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.     

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.     

Synthesis of withanolide A depends on carbon source and medium pH in hairy root cultures of Withania somnifera

Withania somnifera (L.) Dunal. (Indian ginseng) is an important medicinal plant which yields pharmaceutically active compounds called withanolides. The present work deals with optimization of parameters of hairy root culture of W. somnifera for the production of biomass and withanolide A. We also investigated the effects of carbon source [sucrose, glucose, fructose, maltose, glucose + fructose (1:1), fructose + sucrose (1:1) and sucrose + glucose (1:1)], sucrose concentration (1%, 2%, 3%, 4%, 6% and 8%) and the initial medium pH (4.0, 4.5, 5.0, 5.5, 5.8, 6.0 and 6.5) on growth and production of withanolide A in hairy root cultures of W. somnifera. We found that biomass accumulation and production of withanolide A was highest when sucrose was used as the carbon source (11.92 g l⁻¹ DW and 11.96 mg g⁻¹ DW of withanolide A). Further 3% sucrose concentration was found to be optimal for biomass accumulation (11.92 g l⁻¹ DW) and 4% sucrose favoured the production of withanolide A (13.28 mg g⁻¹ DW) in the tested range of concentrations (1-8%). The biomass of hairy roots was optimal when the initial medium pH was 5.8 (12.1 g l⁻¹ DW) and the withanolide A production was highest in the medium pH set at 6.0 (13.84 mg g⁻¹ DW).     

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.     

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.     

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.     

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.     

Screening and quantification of an antiseptic alkylamide, spilanthol from in vitro cell and tissue cultures of Spilanthes acmella Murr.

The study revealed, for the first time, accumulation of spilanthol, an antiseptic alkylamide, in in vitro cultures of Spilanthes acmella Murr., a medicinal plant of immense commercial value. To achieve this, in vitro shoots were regenerated via direct organogenesis from leaf-disc explants of Spilanthes. Shoots were induced in the presence of N⁶-benzylaminopurine (BAP) alone or in combination with either α-naphthalene acetic acid (NAA) or Indole-3-acetic acid (IAA) in Murashige and Skoog medium. The best treatment for shoot regeneration was MS + BAP (5.0 μM) + IAA (5.0 μM), which promoted adventitious shoot proliferation in >82% cultures with an average of 5.3 shoots per explant. Regenerated shoots rooted spontaneously with a frequency of 100% on half strength MS medium (major salts reduced to half strength) containing 50 g l⁻¹ sucrose. The plantlets were acclimatized successfully with 90% survival rate. Additionally, ploidy stability of the regenerated plants was assessed by flow cytometry which showed that all investigated plants had the similar ploidy as that of the mother plant. For spilanthol identification, peaks eluted from HPLC were analyzed by mass spectrometry with its characteristic fragmentation pattern. For quantification studies, calibration curve was generated, which revealed a higher amount of spilanthol content (3294.36 ± 12.4 μg/g DW) in the leaves of in vitro plants compare to those of in vivo plants (2703.66 ± 9.6 μg/g DW of spilanthol). An efficient multiplication frequency, ploidy stability and enhanced spilanthol accumulation ensure the efficacy of the protocol developed for this industrially important medicinal plant.     

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.     

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).     

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.     

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.     

Efficient genetic transformation of Jatropha curcas L. by microprojectile bombardment using embryo axes

