Breeding and growth of Rhizopus in raw cassava by solid state fermentation

Nineteen Rhizopus strains were selected and tested for their growth capacity on raw cassava starch and their ability to produce amylase when grown on solid-state fermentations. Only three strains grew significantly on this natural substrate. Glucoamylase production was higher on raw cassava than on cooked cassava. After 48 h of fermentation, the protein content of cassava was increased from 1.75% to 11.3%. The byproducts of fermentation were fumaric acid, lactid acid and ethanol.     

Two-stage fermentation method for production of protein-enriched feed from cassava

Studies have been carried out into the production of microbial protein from cassava using Trichoderma reesei and yeast. In monoculture studies, T. reesei was grown on whole cassava medium to give 0.74g dry cell/g cassava. The dry material contained 42% protein. The culture filtrate contained 5.8 g/l glucose, which supported the growth of yeast. Mixed culture fermentation was also carried out with the two microorganisms. Besides accelerating the rate of degradation and conversion of cassava to cells (0.85g cell/g cassava) the yeast boosted the protein content of the growth product to 51%.     

Occurrence and distribution of cassava begomoviruses in Kenya

A survey for cassava mosaic disease (CMD) was conducted in Kenya, to investigate the factors contributing to the generally increased incidence and severity of CMD in the cassava growing regions and to study the distribution of the disease's causal begomoviruses, African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV) and their strains. Special emphasis was given to the occurrence of the destructive recombinant Uganda variant strain of EACMV (EACMV-UG2). Samples from 91 farmers' fields in the main cassava-growing areas of coastal and western Kenya were collected and subjected to ELISA and PCR for detection and typing of the begomoviruses present. CMD incidence was highest in western Kenya (80–100%) and lowest in the Coast province (25–50%). In Western and Nyanza provinces, 52% of the samples tested contained EACMV-UG2, 22% ACMV and 17% contained both ACMV and EACMV-UG2. EACMV was found in four cases at different sites. In cassava samples from the coast province, only EACMV with DNA-A sequences similar to EACMV strains present in Kenya and Tanzania was found. East African cassava mosaic Zanzibar virus (EACMZV) was present in several farms in the Kilifi district. In 15% of all cassava samples with CMD symptoms, flexuous, filamentous virus-like particles were also found, providing evidence for a more complex virus situation in cassava grown at the Kenyan coast. In western Kenya, where intense cassava cultivation takes place, CMD is rampant and EACMV-UG2 was found in mixed virus infections with ACMV driving the epidemics. In coastal areas, where farms are scattered and in isolation, EACMV is endemic, however, with a lower disease incidence and with a limited impact to cassava production.     

Progress of cassava mosaic virus disease and whitefly vector populations in single and mixed stands of four cassava varieties grown under epidemic conditions in Uganda

Progress curves of cassava mosaic virus disease (CMD) and populations of the whitefly vector (Bemisia tabaci) were assessed using four cassava varieties grown alone and as a random mixture in two experiments established under epidemic conditions at a site near Kampala in southern Uganda. There were significant differences in final CMD incidence and in the areas under the disease progress curves between varieties when grown alone and as a mixture in both experiments. Variety Ebwanateraka had the highest incidence and SS4 the lowest, even though it supported the largest populations of adult whiteflies. The overall incidence of CMD in the mixture was similar to that in pure stands of the partially resistant Nase 2 and greater than in the resistant Migyera and SS4. Compared to pure stands, incidence of CMD in each component of the mixture was reduced significantly only in Ebwanateraka, whereas vector populations were less only in SS4 and Nase 2. On several observation dates the actual incidence of CMD and populations of adult whiteflies in the mixture were significantly less than expected values estimated from the results for the four varieties when each was grown alone. A highly significant positive relationship was established for each variety between peak populations of adult whitefly and leaf area index at the time. The implications of the findings and the scope for future research on the use of varietal mixtures for the management of CMD are discussed.     

