Sterilized River Sand Research Articles

Symbiotic interaction of bambara groundnut (Vigna subterranea) landraces with rhizobia spp. from other legume hosts reveals promiscuous nodulation

Bambara groundnut (Vigna subterranea) is one of the most underutilized and neglected legume crop used for human consumption worldwide. Unlike the common food and forage legumes, the symbiotic properties of this legume have not been studied sufficiently. This study is designed to investigate the nodulation compatibility and symbiotic performance of twelve landraces of V. subterranea with five different Rhizobia species isolated from the nodules of other legumes under glasshouse conditions. Pre-germinated seeds of each landrace planted in sterile river sand medium were inoculated with 108 cfu/ml of each rhizobium inoculum suspension and monitored with regular watering using nitrogen free Hoagland's solution for six weeks. The taxonomy and phylogeny of the five rhizobia species was determined using nucleotide sequence analysis of the 16S rRNA and recA (DNA recombination protein) genes. A concatenated multiple sequence alignment was used to construct an MLSA tree to further analyze the phylogeny of the strains. Significant differences were observed among individual bambara genotypes and rhizobia strains in terms of nodule numbers and dry weight, as well as plant biomass. Statistical analysis showed that Rhizobia strains XBD2 and XS34 identified as Bradyrhizobium zhangiangens and B. centrosematis respectively exhibited the highest nodulation compatibility in terms of nodule number and nodule dry weight with one or more bambara groundnut landraces. This study demonstrated that V. subterannea does not show preference to unique rhizobia, confirming that they are promiscuously nodulated by more than one species of rhizobium belonging mainly to the cowpea miscellany cross inoculation group. The findings of this study also provide insights into the selection and development of appropriate low cost rhizobia inoculants in the sustainable production of this underutilized legume for use by smallholder farmers in South Africa.

Isolation of Rhizobia From the Nodules of Bambara Groundnuts for Inoculant Production

Rhizobia symbiotic interactions with legumes fix atmospheric nitrogen into the soil, which is essential in amending the characteristically low-nitrogen soils in most farming communities in northern Ghana. A high potential for improvement of Bambara groundnuts production in low-nitrogen soils is by the exploitation of colonization of the plant roots with rhizobial inoculation. This experiment sought to isolate Legume Nodulating Bacteria (LNB) obtained from root nodules of Bambara groundnut (Vigna subterranea) plants and to identify effective strains for improved production of the crop. Roots nodules of Bambara plants used in this study were obtained from preserved plants and the isolates were authenticated for their symbiotic effectiveness under screen house conditions. Nodulation of the isolates was examined in plastic pots containing sterile river sand and test crop (Bambara seeds). The experiment included reference strains, a positive nitrogen control and an un-inoculated control. The results were obtained after two months of data collection. The difference in results was explained via nodulation capacity. Out of the two isolates obtained, 2CL showed a high nodulation capability, rating it as highly effective. The outcome of this study provides stakeholders with the prospect for the use of effective isolates as inoculants to improve Bambara groundnut yield in general and in northern Ghana in particular.

Storage of Terminalia superba Engl. & Diels. Seeds

Teminalia superba is one of the species receiving priority under the on-going Forest Investment Programme in Ghana. A more comprehensive look at the response of the seeds to storage is therefore important for their use in afforestation, reforestation activities, breeding programmes and long term conservation. Seeds of T. superba were harvested in October, 2016 from Amantia in the Ashanti Region of Ghana and immediately processed. Drawn seed samples were dried over silica gel to reduce seed moisture content to 10% and 5% respectively. Moisture content of seeds was determined by drying in an oven at 103° C for 17 hours. Seeds were placed in two types of storage containers namely, hermetic glass bottles and plastic bottles. These were placed at three different temperature conditions namely; ambient temperature (28-32°C), refrigerator temperature (4-5°C) and freezing temperature (-10°C) and stored for a period of 24 months with the conduction of germination tests every two months at the laboratory and plant house of the Forestry Research Institute of Ghana. Germination tests were conducted in a medium of heat sterilized river sand at the plant house with ambient temperature of 27-33°C. The statistical design used in the investigation was a completely randomized design in a 2 x 2 x 3 factorial (moisture levels × storage containers × storage temperatures). Storage of seeds in hermetic glass bottles was found to have maintained seeds at a higher viability level than storage in plastic bottles. Both freezing and refrigeration temperatures were found to be ideal storage conditions. Storage at 5% moisture content resulted in higher viability of seeds. The electrical conductivity test was found to be an efficient test for distinguishing good quality seeds from poor seeds of the species.

