Hydroponic Seedling Bioassay for the Bioherbicides Colletotrichum truncatum and Alternaria cassiae

A rapid bioassay was developed to measure the bioherbicidal efficacy of spore preparations of the pathogens Colletotrichum truncatum (Schwein.) Andrus and W. D. Moore and Alternaria cassiae Jurair and Khan on hemp sesbania (Sesbania exaltata) and sicklepod (Cassia obtusifolia), respectively. The system uses 4-day-old dark-grown seedlings (grown hydroponically in paper towel cylinders) which were sprayed with spore suspensions. Shoot lengths were monitored non-destructively, and recorded over time under conditions of dark growth, 90-100% relative humidity and 25 C. Shoot growth inhibition and stem collapse (mortality) were directly related to the spore concentration applied. Generally, at 10 3 - 10 4 spores ml-1, these pathogens caused significant shoot growth inhibition within 25-30 h and seedling death within 40-50 h. This bioassay has been used to study herbicide-pathogen interactions, and may be extended to determine the bioherbicidal efficacy of different pathogen isolates, pathovars or spore formulations. This technique is more rapid, uses a lower inoculum volume, requires less space and is performed under more controlled conditions than conventional greenhouse bioassay methods. The data obtained are more quantitative than those obtained from bioassays relying on visual rating systems.     

Easily available enzymes as natural retting agents

Easily available commercial enzymes currently have great potential in bast fibre processing and can be modified for different end uses. There are several new technologies using enzymes that are able to modify fibre parameters, achieve requested properties, improve processing results and are more beneficial to the ecology in the area of bast fibre processing and fabrics finishing. Enzymatic methods for retting of flax, "cottonisation" of bast fibres, hemp separation, and processing of flax rovings before wet spinning, etc., fall into this group of new technologies. Such enzymatic biotechnologies can provide benefits in textile, composite, reinforced plastic and other technical applications. Laboratory, pilot and industrial scale results and experiences have demonstrated the ability of selected enzymes to decompose interfibre-bonding layers based on pectin, lignin and hemicelluloses. Texazym SER spray is able to increase flax long fibre yields by more than 40%. Other enzymes in combination with mild mechanical treatment can replace aggressive and energy-intensive processing like Laroche "cottonisation". Texazym SCW and DLG pretreatments of flax rovings are presented.     

Paleoenvironmental study of the Carquefou site (Massif Armoricain, France) from the end of the Sub-boreal

The town of Carquefou, some 10 km northeast of Nantes on the left bank of the river Erdre, occupies a site long associated with human activity. During road construction east of the town, ditches, enclosures and post holes characteristic of the late la Tène were discovered at the locality of “Le Clouet”, which led us to obtain core samples from a nearby peat bog. These investigations indicated the changes in vegetation since 3915±95 uncal B.P., [2828 (2459) 2074 cal B.C.]. The slopes in the surroundings of the bog have been relatively treeless since the Bronze Age, but a very open woodland vegetation composed of Tilia, Corylus and Quercus has been maintained until the present day. In the area around the bog, and Alnus wood with an undergrowth of Cyperaceae was the dominant vegetation, despite some changes probably related to human occupation since the Bronze Age. Beginning at 955±35 uncal B.P. [1004 (1036, 1144, 1146) 1181 cal A.D.], in the Middle Ages, the alders disappeared almost totally, apparently because of clearance or an increase in water level. Human presence led to intensified cultivation of different crops including Cannabis and especially Cerealia. Finally, the presence of a variety of anthropogenic indicator plants (Cichorioideae, Asteraceae, Plantago lanceolata, etc.) suggests that cattle were reared in the vicinity of the site. Received May 22, 2000 / Accepted March 29, 2001     

Enhancement of disease caused by Colletotrichum truncatum in Sesbania exaltata by coinoculating with epiphytic bacteria

Colletotrichum truncatum isolate NRRL 13737 (ARS patent culture No. 18434) is being evaluated for development as a mycoherbicide against the problematic weed Sesbania exaltata. Studies were conducted to determine whether selected phylloplane microorganisms, used as coinoculants, could increase the severity of disease incited by C. truncatum in S. exaltata. Hemp Sesbania seedlings were grown in a variety of soils and environments, sprayed with conidia of C. truncatum, and the formation of appressoria was examined on leaves using epifluorescence microscopy. From hemp sesbania plants that supported high levels of appressoria formation, over 200 phyllosphere microorganisms were isolated. Fifteen of 73 microbial isolates assayed stimulated appressoria formation in vitro on cellophane membranes. Five of 8 superior isolates from the in vitro assay also enhanced disease symptoms induced by C. truncatum on S. exaltata compared to seedlings treated with conidia only. Populations of three selected superior isolates remained high on leaves during plant exposure to dew. The superior isolates initiated no apparent symptoms and rarely decreased seedling growth parameters in the absence of C. truncatum. This is the first instance of utilizing phylloplane microorganisms to increase the level of disease incited by a mycoherbicide agent. Microbial facilitators may provide a means of improving the weed control efficacy of mycoherbicides.     