Amylase synthesis inAspergillus flavus andAspergillus niger grown on cassava peel

Summary Aspergillus flavus andAspergillus niger produce extracellular amylase into the culture medium when grown on basal medium containing 2% (w/v) soluble starch or cassava peel as the sole carbon source. On soluble tarch the highest amylase activities were 1.6 and 5.2 mg of starch hydrolyzed/min per mg protein forA. flavus andA. niger, respectively. When grown on cassava peel, the highest amylase activity in the culture filtrate ofA. flavus was 170-times higher than that on soluble starch, while that ofA. niger was 16-times higher. The mycelial dry weight for both organisms was not significantly affected by the carbon sources. Maximum enzyme activity was obtained at the growth temperature of 29.0±1°C and pH 7 for both organisms. It is concluded that cassava peel might be a better substrate for the production of amylase byA. flavus andA. niger than commercial soluble starch.     

Dependency of cassava (Manihot esculanta Crantz) on vesicular-arbuscular mycorrhizal fungi

Cassava plants were started in the greenhouse either from small cuttings (2.0 mg P/cutting) or large cuttings (20.2 mg P/cutting) in a subsurface Oxisol not inoculated or inoculated with Glomus aggregatum at target soil solution P concentrations of 0.003–0.2 mg l^-1. Vesicular-arbuscular mycorrhizal (VAM) fungal colonization levels in excess of 60% were attained on cassava roots irrespective of the size of cutting material used or target soil solution P status. However, plants started from large cuttings grew faster and better than those started from smaller ones. Cassava was found to be very highly dependent on VAM fungi if grown from small cutting but only marginally dependent if grown from large cuttings. The lower dependence of cassava on VAM fungi when started from larger cuttings appears to be related to the high P reserve in these cuttings and hence the low requirement of the plants for soil P until the P reserve in the cuttings is significantly depleted.A contribution from the Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 3896     

Breeding cassava for apomixis

Apomixis genes have been successfully transferred to cassava (Manihot esculenta) by hybridizing it with the wild species, M. glaziovii. The interspecific hybrid of cassava and M. glaziovii was exposed to open pollination during three subsequent generations. Seven sibs and the maternal progenitor of the fourth generation were genotyped using five microsatellite loci previously developed for cassava. All sibs were identical with each other and with their maternal progenitor. Sibs from M. glaziovii proved to be identical when examined by the same microsatellite loci. This evidence leads to the conclusion that apomixis does occur in wild-cassava relatives and apparently has played an important role in Manihot speciation. This is the first report of nearly 100% apomixis.     

Influence of water-stressed cassava on Phenacoccus herreni and three associated parasitoids

The influence of cassava Manihot esculenta Crantz grown under condition of water-stress on development and reproduction of the cassava mealybug, Phenacoccus herreni Cox & Williams, and levels of parasitism of three encyrtid parasitoids, Apoanagyrus diversicornis Howard, Aenasius vexans Kerrich, and Acerophagus coccois Smith, were studied in the laboratory. Two cassava cultivars were used: CM 507-37 (drought-tolerant) and CMC 40. A 30 day period of water stress, imposed by reducing the irrigation volume, led to a reduction in shoot development and stomatal conductance of leaves of both cassava genotypes. Phenacoccus herreni development and reproduction were favoured by cassava under water shortage. Parasitism decreased and water stress appeared to enhance the encapsulation of parasitoid eggs or larvae by the mealybug. In the case of the parasitoid A. diversicornis, there was a decrease in size of female progeny, suggesting a lower fitness in this species on cassava plants under water stress. All results indicated that cassava grown under low water availability favoured P. herreni development and reproduction, and affected the success of parasitism and, depending on the species, parasitoid development. The drought-tolerance characteristic of cassava genotypes and parasitoid species most suitable for controlling P. herreni in drought-stricken areas are discussed.     