An efficient and reproducible protocol was established for genetic transformation in Jatropha curcas through microprojectile bombardment. Decotyledonated embryos from mature seeds were pre-cultured for 5 days and elongated embryonic axis was subjected to bombardment for the optimization of physical parameters. The frequency of transient gus expression and survival of putative transformants were taken into consideration for the assessment of physical parameters. Statistical analysis reveal that microcarrier size, helium pressure and target distance had significant influence on transformation efficiency. Among different variables evaluated, microcarrier size 1 μm, He pressure 1100 and 1350 psi with a target distance of 9 and 12 cm respectively were found optimum by co-relating microcarrier size, helium pressure and target distance on the frequency of gus expression and survival of putative transformants. Selection of putative transformants was done with increasing concentrations (5-7 mg L⁻¹) of hygromycin. The integration of desired gene into Jatropha genome was confirmed with PCR amplification of 0.96 and 1.28 kb bands of hptII and gus gene respectively from the T₀ transgenics and Southern blot analysis using PCR amplified DIG labeled hptII gene as a probe. A successful attempt of genetic transformation was made with optimized conditions using particle gene gun and establishing a stable transformation in J. curcas with 44.7% transformation efficiency. The procedure described will be very useful for the introgression of desired genes into J. curcas and the molecular analysis of gene function.     

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.     

Optimization of ultrasonic-assisted extraction for herbicidal activity of chicory root extracts

The present study evaluated the herbicidal potential of extracts from chicory (Cichorium intybus L.) roots on the germination of Echinochloa crusgalli L. Beauv and Amaranthus retroflexus L. Eight ultrasonic assisted-extraction (UAE) conditions were optimized, using an orthogonal matrix design. The extract concentrations that would yield the largest allelopathic effects on the plant species were estimated by a modeling analysis. Our results showed that an alcohol solvent extract of chicory root had significant herbicidal activity which depended on the extract concentrations and the target species. The half-inhibitory concentrations of crude extract of chicory root ranged from 0.5 g l⁻¹ to 40.5 g l⁻¹. At a frequency of 40 kHz, the optimum UAE conditions to produce an extract for use as herbicide against E. crusgalli L. included an ethanol content (Ec) of 50% (v/v), a solvent-to-solid ratio (SR) of 16:1, an ultrasound temperature (UT) of 35 °C, an impregnation time (Imt) of 24 h with two rounds of impregnation (Imr), a sonication period (St) of 120 min with two rounds of sonication (Sr) and an ultrasound input power (P) of 200 W. The optimum conditions to produce an extract for use against A. retroflexus L. included an Ec of 100% (v/v), a SR of 16:1, an UT of 20 °C, an Imt of 48 h with two Imr, a St of 30 min with one Sr and a P of 400 W. The extract had the largest inhibitory effects on the germination index and root growth of both E. crusgalli L. and A. retroflexus L. at concentrations ranging from 30.8 to 33.7 g l⁻¹. At a concentration of 4.2 g l⁻¹, the extract significantly enhanced the shoot growth of A. retroflexus L. Overall, chicory root extract has potential for use as a main ingredient in natural herbicides or for development as a novel plant-derived herbicide.     

Large scale in vitro propagation of Stevia rebaudiana (bert) for commercial application: Pharmaceutically important and antidiabetic medicinal herb

Stevia rebaudiana is a valuable medicinal plant species and it is being used for the treatment of diabetes. Currently, there is a high demand for raw material of this medicinal herb due to ever increasing diabetes disorder among the population. In order to meet the increased demand an efficient in vitro propagation of S. rebaudiana was established. Nodal explants collected from the field were cultured on MS basal medium fortified with different concentrations of BAP (0.5-3.0 mg/l) and KIN (0.5-3.0 mg/l) individually for shoot bud induction. In vitro derived nodal buds were cultured on MS medium supplemented with different concentrations (0.5-3.0 mg/l) of BAP and KIN for multiple shoot bud regeneration. In the second experiment, in vitro derived buds were placed on MS medium supplemented with different concentrations of BAP (0.5-3.0 mg/l) in combination with 0.5 mg/l IAA or IBA or NAA for shoot bud multiplication. The highest frequency (94.50%) of multiple shoot regeneration with maximum number of shoots (15.69 shoots/explant) was noticed on MS medium supplemented with 1.0 mg/l BAP. For large scale plant production, in vitro derived nodal bud explants were cultured on MS medium fortified with 1.0 mg/l BAP, in which about 123 shoots/explant were obtained after three subcultures on the same media composition. Elongated shoots (>2 cm) dissected out from the in vitro proliferated shoot clumps were cultured on half-strength MS medium containing different concentrations of NAA (0.1-0.5 mg/l) and/or MS medium fortified with various concentrations (0.5-2.0 mg/l) of auxins (NAA, IAA and IBA) for root induction. Highest frequency of rooting (96%) was noticed on half-strength MS medium augmented with 0.4 mg/l NAA. The rooted plantlets were successfully transferred into plastic cups containing sand and soil in the ratio of 1:2 and subsequently established in the greenhouse. The present in vitro propagation protocol would facilitate an alternative method for rapid and large-scale production of this important antidiabetic medicinal plant.     