Effect of pretreatments on germination of seeds of the timber plant, Terminalia ivorensis and Mansonnia altissima (A. Chev.)

Terminalia ivorensis and Mansonnia altissima is an important tropical timber tree plant, but poor germination is an important factor militating its regeneration. Effects of pretreatments on germination of seeds of Terminalia ivorensis and Mansonia altissima were studied. Four hundered and eighty matured seeds of Terminalia ivorensis and three hundred and sixty seeds of Mansonia altissima were obtained from Forest Reserve Institute of Nigeria (FRIN), Ibadan. The seeds were divided into twenty-one equal portions of 10 seeds each. Twelve pretreatments were investigated on Terminalia ivorensis and nine pretreatments on Mansonia altissima. The seeds were sown inside plastic basket containing sterilized river sand under high humidity propagator with four replicates each. Germination counts were taken daily for eighty days. Percentage germination was computed for each treatment and the data were subjected to analysis of variance. Germination was first observed among the scarified Terminalia ivorensis seeds at 14 days after sowing (DAS) and this treatment group had the highest percentage germination of 82.5%, while seeds soaked in hot water recorded the least germination. Germination was first observed in decoated Mansonia altissima and this treatment group had the highest percentage germination of 45%, whereas the least was observed among the treatments with H­2SO4. The effect of pretreatment was highly significant (p≤0.05) on the germination of both species. It is therefore recommended that mechanical scarification of Terminalia ivorensis and decoating seed of Mansonia altissima will enhance the germination of the two species.

Effects of Stock Age, Hormone Types and Concentrations on Rooting and Early Growth of Vitellaria paradoxa C. F. Gaertn. Stem Cuttings

This study investigated the effects of stock age, hormones and hormone concentrations on survival and rooting of Vitellaria paradoxa stem cuttings with the aim of improving on early development of the species. Single node stem cuttings were obtained from 9 and 15 months old seedlings of V. paradoxa and treated with Naphthalene Acetic Acid (NAA), Indole Butyric Acid (IBA), unripe coconut water (CW) and distilled water (control) at 100 mg/l and 200 mg/l concentrations NAA, IBA and 50% and 100% coconut water. Quick dip method was used and the cuttings set in washed and sterilized river sand medium under non-mist propagation in a 2x4x2 factorial experiment laid out in Completely Randomized Design and replicated 3 times. Percentage rooted and percentage die-back were assessed after eight weeks of setting while shoot height (cm), shoot diameter (mm), leaf production and leaf area (cm2) were assessed for three months. The data collected were subjected to descriptive statistics and analysis of variance (ANOVA). Cuttings from both 9 months and 15 month old stocks recorded higher percentage (90%) with NAA hormone treated cuttings and also produced the highest rooting at (90%) while control recorded the least (50%). Hormone type also significantly influenced the early growth of the rooted cuttings in term of shoot height, shoot diameter, leaf area and leaf production (p≤0.05). The highest shoot height, shoot diameter, leaf area and number of leaves were obtained with NAA with mean values of (4.81 cm, 3.46 mm, 35.08 cm2 and 5.00) respectively while control had the least (3.80 cm, 2.28 mm, 27.81 cm2 and 3.29) respectively. It therefore implies that the use of hormones can improve rooting and early growth of V. paradoxa stem cuttings collected from young stock plants.

Effects of Seed Source, Moisture Content and Duration of Storage on the Viability of Vitellaria paradoxa C.F. Gaertn

