Photosynthesis and Leaf Structure in Domesticated Cassava (Euphorbiaceae) and a Close Wild Relative: Have Leaf Photosynthetic Parameters Evolved Under Domestication?

Broad patterns across wild plant species show that leaf composition and morphology vary predictably among habitats, richer habitats favoring resource‐acquisition strategies and poorer habitats favoring resource‐conservation strategies. Domestication is often accompanied by a shift to richer habitats, and might thus be expected to lead to a shift in leaf composition and morphology and hence in photosynthetic parameters. We compared leaf photosynthetic parameters in domesticated cassava (Manihot esculenta) and a close wild relative, using greenhouse‐grown plants. In domesticated cassava, CO₂ exchange rate expressed per unit mass and specific leaf area (SLA, m²/kg dry mass) were greater than in the wild relative, whereas leaf dry matter content (LDMC, dry mass/fresh mass) was lower in the domesticate. These results suggest that SLA and net photosynthetic rates may both have increased in the evolution of cassava under domestication, enabling more rapid growth in relatively resource‐rich and protected agricultural habitats. Previous comparisons of photosynthetic rates in domesticated plants and wild relatives have usually considered only leaf area‐based measures. Here, we discuss the interest of using mass‐based rates to study the evolution of ecological strategies under domestication.     

Carbon Partitioning and Assimilation as Affected by Nitrogen Deficiency in Cassava

Plants of cassava (Manihot esculenta Crantz) were raised in a sand root medium watered with nutrient solutions, under greenhouse conditions. As the N-supply increased, shoot dry mass was enhanced to a greater extent than root dry mass, thus leading to an increased shoot to root ratio. In leaves, contents of total soluble saccharides, non-reducing saccharides, and inorganic phosphate increased linearly with increasing N-supply. An opposite response was found for reducing saccharides and starch. In general, content of non-reducing saccharides was considerably greater than starch content. Activity of sucrose synthase was not detected, regardless of the N-treatments; by contrast, activity of neutral and acid invertases increased with increasing N-availability. Roots accumulated more total soluble saccharides, but less reducing saccharides and starch, as the N-supply increased. Photosynthetic rates decreased with increasing N-deficiency. Such a decrease was circumstantially associated to reducing saccharide, but not starch, accumulation. Results suggest a limited capacity for carbon export from source leaves under N-limitation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Chlorophyll meter and leaf colour chart to estimate chlorophyll content, leaf colour, and yield of cassava

A field experiment was conducted with two cassava cultivars and eight levels of nitrogen to examine the relationship between extractable chlorophyll (Chl) content of cassava leaves and both the Chl meter value (SPAD) and leaf colour chart (LCC) score. The SPAD, LCC, and Chl a+b content were influenced by leaf position, growth stage, cultivar (cv.), and N fertilization. The cvs. and N fertilization had significant effect on SPAD, LCC, and Chl a+b content of youngest fully expanded leaf (leaf 1) blade in most cases. An F-test indicated that common equations pooled across cvs., N fertilization, and growth stages could be used to describe the relationships between Chl a+b content and LCC and between SPAD and LCC, but not between SPAD and Chl a+b content. Relationships between tuber yield and SPAD, LCC, and Chl a+b content were significant (p<0.05) and positive at 30 and 60 d after planting. Thus LCC and SPAD can be used to estimate leaf Chl content which is an indicator of leaf N status.     

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.     

Attraction of the predatory mites Typhlodromalus manihoti and Typhlodromalus aripo to cassava plants infested by cassava green mite