Water Activity and Other Factors that Affect the Viability of Colletotrichum truncatum Conidia in Wheat Flour-Kaolin Granules ('Pesta')

Optimization of shelf-life is critically important for biocontrol products containing living microorganisms. Conidia of Colletotrichum truncatum, a fungal pathogen of the weed, hemp sesbania (Sesbania exaltata), were produced in shake flasks (corn meal-soya flour medium) and on Emerson Yp Ss agar and formulated in wheat flour-kaolin granules ('Pesta'). The granules were conditioned at water activities of 0, 0.12, 0.33, 0.53 and 0.75 during storage at 25 C over desiccant or saturated salt solutions. The longest shelf-life (conidial inoculum viability) was found in samples in the water activity range 0-0.33, where the water was bound by the matrix and not readily available to the fungus. At a water activity of 0.12, granules were 100% viable (on water agar) for at least 24 weeks, and were 87% viable after 1 year. Sucrose (5% w/w) partially counteracted the detrimental effect of high water activity on the shelf-life of C. truncatum when incorporated in the granules.     

Hemp (Cannabis sativa L.) leaf photosynthesis in relation to nitrogen content and temperature: implications for hemp as a bio‐economically sustainable crop

Hemp (Cannabis sativa L.) may be a suitable crop for the bio‐economy as it requires low inputs while producing a high and valuable biomass yield. With the aim of understanding the physiological basis of hemp's high resource‐use efficiency and yield potential, photosynthesis was analysed on leaves exposed to a range of nitrogen and temperature levels. Light‐saturated net photosynthesis rate (A_max) increased with an increase in leaf nitrogen up to 31.2 ± 1.9 μmol m^?2 s^?1 at 25 °C. The A_max initially increased with an increase in leaf temperature (T_L), levelled off at 25–35 °C and decreased when T_L became higher than 35 °C. Based on a C₃ leaf photosynthesis model, we estimated mesophyll conductance (g_m), efficiency of converting incident irradiance into linear electron transport under limiting light (κ_2LL), linear electron transport capacity (J_max), Rubisco carboxylation capacity (V_cmax), triose phosphate utilization capacity (T_p) and day respiration (R_d), using data obtained from gas exchange and chlorophyll fluorescence measurements at different leaf positions and various levels of incident irradiance, CO₂ and O₂. The effects of leaf nitrogen and temperature on photosynthesis parameters were consistent at different leaf positions and among different growth environments except for κ_2LL, which was higher for plants grown in the glasshouse than for those grown outdoors. Model analysis showed that compared with cotton and kenaf, hemp has higher photosynthetic capacity when leaf nitrogen is <2.0 g N m^?2. The high photosynthetic capacity measured in this study, especially at low nitrogen level, provides additional evidence that hemp can be grown as a sustainable bioenergy crop over a wide range of climatic and agronomic conditions.     

Identification and Expression Analysis of Abscisic Acid Signal Transduction Genes in Hemp Seeds

Abscisic acid (ABA) is involved in regulating diverse biological processes, but its signal transduction genes and roles in hemp seed germination are not well known. Here, the ABA signaling pathway members, PYL, PP2C and SnRK2 gene families, were identified from the hemp reference genome, including 7 CsPYL (pyrab-actin resistance1-like, ABA receptor), 8 CsPP2CA (group A protein phosphatase 2c), and 7 CsSnRK2 (sucrose nonfermenting1-related protein kinase 2). The content of ABA in hemp seeds in germination stage is lower than that in non-germination stage. Exogenous ABA (1 or 10 μM) treatment had a significant regulatory effect on the selected PYL, PP2C, SnRK2 gene families. CsAHG3 and CsHAI1 were most significantly affected by exogenous ABA treatment. Yeast two-hybrid experiments were performed to reveal that CsPYL5, CsSnRK2.2, and CsSnRK2.3 could interact with CsPP2CA7 and demonstrate that this interaction was ABA-independent. Our results indicated that CsPYL5, CsSnRK2.2, CsSnRK2.3 and CsPP2CA7 might involve in the ABA signaling transduction pathway of hemp seeds during the hemp seed germination stages. This study suggested that novel genetic views can be brought into investigation of ABA signaling pathway in hemp seeds and lay the foundation for further exploration of the mechanism of hemp seed germination.     