Viability of tropical seeds is crucial in plantation establishment. Temperature – moisture duration relationship of the seed is of great significance in seed viability. 3000 ripe fruits of V. paradoxa C. F. Gaertn were collected from each of three sources where it is endemic: Eruwa, Sakiand New-Bussa. The fruits were depulped; a sample of 100 seeds was drawn from each of the three sources and sown in washed and sterilized river sand to monitor the germination percentage of the seeds. The seeds were dried under dehumidifier to attain two moisture contents (43% and 32%). Thereafter, each seed lot from the three sources was divided into two batches (A and B) of 1200 seeds each, divided into four and stored at four different temperature regimes: -20°C, 5°C, 22°C and 28± 2°C. Samples were taken monthly for germination test for six months. Data were subjected to percentages and analysis of variance (ANOVA). Results showed that freshly collected seeds of V. paradoxa had 98% germination when sown within 48 hours of fallen and collection which eventually dropped to 4.4% after the six months storage. Storage temperature had significant effect (P > 0.05) on germination of the seeds. Seeds stored at 28±2°C had the highest mean germination (72.2%), those stored at 22°C had 70.2% while seeds stored at -20°C and 5°C failed to germinate. The seeds of V. paradoxa readily lost viability with time, hence fallen fruits should be collected during fruiting season processed and sown immediately or within 7 days of collection.

Isolation and Characterization of Phosphate Solubilising Rhizobia Nodulating Wild Field Pea (Pisum sativum var. abyssinicum) from Southern Tigray, Ethiopia

Hypothesis: Phosphorus is the second limiting nutrients next to nitrogen as well as the least mobile element in the soil. This nutrient is one of the major constraints for low productivity of wild field pea in the study site. Hence, the development of environmental friendly and economically accepted to subsistent farmer is undeniably important.
 Aims: This experiment was initiated to isolate and characterize inorganic phosphate solubilizing rhizobia from root nodules of field pea (Pisum sativum var. abyssinicum) were characterized for their solubilisation ability on Pikovaskaya liquid and solid media.
 Study Design: Laboratory experimental design was used.
 Place and Duration of Study: Field pea root nodules were collected from Southern Tigray and the study was carried out between October, 2015 and June, 2016 at Haremaya University soil microbiology laboratory.
 Methodology: The presumptive test, qualitative and quantitative phosphate solubilisation of isolates was done in triplicates using the standard procedures. Authentication of the isolates was made using autoclaved and sterilized river sand in pot experiment.
 Results: The current results revealed that all isolates were gram negative, failed to grow on peptone glucose agar, ketolactose test and did not absorb congo-red upon incubation period. It also showed that phosphate solubilisation index of root nodulating bacteria on in vitro Pikovskaya’s agar medium varied from 1.54 to 2.70. Inorganic phosphate solubilisation in broth medium dissolved insoluble Ca3 (PO4)2 was within the range of 16.59-23.95 mg plant-1 with pH drop from 7.01 to 5.33. Among the tested rhizobia isolates, HUDRI-8 and HUDRI-25 was found to be highest phosphate solubilisation compared to the remaining isolates, served as efficient phosphate solubilizers and could be used for further test under field condition.
 Conclusion: Finally, the selected isolates which are effective in N2 fixation and able to solubilise inorganic P were found to be effective in promoting nodulation and plant growth under greenhouse condition.

The carboxylate-releasing phosphorus-mobilizing strategy can be proxied by foliar manganese concentration in a large set of chickpea germplasm under low phosphorus supply.

Root foraging and root physiology such as exudation of carboxylates into the rhizosphere are important strategies for plant phosphorus (P) acquisition. We used 100 chickpea (Cicer arietinum) genotypes with diverse genetic backgrounds to study the relative roles of root morphology and physiology in P acquisition. Plants were grown in pots in a low-P sterilized river sand supplied with 10μgPg-1 soil as FePO4 , a poorly soluble form of P. There was a large genotypic variation in root morphology (total root length, root surface area, mean root diameter, specific root length and root hair length), and root physiology (rhizosheath pH, carboxylates and acid phosphatase activity). Shoot P content was correlated with total root length, root surface area and total carboxylates per plant, particularly malonate. A positive correlation was found between mature leaf manganese (Mn) concentration and carboxylate amount in rhizosheath relative to root DW. This is the first study to demonstrate that the mature leaf Mn concentration can be used as an easily measurable proxy for the assessment of belowground carboxylate-releasing processes in a range of chickpea genotypes grown under low-P, and therefore offers an important breeding trait, with potential application in other crops.

Leaf transpiration plays a role in phosphorus acquisition among a large set of chickpea genotypes.