Improvement of Hanseniaspora uvarum biocontrol activity against gray mold by the addition of ammonium molybdate and the possible mechanisms involved

The efficacy of Hanseniaspora uvarum against gray mold by adding ammonium molybdate (NH₄–Mo) and the mode of actions were evaluated. The results showed that H. uvarum at 1 × 10⁶ CFU ml⁻¹ plus 1 mmol l⁻¹ NH₄–Mo greatly reduced gray mold in grape fruits. NH₄–Mo at concentrations of 1, 5, 10 and 15 mmol l⁻¹ significantly inhibited spore germination and mycelium growth of Botrytis cinerea. Population growth of H. uvarum was markedly inhibited by NH₄–Mo at 5 mmol l⁻¹in vitro and not affected by addition of NH₄–Mo at 1 and 5 mmol l⁻¹ in wounds combination of NH₄–Mo and H. uvarum induced higher activities of peroxidase (POD), polyphenoloxidase (PPO), phenylalanine ammonialyase (PAL), superoxide dismutase (SOD), catalase (CAT) and β-1,3-Glucanase than individual application of H. uvarum or NH₄–Mo. The enhancement of disease control may be directly because of the inhibitory effects of NH₄–Mo on spore germination and mycelial growth of B. cinerea in vitro, and indirectly because of the induced defense reactions by NH₄–Mo in grape berries.     

Toxicity of biofungicide Timorex 66 EC to Cladobotryum dendroides and Agaricus bisporus

Twenty Cladobotryum dendroides isolates from Serbian Agaricus bisporus farms collected during 2003–2007, and the F56 strain of A. bisporus were tested in vitro for sensitivity to tea tree oil (Timorex 66 EC), a biofungicide, in comparison with prochloraz–manganese (Octave WP). The efficacies of tea tree oil and prochloraz–manganese were evaluated in a mushroom growing room, after application at standard product application rates and a combination of the two at respective proportion of 20:80%. Tea tree oil was considerably less toxic than prochloraz–manganese in vitro to C. dendroides isolates (ED₅₀ 112.9–335.8 mg l⁻¹) and A. bisporus F56 strain (98.0 mg l⁻¹), although neither fungicide was lethal to the pathogen. The biological efficiency of tea tree oil was higher than in treatments with the reference formulation of prochloraz–manganese. In our in vivo trials, no negative interference of the biofungicide with A. bisporus physiology was observed. Tea tree oil applied at the standard product application rate caused a significant reduction in cobweb disease levels in the A. bisporus growing room. Timorex 66 EC should be tested further in combination with other biofungicides to investigate the effectiveness of various mixtures for A. bisporus disease control.     