In vitro somatic embryogenesis and plant regeneration of cassava

An efficient and reproducible plant regeneration system, initiated in somatic tissues, has been devised for cassava (Manihot esculenta Crantz). Somatic embryogenesis has been induced from shoot tips and immature leaves of in vitro shoot cultures of 15 cassava genotypes. Somatic embryos developed directly on the explants when cultured on a medium containing 4–16 mg/l 2,4-D. Differences were observed with respect to the embryogenic capacity of the explants of different varieties. Secondary embryogenesis has been induced by subculture on solid or liquid induction medium. Long term cultures were established and maintained for up to 18 months by repeated subculture of the proliferating somatic embryos. Plantlets developed from primary and secondary embryos in the presence of 0.1 mg/l BAP, 1mg/l GA₃, and 0.01 mg/l 2,4-D. Regenerated plants were transferred to the field, and were grown to maturity.     

木薯己糖激酶MeHXK5的催化功能鉴定

该研究利用ISSR分子标记,对分布于福建省内5个样地( 邵武、建阳、建瓯、周宁和屏南)的61个野钩锥(Castanopsis tibetana)单株的遗传多样性进行了分析,并采用聚类分析方法探讨了它们的遗传关系。结果表明： 用10条ISSR引物从61个单株的基因组DNA共扩增出158条带,包含145条多态性条带,多态性条带百分率达91.77%,其中引物 UBC817、UBC819与UBC842的多态性条带百分率（PPB）为100.0%。各居群的多态性条带百分率(PPB)、有效等位基因数(Ne)、Nei’s基因多样度(H)和Shannon’s多样性指数（I）等各遗传指数差异较大,其中各项遗传指标中最高的为邵武居群,而周宁居群则最低。5个居群的基因分化系数和基因流分别为0.144 0和2.973 0,说明5个居群总遗传变异的14.40%存在于居群间,85.60%存在于居群内。种间总基因多样度分别为0.395 8,种内基因多样度分别为0.338 8,表明钩锥种间遗传多样性较高,且种间变异大于种内变异。各居群间的遗传距离差异较大; 其中,邵武与建瓯居群的遗传距离最近,仅为0.081 5; 建阳和周宁居群的遗传距离最远,为0.162 9。通过聚类分析可将5个钩锥居群聚为3支,屏南与周宁的居群各自独立聚为2支;来自邵武、建瓯及建阳的居群聚为一支,且可进一步分为两个亚支,建阳居群为1个亚支,邵武和建瓯居群聚为1个亚支。供试的钩锥具有较高的遗传多样性,存在着较为频繁的基因交流。该研究结果较准确地揭示了钩锥种间的遗传多样性。     

Field testing and exploitation of genetically modified cassava with low-amylose or amylose-free starch in Indonesia

The development and testing in the field of genetically modified -so called- orphan crops like cassava in tropical countries is still in its infancy, despite the fact that cassava is not only used for food and feed but is also an important industrial crop. As traditional breeding of cassava is difficult (allodiploid, vegetatively propagated, outbreeding species) it is an ideal crop for improvement through genetic modification. We here report on the results of production and field testing of genetically modified low-amylose transformants of commercial cassava variety Adira4 in Indonesia. Twenty four transformants were produced and selected in the Netherlands based on phenotypic and molecular analyses. Nodal cuttings of these plants were sent to Indonesia where they were grown under biosafety conditions. After two screenhouse tests 15 transformants remained for a field trial. The tuberous root yield of 10 transformants was not significantly different from the control. Starch from transformants in which amylose was very low or absent showed all physical and rheological properties as expected from amylose-free cassava starch. The improved functionality of the starch was shown for an adipate acetate starch which was made into a tomato sauce. This is the first account of a field trial with transgenic cassava which shows that by using genetic modification it is possible to obtain low-amylose cassava plants with commercial potential with good root yield and starch quality.     