Low availability of inorganic phosphorus (P) is considered a major constraint for crop productivity worldwide. A unique set of 266 chickpea (Cicer arietinum L.) genotypes, originating from 29 countries and with diverse genetic background, were used to study P-use efficiency. Plants were grown in pots containing sterilized river sand supplied with P at a rate of 10μgPg-1 soil as FePO4 , a poorly soluble form of P. The results showed large genotypic variation in plant growth, shoot P content, physiological P-use efficiency, and P-utilization efficiency in response to low P supply. Further investigation of a subset of 100 chickpea genotypes with contrasting growth performance showed significant differences in photosynthetic rate and photosynthetic P-use efficiency. A positive correlation was found between leaf P concentration and transpiration rate of the young fully expanded leaves. For the first time, our study has suggested a role of leaf transpiration in P acquisition, consistent with transpiration-driven mass flow in chickpea grown in low-P sandy soils. The identification of 6 genotypes with high plant growth, P-acquisition, and P-utilization efficiency suggests that the chickpea reference set can be used in breeding programmes to improve both P-acquisition and P-utilization efficiency under low-P conditions.

English

The family Verbenaceae includes 36 genera and 1035 species. Among them lemon verbena (Aloysia triphylla) is known to have high medicinal value. Therefore, development of fast and new in vitro micropropagation protocol will have a high importance in lemon verbena mass-propagation. This research study targeted to develop rapid in vitro micropropagation protocol for lemon verbena. Up to 88% of clean survived plantlets were obtained after treating the nodal explants with 0.5% berekina (NaClO) for 10 min. Shoot initiation and multiplication was achieved using node as explant planted on MS medium supplemented with different strength of 6-benzyladeninepourine (BAP) and kinetin (Kin) individually and in combination. Plants were put on root induction ½ strength MS medium fortified with different strength of indole-3-butyric acid (IBA) alone. The best treatment for shoot initiation was 6-benzyladeninepourine (1.5 mg/L) with 84.1% of initiation. The best treatment for shoot multiplication was 6-benzyladeninepourine (2.0 mg/L) with 9.23 shoots per explant. Best rooting (100%) and maximum root number per shoot (14.4) were found at 1.0 mg/L indole-3-butyric acid (IBA). The longest root (3.1 cm) was achieved without supplementing the media with plant growth regulators. The plantlets were hardened and acclimatized in fully automated greenhouse and survival percentage was greater than 70% planting on a combination of sterilized river sand, top forest soil and animal manure in a 1:2:1 (v/v/v) ratio. This in vitro micropropagation protocol can be used instead of conventional propagation techniques, as a fast and economically cheap method to propagate a wide range of similar plants. Key words: Acclimatization, auxin, cytokinin, micropropagation, lemon verbena.

Differential growth costs and nitrogen fixation in Cytisus multiflorus (L'Hér.) Sweet and Cytisus scoparius (L.) Link are mediated by sources of inorganic N.

Shrubby legumes in Mediterranean-type ecosystems face strong nutrient limitations that worsen in summer, when water is absent. Nitrogen-fixing legumes are likely to be able to switch between soil N and atmospheric N (N2 ) sources to adjust the C costs of N acquisition in different seasons. We investigated the utilisation of different inorganic N sources by two indigenous shrubby legumes (Cytisus multiflorus and Cytisus scoparius). Plant performance in terms of photosynthesis and biomass production was also analysed. Plants were cultivated in sterile river sand supplied with Hoagland nutrient solution, grown in N-free nutrient solution and inoculated with effective rhizobial strains from nodules of adult plants of the same species. A second treatment consisted of plants given 500μm NH4 NO3 added into the nutrient solution. In a third treatment, plants were watered with another source of N (500μm NH4 NO3 ) as well as being inoculated with effective rhizobial strains. The application of NH4 NO3 to the legumes resulted in a larger increase in plant dry matter. Carbon construction costs were higher in plants supplied with mineral and symbiotic N sources and always higher in the endemic C.multiflorus. Differences in photosynthesis rates were only observed between species, regardless of the N source. Non-fertilised inoculated plants had more effective root nodules and a clear dependence on N2 fixation. We propose that the ability of C.scoparius to change N source makes it a plastic species, which would account for its broader distribution in nature.

