Improvement of growth and gymnemic acid production by altering the macro elements concentration and nitrogen source supply in cell suspension cultures of Gymnema sylvestre R. Br

Gymnema sylvestre is an important medicinal plant which bears bioactive compound namely gymnemic acids. The present work deals with optimization of cell suspension culture system of G. sylvestre for the production of biomass and gymnemic acid and we investigated effects of macro elements (NH₄NO₃, KNO₃, CaCl₂, MgSO₄ and KH₂PO₄ - 0.0, 0.5, 1.0, 1.5 and 2.0× strength) and nitrogen source [NH₄⁺/NO₃⁻ ratio of: 0.00/18.80, 7.19/18.80, 14.38/18.80, 21.57/18.80, 28.75/18.80, 14.38/0.00, 14.38/9.40, 14.38/18.80, 14.38/28.20 and 14.38/37.60 (mM)] of Murashige and Skoog medium on accumulation of biomass and gymnemic acid content. The highest accumulation of biomass (165.00 g l⁻¹ FW and 15.42 g l⁻¹ DW) was recorded in the medium with 0.5× concentration of NH₄NO₃ and the highest production of gymnemic acid content was recorded in the medium with 2.0× KH₂PO₄ (11.32 mg g⁻¹ DW). The NH₄⁺/NO₃⁻ ratio also influenced cell growth and gymnemic acid production; both parameters were greater when the NO₃⁻ concentration was higher than that of NH₄⁺. Maximum biomass growth (159.72 g l⁻¹ of FW and 14.95 g l⁻¹ of DW) was achieved at an NH₄⁺/NO₃⁻ ratio of 7.19/18.80, and gymnemic acid production was also greatest at the same concentration of NH₄⁺/NO₃⁻ ratio (11.35 mg g⁻¹ DW).     

Chitosan enhances withanolides production in adventitious root cultures of Withania somnifera (L.) Dunal

Calli were obtained from leaf, cotyledon and internode explants of in vitro-grown plants of Indian cultivar of Withania somnifera in MS medium supplemented with 2, 4-D (2.0 mg l⁻¹) and Kinetin (0.2 mg l⁻¹). The brown, semi-friable callus (500 mg FW) derived from leaf explants produced higher number of primary adventitious roots (9 roots/callus) in half strength MS medium fortified with IBA (0.5 mg l⁻¹) and NAA (0.1 mg l⁻¹). The primary adventitious roots with an inoculum mass of 15 g FW were cultured for 6 weeks in the same medium for secondary adventitious root proliferation. Elicitation of abiotic elicitor, aluminium chloride at 10 mg l⁻¹ at the end of 4 weeks culture with 4 h exposure time enhanced withanolides productivity. Under similar culture conditions, the biotic elicitor, chitosan at 100 mg l⁻¹ stimulated higher production of all withanolides when compared to aluminium chloride treatment. This is the first report on the use of callus-derived adventitious root culture for the enhanced production of withanolides upon chitosan elicitation.     

Adventitious root cultures of Castilleja tenuiflora Benth. as a source of phenylethanoid glycosides

Castilleja tenuiflora is a highly valued medicinal plant that grows in pine-oak woods in Mexico. In this study, we identified for the first time verbascoside and isoverbascoside as the major phenylethanoid glycosides (PhGs) in C. tenuiflora. These compounds have proven biological activities, including anti-inflammatory, antioxidant, and cytotoxic activities, which may be related to the traditional uses of this plant. We developed a reverse-phase high-performance liquid chromatography (RP-HPLC) procedure to analyze PhGs, and determined their concentrations in various different tissues of wild plants. Verbascoside accumulated mainly in roots and inflorescences (9.23 and 7.88 mg g⁻¹ dry biomass, respectively), while isoverbascoside accumulated mainly in the roots (7.13 mg g⁻¹ dry biomass). To provide an alternative source of material for production of bioactive compounds, we established in vitro adventitious root cultures in which roots were grown in B5 medium containing either 10 μM indole 3-acetic acid (IAA) or 10 μM α-naphthaleneacetic acid (NAA). The greatest dry biomass yield (30 g L⁻¹) was achieved at 30 days after transfer of roots into IAA-containing medium. The highest specific yields of PhGs were also obtained using this auxin; the maximum level of verbascoside was 14.62 mg g⁻¹ dry root biomass (438.6 mg L⁻¹) at 30 days after root transfer, and the maximum yield of isoverbascoside was 37.32 mg g⁻¹ dry root biomass (522.48 mg L⁻¹) at 23 days after root transfer. Adventitious root cultures of C. tenuiflora are a promising system for further studies on scale-up and phenylethanoid glycosides biosynthesis.     