The attraction of the predatory mites, Typhlodromalus manihoti and Typhlodromalus aripo, to the host plant-spider mite complex, Manihot esculenta – Mononychellus tanajoa, was investigated with a Y-tube olfactometer. Factors examined included predator starvation period, several combinations of cassava leaf biomass and initial M. tanajoa infestations, M. tanajoa-damaged leaves with mites and/or their residues removed, M. tanajoa alone, and mechanically damaged cassava leaves. We found that females of T. manihoti and T. aripo were significantly attracted to M. tanajoa-infested cassava leaves when the predators were starved for 2, 6, or 10 h. Satiated T. aripo was significantly attracted to infested cassava leaves whereas satiated T. manihoti did not discriminate between infested and non-infested leaves. When a choice was given between either two or four leaves infested with 200 female M. tanajoa and an equivalent number of non-infested leaves, 2 h-starved T. manihoti and T. aripo were significantly attracted to each of the infested groups of cassava leaves. At a density of 12 female M. tanajoa per leaf on four leaves, 2 h-starved T. manihoti was still attracted to M. tanajoa-infested leaves whereas 2 h-starved T. aripo was not attracted. When a choice was given between non-infested cassava leaves and either infested leaves from which only M. tanajoa females had been removed, or infested leaves from which all M. tanajoa and their visible products (web, feces) had been wiped off, T. aripo preferred odors from both types of previously infested leaves. Typhlodromalus manihoti was only attracted to infested leaves from which the M. tanajoa females only had been removed. Finally, the two predators were not attracted to 400 female M. tanajoa on clean cotton wool or to mechanically wounded leaves. This supports the hypothesis that M. tanajoa damage induces volatile cues in cassava leaves that attract T. manihoti and T. aripo to M. tanajoa-infested leaves.     

Isozyme Diversity in Cassava Cultivars (Manihot esculenta Crantz)

Isoenzyme electrophoresis was used as a method to determine genetic diversity in various M. esculenta cultivars collected in the Southwestern (SW) and Northwestern (NW) regions of the State of Parana, in the South region of Brazil, and in cultivars produced at the Agronomic Institute of Campinas (IAC), S~ao Paulo State, Southeastern region of Brazil. The cultivars have been maintained by vegetative propagation for 5 years and are useful in production programs. A total of 28 loci in the acid phosphatase (ACP; EC 3.1.3.2), esterases (EST; EC 3.1.1.1), malate dehydrogenase (MDH; EC 1.1.1.37), and shikimate dehydrogenase (SKDH; EC 1.1.1.15) isozymes was analyzed. The proportion of polymorphic loci for NW, SW, and IAC cultivars was 57.14, 50.0, and 53.6%, respectively. Genetic diversity calculated by Nei's genetic identity (I) showed high I values for the three M. esculenta subpopulations. The high degree of polymorphism expressed by cassava cultivars is highly relevant to stimulate breeding programs with M. esculenta species.     

Efficient Production of Transgenic Cassava Using Negative and Positive Selection

In order to improve the efficiency of cassava (Manihot esculenta Crantz) transformation, two different selection systems were assessed, a positive one based on the use of mannose as the selective agent, and a negative one based on hygromycin resistance encoded by an intron-containing hph gene. Transgenic plants selected on mannose or hygromycin were regenerated for the first time from embryogenic suspensions cocultivated with Agrobacterium. After the initial selection using mannose and hygromycin, 82.6% and 100% of the respective developing embryogenic callus lines were transgenic. A system allowing plant regeneration from only transgenic lines was designed by combining chemical selection with histochemical GUS assays. In total, 12 morphologically normal transgenic plant lines were produced, five using mannose and seven using hygromycin. The stable integration of the transgenes into the nuclear genome was verified using PCR and Southern analysis. RT-PCR and northern analyses confirmed the transgene expression in the regenerated plants. A rooting test on mannose containing medium was developed as an alternative to GUS assays in order to eliminate escapes from the positive selection system. Our results show that transgenic cassava plants can be obtained by using either antibiotic resistance genes that are not expressed in the micro-organisms or an antibiotic-free positive selection system.     

Living at the threshold: Where does the neotropical phytoseiid mite Typhlodromalus aripo survive the dry season?