Respuesta del ají (Capsicum annuum L. Var. Cayena) a concentraciones de N, P, K, Ca y Mg en Palmira, Valle Del Cauca, Colombia

En la Universidad Nacional de Colombia, Sede Palmira, se estableció un experimento para determinar los requerimientos nutricionales (N, P, K, Ca y Mg) del ají Capsicum Annuum L., con 16 tratamientos (soluciones nutritivas), cuatro repeticiones, y cinco muestreos a los 20, 40, 70, 100 y 140 días después del trasplante. Los semilleros se establecieron en paneles con arena de río esterilizada, las plántulas fueron regadas con las soluciones nutritivas correspondientes a cada tratamiento y trasplantadas 20 días después de la siembra (dds) amacetas de 6 Kg de capacidad, se empleó arena de cuarzo como sustrato. Se tomó como referencia la solución de Hoagland y Arnon; los demás tratamientos se obtuvieron variando las concentraciones de N, P, K, Ca, y Mg. La concentración alta correspondió a la del testigo más 50% de la misma; la media, el 25% de la concentración alta y la baja, un cuarto de la concentración media. El nivel crítico de los elementos nutritivos se determinó mediante la construcción de curvas de calibración, relacionando la concentración de cada uno de ellos con la producción de biomasa. Se trazó una línea paralela al eje de las X, tomado como referencia el 95% de la biomasa máxima. Los puntos que cortan la curva de respuesta corresponden al nivel crítico o rango de concentración adecuada de cada elemento nutritivo. Los rangos de concentración adecuados en la solución nutritiva para el ají variedad cayena son: N 16,2 - 21,5 mmol.L-1. P 1,1 – 1,4 mmol.L-1, K 4,4 – 8,2 mmol.L-1. No se encontraron los requerimientos para Ca y Mg.

Short-Term Biochemical Impacts in <i>Cyphoderus javanus</i> Borner (Collembola) as Potential Biomarkers of Fly Ash and Heavy Metal Pollution in Tropical Lateritic Soil

The detritivorous soil microarthropods are ideal models for environmental health assessment, but only scant information is available on their biomarker potentials against xenobiotics in the tropical edaphic conditions. This microcosm study has estimated short-term biochemical changes with respect to tissue nutrient levels and digestive enzyme activities in a near cosmopolitan Collembola Cyphoderus javanus Borner exposed to coal fly ash amended lateritic cropland soil (5% and 20% w/w i.e. 50 or 200 t ·ha-1), and cadmium sulphate (200 mg/kg soil)/lead acetate (200 mg/kg soil) treated sterile river sand. The levels of total tissue carbohydrates, proteins, free amino acids, and lipids decrease within 7 days in the specimens of fly ash (P α-amylase, cellulase and protease activities are also down regulated by fly ash and heavy metals (P < 0.05). It is also observed the enzyme: protein ratios increase in fly ash treated sets and decrease in lead and cadmium treated specimens. Specimens in treated sets accumulate high levels of lead and cadmium within 15 days, which decrease subsequently probably due to elimination through frequent moulting. These short-term biochemical effects in Collembola have applied importance in C. javanus, as biomarkers for quick ecotoxicological assessments in the tropical agricultural soils.

Arbuscular mycorrhizal symbiosis alters the expression patterns of three key iron homeostasis genes, ZmNAS1, ZmNAS3, and ZmYS1, in S deprived maize plants.

Nicotianamine is an essential molecule for Fe homeostasis in plants, its primary precursor is the S-containing compound methionine, and it is biosynthesized by the enzyme family of nicotianamine synthases (NASs). In maize, a graminaceous plant that follows Strategy II for Fe uptake, ZmNAS genes can be subgrouped into two classes, according to their roles and tissue specific expression profiles. In roots, the genes of class I provide NA for the production of deoxymugineic acid (DMA), which is secreted to the rhizosphere and chelates Fe(III). The Fe(III)-DMA complex is then inserted to the root via a ZmYS1 transporter. The genes of class II provide NA for local translocation and detoxification of Fe in the leaves. Due to the connection between S and Fe homeostasis, S deficiency causes Fe deprivation responses to graminaceous plants and when S is supplied, these responses are inverted. In this study, maize plants were grown in pots with sterile river sand containing FePO4 and were inoculated with the mycorrhizal fungus Rhizophagus irregularis. The plants were grown under S deficient conditions until day 60 from sowing and on that day sulfate was provided to the plants. In order to assess the impact of AM symbiosis on Fe homeostasis, the expression patterns of ZmNAS1, ZmNAS3 (representatives of ZmNAS class I and class II), and ZmYS1 were monitored before and after S supply by means of real time RT-PCR and they were used as indicators of the plant Fe status. In addition, total shoot Fe concentration was determined before and after S supply. AM symbiosis prevented Fe deprivation responses in the S deprived maize plants and iron was possibly provided directly to the mycorrhizal plants through the fungal network. Furthermore, sulfate possibly regulated the expression of all three genes revealing its potential role as signal molecule for Fe homeostasis.