Role of exogenous phytohormones on growth and plumbagin accumulation in Plumbago indica hairy roots and conservation of elite root clones via synthetic seeds

The present study describes the role of different exogenous hormones on morphology and plumbagin production in Plumbago indica hairy roots. It was also aimed to conserve elite root clones via synthetic seed technology. Insertion of rolB gene in transformed roots was confirmed by polymerase chain reaction followed by southern blot analysis. Hairy roots were treated with single or in combination of different phytohormones viz. IAA, IBA, 2, 4-D, NAA, BAP, GA₃ and ABA. Cultures incubated with GA₃ (0.5 mg l⁻¹) yielded highest root growth due to formation of profuse lateral branching while NAA (0.5 mg l⁻¹) treatment caused highest plumbagin accumulation. Cultures incubated with 2, 4-D exhibited highest inhibitory effect in terms of both root growth and plumbagin production. All phytohormones were found to be effective at lower concentration. In combinatorial study, GA₃ + NAA (0.5 mg l⁻¹, each) was found optimum for root biomass and plumbagin production at earlier stage of culture. Different combinations of auxins and BAP induced different morphologies ranging from reduction of lateral branching to rapid disorganization of root matrix. The combinations of ABA and selected auxins were not found promising at any of selected concentration. Based on the effect of exogenous hormones on hairy root culture, elite root clones were selected and encapsulated with sodium alginate matrix. Uniform shaped alginate coated synthetic seeds were conserved up to 6 months exhibited high regeneration potential without disturbing plumbagin content.     

Antioxidant activity and phenolic composition of the medicinal and edible halophyte Mesembryanthemum edule L.

Mesembryanthemum edule L. (sourfig, Aizoaceae) has long been used as food and in traditional medicine. This study was intended to characterize the antioxidant properties and the phenolic compounds of M. edule leaf, stem and root. The approach consisted to evaluate these organs for their antioxidant activities through several in vitro tests, to determine tissue contents in total phenolics, flavonoids and proanthocyanidins and to establish the phenolic composition through RP-HPLC analysis. All studied organs showed a high antioxidant activity as compared to positive control BHT, with maximal efficiency for stems followed by leaves and roots. The highest polyphenolic levels were found in stems and leaves (86.5 and 68.7 mg GAE g⁻¹ DW, respectively), suggesting that their strong antioxidant activity could be attributed to these phytochemicals. The HPLC analysis revealed that the main phenolic compounds were quercitrin and avicularin (1.4 and 1.15 mg g⁻¹ DW, respectively) in the leaves, while catechin and procyanidin B2 (1.66 and 1.54 mg g⁻¹ DW, respectively) were the most abundant phenolics in the stems. Overall, the strong antioxidant activity and richness of M. edule aerial tissues suggest that it could be advantageously used as a functional or nutraceutical food, to prevent or moderate oxidative stress-related diseases.     

Regulation of tissue differentiation by plant growth regulators on tTCLs of Panax ginseng adventitious roots

Differentiated tissue in Panax ginseng cultures was found to be very efficacious for saponin production. In order to increase the yield of saponins and preserve culture stability we were testing different plant growth regulators (PGR) and auxin/cytokinin combinations to regulate a level of tissue differentiation. For this purpose we used transverse thin cell layers (tTCLs) of adventitious roots of Panax ginseng. Adventitious roots were cultivated in Shenk and Hildebrand (SH) liquid medium supplemented with IBA (24.6 μM). Callus formation and root multiplication of adventitious root tTCLs was evaluated after 4 and following 12 weeks of cultivation, respectively, on SH basal medium containing various auxins (3 mg l⁻¹) or cytokinins (0.2 or 0.02 mg l⁻¹) or their combinations. We found that kinetin (Kin) in combination with auxin benzo[b]selenienyl acetic acid (BSAA), naphthalene acetic acid or indole-3-butric acidis the best for biomass production and following root multiplication. These combinations were tested in previously selected most suitable large-scale system—a temporary immersion system RITA. The best saponin production (15.94 ± 1.89 mg g⁻¹ dry weight) and growth value (5.62 ± 0.34) was reached on medium containing BSAA and Kin combination.     