Relationship between P and N concentrations in maize and wheat leaves

Simple plant-based diagnostic tools can be used to determine crop P status. Our objectives were to establish the relationships between P and N concentrations of the uppermost collared leaf (P_L and N_L) of spring wheat (Triticum aestivum L.) and maize (Zea mays L.) during the growing season and, in particular, to determine the critical leaf P concentrations required to diagnose P deficiencies. Various N applications were evaluated over six site-years for wheat and eight site-years for maize (2004-2006) with adequate soil P for growth. Phosphorus and N concentrations of the uppermost collared leaf were determined weekly and the relationships between leaf N and P concentrations were established using only the sampling dates from the stem elongation stage for wheat and from the V8 stage of development for maize. Leaf P concentration generally decreased with decreasing N fertilization. Relationships between P_L and N_L concentrations (mg g⁻¹ DM) using all site-years and sampling dates were described by significant linear-plateau functions in both maize (P_L = 0.82 + 0.089 N_L if N_L ≤ 32.1 and P_L = 3.7 if N_L > 32.1; R² = 0.41; P < 0.001) and wheat (P_L = 0.02 + 0.106 N_L if N_L ≤ 33.2 and P_L = 3.5 if N_L > 33.2; R² = 0.42; P < 0.001). Variation among sampling dates in the relationships were noted. By restricting the sampling dates [413-496 growing degree days (5 °C basis) in wheat (i.e., stem elongation) and 1494-1579 crop heat units in maize (i.e., silking), relationships for wheat (P_L = 0.29 + 0.073 N_L, R² = 0.66; P < 0.001) and maize (P_L = 1.04 + 0.084 N_L, R² = 0.66; P < 0.001) were improved. In maize, expressing P and N concentrations on a leaf area basis (P_LA and N_LA) at silking further improved the relationship (P_LA = 0.002 + 0.101 N_LA, R² = 0.80; P < 0.001). Predictive models of critical P concentration as a function of N concentration in the uppermost collared leaf of wheat and maize were established which could be used for diagnostic purposes.     

Indigenous legume fallows (indifallows) as an alternative soil fertility resource in smallholder maize cropping systems

Widening the range of organic nutrient resources, especially N sources, is a major challenge for improving crop productivity of smallholder farms in southern Africa. A study was conducted over three seasons to evaluate different species of indigenous legumes for their biomass productivity, N₂-fixation and residual effects on subsequent maize crops on nutrient-depleted fields belonging to smallholder farmers under contrasting rainfall zones in Zimbabwe. Under high rainfall (>800 mm yr⁻¹), 1-year indigenous legume fallows (indifallows), comprising mostly species of the genera Crotalaria, Indigofera and Tephrosia, yielded 8.6 t ha⁻¹ of biomass within 6 months, out-performing sunnhemp (Crotalaria juncea L.) green manure and grass (natural) fallows by 41% and 74%, respectively. A similar trend was observed under medium (650–750 mm yr⁻¹) rainfall in Chinyika, where the indifallow attained a biomass yield of 6.6 t ha⁻¹ compared with 2.2 t ha⁻¹ for natural fallows. Cumulatively, over two growing seasons, the indifallow treatment under high rainfall at Domboshawa produced biomass as high as 28 t ha⁻¹ compared with ∼7 t ha⁻¹ under natural fallow. The mean total N₂ fixed under indifallows ranged from 125 kg ha⁻¹ under soils exhibiting severe nutrient depletion in Chikwaka, to 205 kg ha⁻¹ at Domboshawa. Indifallow biomass accumulated up to 210 kg N ha⁻¹, eleven-fold higher than the N contained in corresponding natural fallow biomass at time of incorporation. Application of P to indifallows significantly increased both biomass productivity and N₂-fixation, translating into positive yield responses by subsequent maize. Differences in maize biomass productivity between indifallow and natural fallow treatments were already apparent at 2 weeks after maize emergence, with the former yielding significantly (P < 0.05) more maize biomass than the latter. The first maize crop following termination of 1-year indifallows yielded grain averaging 2.3 t ha⁻¹, significantly out-yielding 1-year natural fallows by >1 t ha⁻¹. In the second season, maize yields were consistently better under indifallows compared with natural fallows in terms of both grain and total biomass. The first maize crop following 2-year indifallows yielded ∼3 t ha⁻¹ of grain, significantly higher than the second maize crop after 1-year indifallows and natural fallows. The study demonstrated that indigenous legumes can generate N-rich biomass in sufficient quantities to make a significant influence on maize productivity for more than a single season. Maize yield gains under indifallow systems on low fertility sandy soils exceeded the yields attained with either mineral fertilizer alone or traditional green manure crop of sunnhemp.