Soil fungi of three native tree species inhibit biomass production and shift biomass allocation of invasive Mikania micrantha Kunth

Soil microbes contribute to native plant species successful resistance against invasive plant. Three native tree species, Heteropanax fragrans (HF), Cinnamomum burmanii (CB), and Macaranga tanarius (MT) were effective in controlling the notorious invasive vine Mikania micrantha (MM). Biomass production and allocation patterns (shoot/root biomass ratio (shoot/root)) are important indicators of MM climbing coverage and competitive light-capturing capacity. An investigation was conducted to test the role of soil microbes associated with the three native tree species to inhibit MM biomass production and shift MM shoot/root. Rhizosphere soils originating from preculture HF, CB, MT, and MM plots were collected separately for use as inocula. The inocula were mixed with sterilized river sand at a 1:9 (w/w) ratio to grow MM. The fungicide carbendazim (methyl benzimidazol-2-ylcarbamate) was applied to half the treatments to kill pathogenic soil fungi. Two nutrient levels were established based on the natural soil nutrient concentration from a field stand invaded by MM. MM were grown from seeds in a glasshouse, harvested 15 weeks after sowing, and separated into shoot and root portions. Results showed that under interaction of soil origin and nutrient levels, MM biomass production was unchanged, but biomass allocation patterns were significantly different. MM biomass production grown in the three native tree soils under two nutrient levels was similar or higher than MM biomass production in MM conspecific soil, indicating the absence of species-specific pathogens that inhibited MM biomass production in native tree soils. However, in both conspecific and tree soils, MM biomass production was significantly reduced in the presence of pathogenic soil fungi, i.e. MM experienced significant fungal inhibition, demonstrating the pathogenic soil fungi promoted native tree resistence to MM. MM exhibited decreased shoot biomass allocation when cultivated in native tree soil relative to MM conspecific soil under field stand nutrient level conditions. Reduced resource allocation to shoot biomass could result in diminished capacity to climb, cover, and subsequent smother to native trees, and reduced surface area exposed to available light. Following fungicide application, significant biomass allocation differences disappeared, suggesting the native tree soil fungi were responsible for decreasing MM shoot biomass. The overall results indicated tree soil fungi serve an integral role in controlling invasive MM through fungal inhibition on MM biomass production, and shifts in MM biomass allocation patterns.

Scarification and Environmental Factors that Enhance Carpogenic Germination of Sclerotia of Sclerotinia sclerotiorum.

Ascospores of Sclerotinia sclerotiorum are the primary source of inoculum for disease epidemics in many economically important crops. Mass production of ascospores under laboratory conditions is required to prepare inoculum for use in selection of genotypes with resistance against Sclerotinia diseases. A study was undertaken, first, to investigate the effect on carpogenic germination of scarifying sclerotia from two S. sclerotiorum isolates taken from canola (Brassica napus) and, second, to identify environmental factors that enhance carpogenic germination. Seven different environmental treatments were applied to scarified and unscarified sclerotia: (i) sterilized distilled water for 4 months at 15°C, (ii) aerated water for 4 months at 4°C, (iii) constant rinsing with tap water for 8 weeks at 4°C, (iv) cold-conditioning for 4 weeks at 4°C and subsequent transfer into moist unsterilized compost at 15°C, (v) incubation in sterilized river sand at 15°C, (vi) air drying for 2 weeks followed by subsequent transfer into sterilized moist river sand at 15°C, or (vii) placed into 0.5% water agar and incubated at 15°C. Carpogenic germination of scarified sclerotia was significantly greater (P < 0.05) than for unscarified sclerotia. There was significant interaction (P < 0.001) between scarification and the different environmental treatments in relation to the carpogenic germination. Carpogenic germination of scarified sclerotia was enhanced by incubation of sclerotia in compost or in sterilized river sand. Further, overall carpogenic germination of both scarified and unscarified sclerotia occurred to the greatest extent when sclerotia of either of the two isolates were subjected to constant rinsing with tap water. We believe this to be the first report of both the enhanced carpogenic germination by scarification in S. sclerotiorum and the environmental factors we report that enhance carpogenic germination of scarified sclerotia. The progression of carpogenic germination in all the environmental treatments was also monitored as a part of this study across the two consecutive years for the same two isolates. The majority of sclerotia of both isolates germinated between the months of June and September in both years, a period which coincides with the main part of the cropping season when Sclerotinia stem rot is normally observed in rainfed canola in Western Australia. These data suggested the existence of a seasonal rhythm-like pattern in relation to the carpogenic germination of this pathogen.