In vitro plant regeneration from decapitated embryonic axes of Clitoria ternatea L.—An important medicinal plant

In vitro clonal propagation of Clitoria ternatea has been achieved by employing decapitated embryonic axes (DEAs) explants. The explants induced multiple shoots on cytokinin-containing medium. Several cytokinins [6-benzylaminopurine (BAP), 6-furfuryl aminopurine (KIN) and thidiazuron (TDZ)] were assayed. The best response was achieved with 2 mg l⁻¹ BAP in which 100% of cultures produced 6.0 ± 0.14 shoots per explant. MS + 1 mg l⁻¹ gibberellic acid (GA₃) was the most suitable for shoot elongation. Regenerated shoots were rooted in half-strength Murashige and Skoog (MS) medium with 0.2 mg l⁻¹ indole-3-butyric acid (IBA). Plantlets were successfully acclimatized and established in soil, and they were morphologically indistinguishable from the source plant. The plantlets attained maturity and flowered normally. The efficient regeneration protocol reported here provides an important method of micropropagation of this plant. Furthermore, this protocol may be used for genetic transformation of this valuable medicinal plant for its further improvement.     

Growth and accumulation of caffeic acid derivatives in Echinacea angustifolia DC. var. angustifolia grown in hydroponic culture

In separate experiments conducted in 2007 and 2008, growth and accumulation of selected caffeic acid derivatives (CADs; i.e., caftaric acid, chlorogenic acid, echinacoside, caffeic acid, cynarin, p-coumaric acid, ferulic acid and cichoric acid) were determined in Echinacea angustifolia DC. var. angustifolia seedlings grown in hydroponic culture (floating raft system) at a density of 122 plant m⁻² (at planting). Plants were harvested 11 (2007) or 16 (2008) weeks after transplanting (i.e., 15 or 20 weeks after sowing). In both years, plants grew vigorously and at harvest approximately half of the plants under observation had developed one to three inflorescences. In 2008, the root yield (2940 kg ha⁻¹) harvested in nearly eight months from two consecutive hydroponic cultures was within the yield reported in the literature for field cultivations lasting two to four years. None of the selected CADs was found in the leaves, while the inflorescences (stem and capitulum) contained only caftaric acid and echinacoside at concentrations higher than the detection limits (0.05 mg g⁻¹ dry weight). Echinacoside, cynarin and chlorogenic acid were found in root tissues at concentrations ranging from 0.36 to 5.25 mg g⁻¹ dry weight. In all plant samples, echinacoside, which is the marker compound for E. angustifolia material, did not reach the minimum quality standard (10 mg g⁻¹ dry weight) for the production of standardized extract. We concluded that short-cycle, high-density greenhouse hydroponic culture stimulates plant growth and root production in E. angustifolia, but it does not ensure sufficient CADs accumulation in dried roots.     

Highly efficient in vitro regeneration of the industrial oilseed crop Crambe abyssinica

