersal increased with host infection levels, independent of host densities, whereas mite dispersal varied between isolates. These results demonstrate that epizootiology of N. tanajoae can be studied under controlled conditions and suggest that virulence tests at the population level may help to better predict performance of fungal isolates than individual-level tests.     

Induced mutations in cassava using somatic embryos and the identification of mutant plants with altered starch yield and composition

A cyclic somatic embryogenic system was used to induce mutations in cassava variety PRC 60a in vitro. Globular-stage somatic embryos were selected as suitable experimental materials, and 50 Gy of -rays was determined to be the optimal dose for inducing mutations. During subsequent field trials, more than 50% of the regenerated mutant lines varied morphologically from wild-type plants. Consequently, we used this approach to induce genetic variability for obtaining novel cassava cultivars. Among the different mutant lines obtained, lines S14 and S15 showed large morphological variations. In 10-month-old S14 and S15 mutant lines, storage root yield was reduced 17-fold and 60-fold, respectively, compared to wild-type plants, while the storage roots of S15 mutant plants also exhibited an almost 50% decrease in starch content and a significant reduction (30%) in amylose content. These two features were observed throughout the different developmental stages of the storage roots in S15 plants.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - EMS Ethylmethanesulfonate - IBA Indole-3-butyric acid     

Somaclonal variation in celery: screening for resistance to Fusarium oxysporum f. sp. apii

Summary Two methods were used to screen putative Fusarium-resistant celery (Apium graveolens L.) plantlets from cell culture: placing plantlets on a mycelial mat for one month or planting them directly in Fusarium-infested soil. Resistant phenotypes were identified with both methods, but the plants grown on the mycelial mat died before they reached reproductive maturity. Four plants, K, T-2, T-3, and R-R₁ from the soil screen, survived and produced viable seed. Tests of self-pollinated progeny, in field and greenhouse conditions, showed that T-2, T-3, and R-R₁ were superior to the original cultivar, 5270R, with respect to disease resistance, as measured by vascular discoloration and plant height. Chi-square analysis of progeny scores for root and crown decay showed that the new variation was heritable and appeared to be conditioned by more than one locus.     

Progeny tests of barley,wheat, and potato regenerated from cell cultures after in vitro selection for disease resistance

Summary Because plant cells cultured in vitro express genetic variability and since they can be regenerated into functional plants, procedures have been designed to use this system for the production of plants with new important agronomic characteristics, particularly for disease resistance. For barley, wheat, and potato somaclones have been found that were less susceptible to a toxin of Helminthosporium, fusaric acid, Fusarium coeruleum, F. sulphureum, or Phytophthora infestans, when screened in the first in-vitro-derived generation. Here the progeny of such somaclones is evaluated after natural and artificial infection, using greenhouse-grown or field material. The progenies of the same somaclones did not express detectable differences, which indicated that no heterozygous mutations occurred. Most lines and clones differed in their level of susceptibility to the pathogen compared to the level of the starting material, but these data were in no instance significant. It is discussed here whether this lack of significance is due to a lack of genetic differences or whether the test procedures are in adequate for detecting and securing the slight, probably quantitative, alterations.     

The potential of Ulocladium botrytis for biological control of Orobanche spp.

A strain of Ulocladium botrytis isolated from diseased Orobanche crenata shoots caused disease on the parasitic weed in pathogenicity tests. The potential of the fungus to be developed as a mycoherbicide for Orobanche spp. was further investigated. Although the fungus significantly decreased O. crenata germination in vitro by 80%, it did not generally lead to a decreased number of O. crenata shoots or tubercles in inoculated root chambers or pots. However, the number of diseased or dead tubercles and underground shoots was significantly increased compared to the noninoculated treatments. Postemergence inoculation of O. crenata shoots with a conidial suspension resulted in the death of almost all inoculated plants 14 days after application under greenhouse conditions. In preliminary host-range studies, the pathogen caused disease on Orobanche cumana on sunflower whereas on Orobanche aegyptiaca shoots parasitizing tomato only minimal disease symptoms could be detected after postemergence inoculation. Based on the results of our investigations, we conclude that Ulocladium botrytis has only a limited potential to be used as a biocontrol agent against Orobanche spp.