木薯纤维素乙醇发酵的纤维素酶成本评价

木薯中的纤维素成分约占木薯干重的10％(W/W).文中以木薯燃料乙醇生产的木薯纤维素酒渣为原料,从纤维素酶成本角度评估了三种利用木薯纤维素组分发酵生产乙醇的方法,包括木薯纤维素酒渣的直接糖化和乙醇发酵、木薯纤维素酒渣预处理后的糖化与乙醇发酵、木薯乙醇发酵中同步淀粉与纤维素糖化以及乙醇发酵.结果表明,前两种方法的纤维素利用效率不高,酶成本分别达到13602、11659元/吨乙醇.第三种方法,即在木薯乙醇发酵过程同时加入糖化酶和纤维素酶,进行同步淀粉与纤维素糖化,进而进行乙醇发酵,木薯纤维素乙醇的收益最高.发酵结束时的乙醇浓度从101.5g/L提高到107.0g/L,纤维素酶成本为3 589元/吨乙醇.此方法利用木薯纤维素与木薯淀粉同时进行,不会带来额外的设备及操作投入,酶成本低于产品乙醇价格,可实现盈利,因此第三种方法为木薯纤维用于乙醇发酵的最适方法,本研究结果将为木薯乙醇产业深度利用木薯纤维提供依据.     

Genetic variation of cassava (Manihot esculenta Crantz) cultivated by Chibchan Amerindians of Costa Rica

This study focuses on the genetic diversity of the cassava (Manihot esculenta Crantz) grown by the Chibchan Amerindians of Costa Rica. The authors collected cassava in various locations within two Amerindian Reserves: Talamanca, inhabited by Cabecares and Bribris, and Coto Brus, inhabited by Guaymi. Through the use of isozyme electrophoretic techniques we found variation for six out of nine systems analyzed, namely DIA, EST, IDH, MDH, PGI, and SKD. No variation was found in ADH, PGD, and PGM systems. We analyzed the distribution of variation within and between the reserves, and found most of the variation occurred within reserves (Gst = 0.1084). Only low levels of genetic differentiation were found between reserves (Nei’s genetic distance = 0.0088). The high levels of genetic variation within reserves that we found concur with results of previous studies on cassava grown by South American Amerindians and farmers. The role of the breeding system of cassava and the agricultural practices of Amerindians in the maintenance of high levels of genetic diversity are discussed.     

Virus content of leaves of cassava infected by African cassava mosaic virus

African cassava mosaic virus (ACMV) was detected in cassava leaves by ELISA. Some normal constituents of cassava leaves interfered with virus detection but leaf extracts of Nicotiana benthamiana did not. The symptom pattern was determined early in the growth of a leaf and subsequently changed little. ACMV was found only in the yellow or yellow green areas of the mosaic pattern. Virus content of the leaves increased with increasing symptom intensity, but decreased with leaf age and ACMV was not detected in mature leaves. Most whiteflies were found on young growing cassava leaves and the number decreased progressively with leaf age. This distribution will aid both the acquisition and inoculation of the virus.     

Customisation of AFLP analysis for cassava varietal identification.

Amplified fragment length polymorphism (AFLP) markers were used in the characterization of eight cassava varieties. This nonradioactive AFLP system was customized in terms of the choice of restriction enzymes used and the selection of nucleotides added to the 3' end of primers. EcoRI/MseI and HindIII/MseI fragments generally gave monomorphic profiles while ApaI/TaqI fragments produced polymorphic profiles suggesting a genome with high G + C content. It was possible to identify the eight cassava varieties used in this study using CTG as selective bases at the TaqI primer. For cassava, the AFLP system provided a higher number of loci detected per run when compared to RAPD. The reliability accompanying AFLP analysis would thus make it suitable for the characterization of cassava varieties.     