A highly efficient regeneration protocol for oilseed crop Crambe abyssinica has been developed using hypocotyls as explants in this study. Crambe is a potential engineering oilseed crop for industrial purposes as it contains 55-60% erucic acid in its oil and, more importantly, it does not outcross with any food oil seed crops. However, the low regeneration frequency with the currently available protocols is still a limiting factor for genetic modification of Crambe. In this study, we investigated the effects of N-source, C-source, AgNO₃, cultural conditions as well as the concentration and combination of plant growth regulators (PGR) on the regeneration frequency of C. abyssinica. The results showed that all these factors, especially the N-source and PGR concentrations and combinations, played an important role in shoot regeneration. Among all the factors tested, the combination of using hypocotyls from C. abyssinica cv. galactica, the Lepiovre basal medium supplemented with 16 g l⁻¹ glucose, 0.5 g l⁻¹ AgNO₃, 2.2 mg l⁻¹ thidiazuron (TDZ), 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), 2.5 g l⁻¹ Gelrite, seeds germinated in dark for 3 days and explants cultured in light, gave the best regeneration frequency (over 95%). The results also suggest that reducing the content of NH₄⁺ or keeping a suitable NO₃⁻/NH₄⁺ ratio in the regeneration medium would be crucial to Crambe shoot regeneration.     

Foliar application of chitosan activates artemisinin biosynthesis in Artemisia annua L.

There has been much interest in artemisinin owing to its excellent activity against malaria, an infectious disease threatening the tropical world. However, the low artemisinin content (0.01-0.8%, DW) in Artemisia annua, which is the only commercial source of artemisinin, makes artemisinin expensive to produce and not yet available on a global scale. Here we show that foliar application of 100 mg l⁻¹ chitosan improved artemisinin biosynthesis in A. annua. The content of dihydroartemisinic acid and artemisinin in chitosan-treated leaves increased by 72% and 53% compared with control values, respectively. Chitosan induced the expression of ADS and DBR2, which could explain the increase in level of artemisinic metabolites. After chitosan treatment, the amounts of hydrogen peroxide (H₂O₂) and superoxide anion (O₂⁻) in leaves of A. annua were 1.4 and 3.0 times higher than those of the control, respectively. Accumulation of reactive oxygen species (ROS) probably accelerated the conversion of dihydroartemisinic acid to artemisinin. Foliar application of 100 mg l⁻¹ chitosan had no harmful effect on A. annua growth. The simple method described here could be an effective method to improve artemisinin production in A. annua field cultivation.     

Regeneration in Jatropha curcas: Factors affecting the efficiency of in vitro regeneration

Factors influencing in vitro regeneration through direct shoot bud induction from hypocotyl explants of Jatropha curcas were studied in the present investigation. Regeneration in J. curcas was found to be genotype dependent and out of four toxic and one non-toxic genotype studied, non-toxic was least responsive. The best results irrespective of genotype were obtained on the medium containing 0.5 mg L⁻¹ TDZ (Thidiazuron) and in vitro hypocotyl explants were observed to have higher regeneration efficiency as compared to ex vitro explant in both toxic and non-toxic genotypes. Adventitious shoot buds could be induced from the distal end of explants in all the genotypes. The number of shoot buds formed and not the number of explants responding to TDZ treatment were significantly affected by the position of the explant on the seedling axis. Explants from younger seedlings (≤15 days) were still juvenile and formed callus easily, whereas the regeneration response declined with increase in age of seedlings after 30 days. Transient reduction of Ca²⁺ concentrations to 0.22 g L⁻¹ in the germination medium increased the number of responding explants.Induced shoot buds, upon transfer to MS medium containing 2 mg L⁻¹ Kn (Kinetin) and 1 mg L⁻¹ BAP (6-benzylamino purine) elongated. These elongated shoots were further proliferated on MS medium supplemented with 1.5 mg L⁻¹ IAA (indole-3-acetic acid) and 0.5 mg L⁻¹ BAP and 3.01-3.91 cm elongation was achieved after 6 weeks. No genotype specific variance in shoot elongation was observed among the toxic genotypes except the CSMCRI-JC2, which showed reduced response. And for proliferation among the toxic genotypes, CSMCRI-JC4 showed highest number of shoots formed. Among the rest, no significant differences were observed. The elongated shoot could be rooted by pulse treatment on half-strength MS medium supplemented with 2% sucrose, 3 mg L⁻¹ IBA (indole-3-butyric acid), 1 mg L⁻¹ IAA, 1 mg L⁻¹ NAA (α-naphthalene acetic acid) and subsequent transfer on 0.25 mg L⁻¹ activated charcoal medium. The rooted plants could be established in soil with more than 90% success. No significant differences were observed in rooting of shoots in the different toxic genotypes. However, rooting response was reduced in non-toxic genotype as compared to toxic genotypes.     