The establishment of the neotropical predatory mite Typhlodromalus aripo in sub-Saharan Africa has resulted in broadly successful biological control of the cassava green mite Mononychellus tanajoa throughout the cassava belt of Africa. In some mid-altitude areas and drier lowland savannahs of sub-Saharan Africa, which are characterized by cool or hot long (≥5 months) dry seasons, the predator disappears from its habitat in the cassava apex during the dry season and reappears after the onset of rains. It is not known, however, where the predator remains during this time period. In this study, we conducted a field enclosure experiment of cassava plants with the objectives to determine if (a) T. aripo survives at very low densities in the apex, if (b) it survives in the soil or leaf litter below the cassava plant, and if (c) it recolonizes the cassava plant from the surrounding vegetation. Towards the end of the dry season, when the predators had disappeared from all cassava plants included in the experiment, five treatments were applied: (1) plants without enclosure; (2) plants with enclosure; (3) plants with enclosure, apices removed; (4) plants with enclosure, glue barrier around stem; and (5) plants kept free of T. aripo, without enclosure. Predator (re)appearance on cassava apices was monitored non-destructively at weekly intervals and was expressed as the proportion of plants with at least one apex with T. aripo per total number of plants of the treatment. The predators reappeared first on the plants of the treatments (1), (2), and (4). With a time lag of 7–8 weeks, the predators appeared also on the plants of the treatments (3) and (5). The time pattern of the predator’s (re)appearance in the cassava apex of the different treatments suggests that (a) T. aripo survives the dry season in very low densities in the cassava apex; this result is supported by an assessment of the efficiency of non-destructive visual in-field apex inspections which proved that about 10% of the cassava apices that had T. aripo were not recognized as such; (b) T. aripo does not survive in the soil or leaf litter, but we did document cases in a screenhouse experiment, where few individuals migrated down to the ground and walked over exposed soil until they reached the apex bouquet traps; additionally, microclimate measurements in various cassava plant strata proved that the cassava apex and the cassava stem base are the locations with the highest relative humidity during the dry season—which makes the stem base a potentially interesting refuge; (c) T. aripo does not survive in the surrounding vegetation, which is supported by a vegetation survey, where T. aripo was not found on any other plant species than cassava.     

Photosynthesis in Drought-Adapted Cassava

After 45 d of limited water supply, cassava (Manihot esculenta Crantz) exhibited pronounced reduction in shoot growth, high leaf fall, and decreased stomatal conductance. However, the water status of the remaining leaves was unaffected. This was combined with an amplified heliotropic response and drooping which minimises radiant energy interception at mid-day, suggesting that leaves are sensitive to high irradiance (I). In well-irrigated plants, CO₂-saturated oxygen evolution and net photosynthetic rate (P _N) in air were markedly higher (5-fold) in young (expanding) leaves than in mature leaves. Water limitation did not strongly modify CO₂-saturated oxygen evolution but it altered P _N in air for both types of leaves, although differently. The mature leaves of drought-adapted plants displayed residual rate of P _N and deteriorated photosystem 2 (PS2) photochemistry estimated from chlorophyll (Chl) a fluorescence measurements. In young leaves at moderate I, P _N was depressed by only 66 % in stressed plants. Moreover, the photochemical quenching of Chl a fluorescence and the quantum efficiency of PS2 photochemistry in young leaves were comparable in both control and stressed plants. In contrast at high I, P _N was almost null and marked decreases in the two fluorescence parameters were apparent. Hence the strong heliotropic response and drooping displayed by young leaves under water limitation is an important strategy for avoiding inactivation of P _N by high I and therefore for cassava tolerance to drought.     

Transgenic plants of cassava (Manihot esculenta) with resistance to Basta obtained by Agrobacterium-mediated transformation

 Transgenic plants of cassava (Manihot esculenta) resistant to the herbicide Basta were obtained through Agrobacterium-mediated transformation. The plants also expressed the uidA gene and two were positive for PCR- and/or Southern-based detection of the nptII gene. Somatic-embryo-derived cotyledons were used as source of explants. A non-disarmed Agrobacterium strain (CIAT 1182) was used to transfer the genes of interest into cassava cultivar MPer183. Greenhouse tests of resistance to Basta (Hoechst) showed three plant lines with different levels of tolerance to the herbicide. Based on Southern tests of transgenesis, the transformation efficiency was 1%. The results constitute the first report of the bar gene conferring herbicide resistance to cassava plants. Received: 9 January 1999 / Revision received: 10 May 1999 / Accepted: 15 June 1999