Effect of pre-sowing and incubation treatment on germination ofGarcinia kola(Heckel) seeds

Introduction. Garcinia kola, a multi-purpose fruit tree, produces fruit, seeds, roots and stem which are extensively used in Nigeria, Ghana and other West African countries for dental care. Cultivation of this fruit tree is limited because of poor seed germination. Our study therefore aimed at improving seed germination properties which will promote cultivation of G. kola by rural farmers. Materials and methods. Seeds taken from a single tree of G. kola were treated with cold water (22 °C), hot water (60 °C) and gibberelic acid (0.5 g 〈 L-1 ) for various lengths of time. They were then put to incubate in sterilized river sand or a thick transparent polyethylene bag to eval- uate the capacities of seed to be germinated under such conditions. In addition, the seed moisture content at harvest was also reduced to various levels before incubation in polyethylene bags to determine the critical moisture rate necessary for seeds to germinate. Results. The seeds incubated in the river sand without any treatment before sowing had an average time of germination of 71.2 d and a total percentage of germination of 28% whereas those incubated in polyethylene bags had an average time of germination of 25.8 d and a total percentage of germination of 62%. The cold water treatment, irrespective of the incubation method, did not significantly affect either the mean germination time or the total germination percentage. However, it significantly reduced the spreading out of the seed germination to 5.4 d. The seed pretreatment with gibberelic acid pro- longed at the same time the average time of germination and the spreading out of the germination. The seed water content at harvest (50.4%) allowed the best rate of seed germination and the shortest spreading out of the germination period. Conclusion. The pretreatment of freshly har- vested seeds with cold water followed by an incubation in a thick transparent polyethylene bag proved most effective in enhancing the germination of G. kola seeds. This procedure has to be recommended to promote the cultivation of this species. Nigeria / Garcinia kola / plant propagation / seed / germination / germinability / seed treatment

Effect of feeding clinoptilolite (zeolite) on the performance of three strains of laying hens

1. One hundred and twenty 16‐week‐old single combed pullets of three strains were fed on a diet containing 135 g protein/kg with or without 50 g clinoptilolite/kg in a trial with 20 hens per treatment. Sterile river sand replaced clinoptilolite in the control diet in order to keep the diets isoenergetic. The hens were individually caged in a naturally ventilated laying house and fed one of the two diets for ten 28‐d periods. 2. Significant dietary effects of feeding clinoptilolite were observed with number of eggs laid per hen, shell thickness, efficiency of food utilisation and droppings moisture content. No significant dietary effects between treatments were observed with body weight, age at first egg, egg weight, Haugh units, food intake/hen and rate of amino acid absorption of radioactive lysine and methionine into the bloodstream. Significant differences between strains were observed with regard to all parameters except food intake/hen. There were no significant strain × clinoptilolite interactions.

Effect of feeding clinoptilolite (zeolite) to three strains of laying hens

1. One hundred and twenty 16-week-old, single combed pullets of three strains were fed on a diet containing 160 g protein/kg with or without 50 g clinoptilolite/kg in a trial with 20 hens per treatment. Sterile river sand replaced clinoptilolite in the control diet in order to keep the diets isonergetic. 2. The hens were individually caged in a naturally-ventilated laying house and fed on one of the two diets for ten 28-d periods. 3. No significant dietary effects between treatments were observed with respect to body weight, age at first egg, egg weight, Haugh scores or food intake/hen. 4. Significant dietary effects in favour of clinoptilolite feeding were noticed with the number of eggs laid per hen, shell thickness, efficiency of food utilisation, droppings moisture content and mortality. 5. Significant differences between strains were observed with respect to all measurements taken except food intake/hen d.