Tocochromanol content and composition in Jatropha curcas seeds

Physic nut (Jatropha curcas L.) is a promising seed oil source for biodiesel production. Natural antioxidants play a major role in maintaining oxidative stability of oils and they also have important food and industrial applications. Among them, tocochromanols are the most abundant in seeds. The objective of this research was to evaluate the variation for tocochromanol content and profile in a germplasm collection of 52 accessions of J. curcas. Seeds collected in two different periods, August and November of 2009, were analysed for tocochromanol content. Additionally, the dynamics of tocochromanol accumulation in developing seeds was studied. Total seed tocochromanol content averaged 307.2 mg kg⁻¹ in August and 303.7 mg kg⁻¹ in November, whereas total oil tocochromanol content averaged 507.4 mg kg⁻¹ in August and 500.8 mg kg⁻¹ in November. The tocochromanol fraction was made up of 15.4% gamma-tocopherol, 83.8% gamma-tocotrienol, and 0.8% delta-tocotrienol in August and 18.0% gamma-tocopherol, 80.4% gamma-tocotrienol, and 1.6% delta-tocotrienol in November. Genotype × environment effects were identified for tocochromanol content but not for the proportion of major tocochromanol homologues, which showed a high positive correlation between both environments. Developing seeds contained primarily alpha-tocopherol and gamma-tocopherol at early stages of development, with gamma-tocotrienol and delta-tocotrienol being practically undetectable. Gamma-tocotrienol content remained practically undetectable till 66 DAP and then increased pronouncedly to final levels of 177.1 mg kg⁻¹ (74.8% of the total tocochromanol content). The powerful antioxidant and health-promoting properties of gamma-tocotrienol encourages further studies on selection for the tocopherol/tocotrienol ratio in Jatropha and on the potential of tocochromanols as high added-value products derived from Jatropha seed oil production.     

Increases in leaf artemisinin concentration in Artemisia annua in response to the application of phosphorus and boron

Malaria resurgence particularly in the third world is considerable and exacerbated by the development of multi-drug resistances to chemicals such as chloroquinone. Drug therapies, as recommended by WHO include the use of antimalarial compounds derived from Artemisia annua L., i.e. artemisinin-based therapies. This work aims to determine how A. annua plant dry matter can be enhanced while maximising artemisinin concentration from understanding the plant's mineral requirements for P and B. Experiments with differing of P, from 5 to 120 mg L⁻¹ and B from 0.1 to 0.9 mg L⁻¹ were undertaken. Mineral nutrients were supplied in irrigation water to potted plants and after a period of growth, dry matter production and leaf artemisinin concentration were determined. Increases in P application enhanced plant growth and total dry matter production. An optimal application rate, with respect to dry matter, was apparent around 30 mg P L⁻¹. Despite increases in P application having no influence on leaf artemisinin concentration, optimal yields of artemisinin, on a per plant basis, were again achieved at supply rate around 30-60 mg L⁻¹. Increasing B application rate had little influence on dry matter production despite increases in B leaf tissue concentration promoting the total amount of B per plant. Leaf artemisinin concentration significantly increased with B application rate up to 0.6 mg B L⁻¹. The higher artemisinin concentrations when multiplied by total leaf dry matter at the higher B application rates produced an increase in total artemisinin production per plant. There was however no further significant effect on leaf artemisinin concentration when B supply concentrations increased further (0.9 mg L⁻¹). Artemisinin production varied between the two experiments to a greater extent than plant dry matter production and the reasons for this are discussed in relation to growing environments and their possible impacts on artemisinin biosynthesis.     