Production of raw cassava starch-degrading enzyme by Penicillium and its use in conversion of raw cassava flour to ethanol

A newly isolated strain Penicillium sp. GXU20 produced a raw starch-degrading enzyme which showed optimum activity towards raw cassava starch at pH 4.5 and 50 °C. Maximum raw cassava starch-degrading enzyme (RCSDE) activity of 20 U/ml was achieved when GXU20 was cultivated under optimized conditions using wheat bran (3.0% w/v) and soybean meal (2.5% w/v) as carbon and nitrogen sources at pH 5.0 and 28 °C. This represented about a sixfold increment as compared with the activity obtained under basal conditions. Starch hydrolysis degree of 95% of raw cassava flour (150 g/l) was achieved after 72 h of digestion by crude RCSDE (30 U/g flour). Ethanol yield reached 53.3 g/l with fermentation efficiency of 92% after 48 h of simultaneous saccharification and fermentation of raw cassava flour at 150 g/l using the RCSDE (30 U/g flour), carried out at pH 4.0 and 40 °C. This strain and its RCSDE have potential applications in processing of raw cassava starch to ethanol.     

Genetic modification of cassava for enhanced starch production

To date, transgenic approaches to biofortify subsistence crops have been rather limited. This is particularly true for the starchy root crop cassava ( Manihot esculenta Crantz). Cassava has one of the highest rates of CO₂ fixation and sucrose synthesis for any C3 plant, but rarely reaches its yield potentials in the field. It was our hypothesis that starch production in cassava tuberous roots could be increased substantially by increasing the sink strength for carbohydrate. To test this hypothesis, we generated transgenic plants with enhanced tuberous root ADP-glucose pyrophosphorylase (AGPase) activity. This was achieved by expressing a modified form of the bacterial glgC gene under the control of a Class I patatin promoter. AGPase catalyses the rate-limiting step in starch biosynthesis, and therefore the expression of a more active bacterial form of the enzyme was expected to lead to increased starch production. To facilitate maximal AGPase activity, we modified the Escherichia coli glgC gene (encoding AGPase) by site-directed mutagenesis (G336D) to reduce allosteric feedback regulation by fructose-1,6-bisphosphate. Transgenic plants (three) expressing the glgC gene had up to 70% higher AGPase activity than control plants when assayed under conditions optimal for plant and not bacterial AGPase activity. Plants having the highest AGPase activities had up to a 2.6-fold increase in total tuberous root biomass when grown under glasshouse conditions. In addition, plants with the highest tuberous root AGPase activity had significant increases in above-ground biomass, consistent with a possible reduction in feedback inhibition on photosynthetic carbon fixation. These results demonstrate that targeted modification of enzymes regulating source–sink relationships in crop plants having high carbohydrate source strengths is an effective strategy for increasing carbohydrate yields in sink tissues.     

Microsatellite markers confirm high apomixis level in cassava bred clones

Apomixis genes have successfully been transferred to cassava by hybridization with the wild species, Manihot glaziovii. The interspecific hybrid of cassava and M. glaziovii was left for open pollination during the subsequent three generations. Seven sibs and their maternal progenitor of the fourth generation were genotyped using five microsatellite loci previously developed for cassava. All sibs were identical with each other and with their maternal progenitor. Sibs from M. glaziovii itself proved to be identical when examined by the same microsatellite loci. These results lead to the conclusion that apomixis do occur in wild cassava relatives and apparently has played an important role in Manihot speciation.     

木薯发酵产丁醇的研究

对丙酮丁醇梭菌发酵木薯产溶剂进行研究,分别考察了N源、木薯含量、酶处理条件和培养基pH对发酵产丁醇的影响。结果表明：最佳的产丁醇发酵培养基为木薯粉120g／L,乙酸铵6g／L;木薯粉先用高温淀粉酶按酶量20U／g、90℃水解60min,再糊化30min;发酵初始pH为6．0,发酵96h。在此条件下,5L发酵罐中丁醇产量达到13．5g／L,总溶剂达到22．8g／L。