Evaluation of CIMMYT conventional and synthetic spring wheat germplasm in rainfed sub-tropical environments. II. Grain yield components and physiological traits

CIMMYT hexaploid spring wheat (Triticum aestivum L.) germplasm has played a global role in assisting wheat improvement. This study evaluated four classes of CIMMYT germplasm (encompassing a total of 273 lines), along with 15 Australian cultivars (Oz lines) for grain yield, yield components and physiological traits in up to 27 environments in Australia's north-eastern region, where terminal drought frequently reduces grain yield and grain size.Broadly-adapted CIMMYT germplasm selected for grain yield had greater yield potential and improved performance under drought stress, being up to 5% greater yielding in High-yielding (mean yield 429 g m⁻²) and 4-10% greater yielding than adapted Oz lines in Low-yielding environments (mean yield 185 g m⁻²). Whilst maintaining statistically similar harvest index and spikes m⁻² compared to broadly-adapted Oz lines across all environments, sets of selected CIMMYT lines had greater canopy temperature depression (0.18-0.27 °C), dry weight stem⁻¹ (0.20-0.37 g), increased grains spike⁻¹ (0.8-3.4 grains), grain number m⁻² (ca. 20-800 grains), and maturity biomass (56-83 g m⁻²). Compared to selected Oz lines, broadly-adapted CIMMYT lines had a smaller reduction in Low compared to High-yielding environments for these traits, especially dry weight stem⁻¹, such that CIMMYT lines had ca. 25% and 10% greater dry weight stem⁻¹ than the Oz lines in Low- and High-yielding environment groups, respectively. Broadly-adapted CIMMYT germplasm also had slightly higher stem water soluble carbohydrate concentration at anthesis (ca. 6 mg g⁻¹), which contributed to their higher grain weight (ca. 0.5 mg grain⁻¹), and maintained an agronomically appropriate time to anthesis and plant height. Thus current CIMMYT germplasm should be useful donor sources of traits to enrich breeding programs targeting variable production environments where there is a high probability of water deficit during grain filling. However, as multiple traits were important, efficient introgression of these traits in breeding programs will be complex.     

Evaluation of bioethanol production from five different varieties of sweet and forage sorghums (Sorghum bicolor (L) Moench)

Cultivars of sweet (Rio, M81E and Della) and forage sorghums (Tato and Thor) were planted in Northeast Mexico in order to estimate optimum harvesting time, sugar production, biomass composition and ethanol yields. The juices were characterized in terms of sugar composition, free amino nitrogen (FAN) and phenolics and then yeast (Saccharomyces cerevisiae)-fermented into ethanol. The cultivars yielded different volumes of sweet juice and total sugars. They also had different optimum harvesting times. Glucose was the most abundant sugar in raw juices, followed by fructose and sucrose. FAN concentration ranged from 19 to 36 mg L⁻¹ therefore, nitrogen supplementation was required for adequate fermentation. After 18 h fermentation, there were no differences in efficiencies among cultivars but the sweet sorghums yielded more ethanol Ha⁻¹ compared to the two forage sorghums (approximately 1000 L Ha⁻¹ versus 770 L Ha⁻¹). Della was the cultivar with the highest productivity with 1051 L Ha⁻¹ ethanol produced after the first cut.     

Effect of boron on the development of brown rot (Monilinia laxa) on peaches

Requirements of consumers for products with low residues of pesticides have increased the need for alternative disease management practices. The concentration of boron in fruit affects its quality, shelf life and the development of physiological disorders. However, the effect of boron on the susceptibility of peach to fruit rots has not been reported. This study investigated the effect of boron (Power B and Borax) on the development of Monilinia laxa on peaches (cv Andross). Mycelial growth of M. laxa was inhibited on potato dextrose agar supplemented with 750 μg ml⁻¹ of Borax or 1000 μg ml⁻¹ of Power B. The EC 50 values were 107.9 and 522.4 for Borax and Power B respectively. Field investigations showed that the incidence of peach infections by M. laxa was negatively correlated with the content of Boron in the leaves. Post-harvest dipping of peaches in Power B or Borax solution, at concentrations recommended by manufacturer (2 μg ml⁻¹ for Power B and 1 mg ml⁻¹ for Borax), significantly reduced the development of M. laxa. Power B, at rates of 6 μg ml⁻¹, and Borax at rates of 3 mg ml⁻¹ were the most effective in reducing infections by M. laxa. Finally, post-harvest dipping of fruit in Power B or Borax reduced losses of fruit weight and improved fruit firmness one month after storage, showing that boron increased the maintainability of peaches in cold storage. Peaches treated with 6 μg ml⁻¹ Power B or 3 mg ml⁻¹ Borax had the highest flesh firmness and the lowest water losses, while untreated control peaches were the least firm. Generally, Borax was significantly less effective than Power B, but better than the control treatment.     

Interactions of sunflower (Helianthus annuus) and sunflower broomrape (Orobanche cumana) as affected by sowing date,resource supply and infestation level

The holoparasitic weed Orobanche cumana (sunflower broomrape) constrains sunflower (Helianthus annuus) production in many countries. The development of efficient control strategies requires an understanding of the processes underlying the complex environment–host–parasite interrelations. Growth and development of O. cumana and sunflower were quantified under field conditions in southeastern Romania. Sunflower hybrid Florom 350 was sown at two dates, in plots infested with 0, 50, 200 and 1600 viable O. cumana seeds kg⁻¹ dry soil, under low-input (rainfed, low nitrogen supply) and high-input (irrigated, high nitrogen supply) conditions. Sunflower shoot biomass reached peak values of 760–1287 g m⁻² between the end of anthesis and physiological maturity. Seed yield varied from 221 to 446 g m⁻². Sunflower biomass and yield were affected by all experimental factors. Seed yield responded positively to delaying sowing from early April to late May as well as to irrigation and fertilisation, and negatively to O. cumana infestation. Yield reductions, which were a product of reduced seed number and size, amounted to 13%, 25% and 37% at parasite seed densities of 50, 200 and 1600 viable seeds kg⁻¹ soil, respectively. Maximum O. cumana attachment numbers, recorded in late-sown high-input crops in 2004, ranged from 11 m⁻² in plots with 50 parasite seeds kg⁻¹ soil to 188 m⁻² with 1600 seeds kg⁻¹ soil. Parasite attachment number was a function of crop sowing date, water and nutrient supply, seedbank density, and sunflower biomass and root length density, via mechanisms of parasite seed stimulation, host carrying capacity and intraspecific competition. Delayed sowing and improved water and nitrogen supply were associated with increases in parasite number that neutralised yield-boosting effects of irrigation and fertilisation at the highest infestation level. Sunflower shoot biomass was significantly reduced by O. cumana infection, with reductions affecting organs in the order head > stem > leaves. Most of the discrepancy between infected and non-infected plants was accounted for by O. cumana biomass. Parasites mainly acted as an extra sink for assimilates during sunflower generative growth and impaired host photosynthesis to a much lesser degree. Results suggest that similar mechanisms govern infection level and host–parasite biomass partitioning across different Orobanche–host systems.     

Postharvest harpin or Bacillus thuringiensis treatments suppress citrus black spot in ‘Valencia’ oranges

Citrus black spot (CBS) caused by Guignardia citricarpa represents an important threat to citriculture in Brazil. Limited information is available regarding potential biological control agents and new alternative compounds that may provide protection of orange fruits against the disease. In this study, the effects of commercial products based on Bacillus thuringiensis var. kurstaki (Bt) bacterium, Bt pure isolates and Harpin protein (Messenger^®) on the postharvest control of CBS, were evaluated in ‘Valencia’ sweet orange fruits harvested for three consecutive years in a citrus grove. The fruits were sprayed with the following products: DiPel^® WP (Bt, subspecies, kurstaki strain HD-1, 16,000 International Units mg⁻¹, 32 g active ingredient kg⁻¹) (1, 20 and 50 mg ml⁻¹), Dimy Pel^® WP (Bt, subspecies, kurstaki, strain HD-1, 17,600 IU mg⁻¹, 26 g active ingredient l⁻¹) (2, 20 and 50 mg ml⁻¹), Messenger^® (3% harpin protein) (1 and 2 mg ml⁻¹) and fungicide Tecto^® Flowable SC (thiabendazole, 485 g l⁻¹) (0.8 g active ingredient l⁻¹), besides the Bt isolates, Bt- HD-567, Bt- DiPel and Bt- Dimy (9 × 10⁸ CFU ml⁻¹). Ten days after treatment, the number of newly developed CBS lesions and pycnidia produced were evaluated using fifty fruits per treatment. The Dimy Pel^® and Messenger^® reduced the number of new developed CBS lesions on fruits in up to 67% and 62%, respectively. All applied treatments drastically decreased the number of pycnidia produced in the CBS lesions on orange fruits with 85% to 96% reductions compared to the untreated control. Volatile compounds produced by the isolates Bt- HD-567, Bt- Dimy and Bt- DiPel, reduced the number of lesions on treated fruits by 70%, 65% and 71% compared to the control, respectively. In addition, the survival of Bt isolates on orange fruit surfaces were evaluated by recovering and quantifying the number of CFU every seven days for up to 28 days. The declines in survival rates on orange fruit surfaces were drastic for the three strains of Bt in the first week. The CFU numbers of all applied isolates declined by 4 to 5 orders of magnitude after storage at room temperature for 28 days. In vitro assays revealed that the Bt isolates significantly reduced the mycelial growth of the pathogen, ranging from 32% to 51%, compared to the control, whereas no inhibitory effect was observed in the presence of Messenger^®.     

Kernel number determination differs among maize hybrids in response to nitrogen

In maize, the effects of nitrogen (N) deficiencies on the determination of kernel number per plant (KNP) have been described only by changes in plant growth rate during the critical period for kernel set (PGRcp). We hypothesize that N availability affects KNP also through variations in biomass allocation to the ear, which determines a stable N concentration in this organ. Six maize hybrids of different breeding origin were evaluated in field experiments at two N levels (0 and 400 kg N ha⁻¹ applied). Traits included were KNP and per apical ear (KN_E1), and the allometric estimation of PGRcp, ear growth rate during the critical period (EGRcp), and N content and N concentration in different plant organs. We demonstrated that (i) N availability promoted differences among genotypes (G) in the response of EGRcp and KNP to PGRcp, (ii) variations in KN_E1 were explained by EGRcp (r² = 0.64) and by ear N content at silking + 12 d (r² = 0.64), and (iii) ear N concentration was a highly conservative trait (range between 10.47 and 15.98 mg N g biomass⁻¹) as compared to N concentration in vegetative tissues (range between 4.94 and 18.04 mg N g biomass⁻¹). Three response patterns were detected among hybrids, one for which the relationship between EGRcp and PGRcp did not vary between N levels and experiments, a second one for which N availability affected this relationship, and a third one for which the response was affected by the year (Y) effect. These results, together with the high correlation between EGRcp and ear N content (r² = 0.88), evidenced the importance of both photo-assimilate and N availability on EGRcp and KNP determination. Values of 1.5–2.3 g ear⁻¹ d⁻¹ during the critical period and 0.49–0.70 g of N ear⁻¹ at silking + 12 d were determined as thresholds for maximizing KN_E1, and both could be easily estimated by means of allometric models.     

Processing of Lespedeza stalks by pretreatment with low severity steam and post-treatment with alkaline peroxide

The steam pre-treatment with low severity preserves valuable biomass components, and further delignification with alkaline peroxide could improve hydrolysis. A combination of low severity steam pretreatment and alkaline peroxide post-treatment of Lespedeza stalks was investigated. The post-treatment of steam-pretreated Lespedeza stalks with alkaline peroxide significantly increased the cellulose content and changed the structure of the cellulose-rich fractions. A glucose yield of 503.5 mg g⁻¹ raw material from enzyme hydrolysis was obtained when the steam-pretreated material (184 °C for 4 min) was post-treated with 2% hydrogen peroxide at 60 °C for 24 h with a substrate concentration of 3.3%. Its hydrolysis yield is 88.8%, which is higher than that of samples processed by steam pretreatment alone (63.7%). The samples obtained by post-treatment with alkaline peroxide were found to have a smoother surface and looser structure in scanning electron microscopy images. The isolated lignin preparations had a yield range from 10.9 to 14.7 (% dry matter). The lignin was characterized by thermogravimetric analysis/differential thermal analysis, Fourier transform infrared spectroscopy, and gel permeation chromatography. Alkaline peroxide treatment increased the thermal stability of lignin, and decreased the amounts of all functional groups. Depolymerization and repolymerization occurred during the alkaline peroxide treatment.