Nitrogen fertilization to optimize the greenhouse gas balance of hemp crops grown for biomass

Nitrogen trials were carried out on hemp crops grown in Ireland over a 3 year period to identify nitrogen fertilization strategies which optimize the greenhouse gas (GHG) and energy balances of hemp crops grown for biomass. Nitrogen rates up to 150 kg N ha^?1 were used in the study. Yield increased with nitrogen rate up to 120 kg N ha^?1 for early (Ferimon), mid (Felina 32) and late maturing (Futura 75) varieties. Variety had a significant effect on yield with yields increasing with maturation date. In 2 years of the study, certain application rates of nitrogen were applied either at sowing, at emergence, after emergence or split between these dates to determine if nitrogen rates could be reduced by delaying or splitting the applications. The application of nitrogen at times later than sowing or in splits during the early part of the growing season had no significant effect on biomass yield compared with the practice of applying nitrogen at the time of sowing. Late applications of nitrogen reduced leaf chlorophyll content and height early in the growing season. Later in the growing season, there was no difference in height between treatments although the highest concentrations of chlorophyll were found in the leaves of the late application treatment. Nitrogen rate and the timing of nitrogen application had no effect on plant density. Biomass yield, net energy and net GHG mitigation increased up to an application rate of 120 kg N ha^?1, this result was independent of soil type or soil nitrogen level. Net GHG and energy balance of hemp crops grown for biomass are optimized if late maturing varieties are used for biomass production and a nitrogen rate of 120 kg ha^?1 is applied at sowing.     

滇南农家大麻品种中大麻素化学型及基因型研究

以1个滇南农家大麻品种群体为研究对象,通过化学分析及同源克隆方法,研究了21个单株中2种主要大麻素——四氢大麻酚(THC)和大麻二酚(CBD)的化学型和基因型,以揭示大麻素含量、化学型以及基因型三者之间的关系,为工业大麻新品种选育提供理论依据。研究表明:(1)化学检测结果显示,21个单株均含有THC,THC含量在0.07%~1.35%之间,其中7个单株仅含THC,5个单株含THC和微量CBD,9个单株同时含有THC和CBD,CBD含量范围为0~0.58%。(2)CBD/THC比值显示,该群体仅存在毒品型和中间型2种化学型,且中间型大麻中THC和CBD含量显著正相关。(3)基因扩增及测序分析结果显示,该群体为基因型杂合群体,群体内THCA合成酶基因存在5个变异位点,CBDA合成酶基因存在2个变异位点,但变异位点和THC及CBD的含量无直接关系。(4)群体内单株的基因型和化学型完全对应,且THCA合成酶基因及CBDA合成酶基因可作为分子标记来鉴定单株化学型。     

A mechanical investigation of the retting process in dew-retted hemp (Cannabis sativa)

Hemp (Cannabis sativa) produces phloem fibres, which are formed in bundles towards the periphery of the stem. This study investigated one aspect of the retting process, the dissociation of these fibres from the core of the stem, which is influenced by the action of micro-organisms. Peel tests were used to investigate the work required to remove a tissue peel, rich in fibres, from the core of the stem. Two separate experiments studied (1) the effects of tissue dehydration as stems were dried out in the oven; and (2) the progress of retting in stems that were dew-retted in the field. A comparison between two different types of peel tests, where the stems were clamped either vertically or horizontally in the jaws of a tensile testing machine and the load applied vertically, showed that the work required to peel stems in the vertical position was over 20% greater than that for the horizontal position. Freshly harvested stems were dehydrated in an oven at 40°C over a period of 42 h and as the moisture content decreased there was a significant increase in the work to peel. In contrast, stems that had been dew-retted in the field showed a significant decrease in work to peel. When the moisture content of stems dried in the oven decreased below 12%, the work to peel rapidly decreased. As the plant tissues dehydrated their fracture characteristics changed and this was reflected in the increase in the work to peel seen in the oven-dried samples. This, however, was not evident in the field-retted samples, in which there was a decrease in work to peel. The field-retted samples were exposed to the activity of micro-organisms for a prolonged period of time compared with the oven-dried samples; this increased the potential degradation caused by microbial enzymes. Thus, the reduced work to peel in dew-retted stems is thought to be due to the colonisation of the stem by micro-organisms and the subsequent retting process. The dramatic decrease in work to peel at low moisture content in the oven-dried stems is thought to result from mechanical changes leading to brittle fracture characteristics. This study indicates that peel tests can be used to objectively monitor the reduction in work to peel for dew-retted stems and relate this reduction to the progress of retting. Thus, peel tests may be used to monitor the effects of different factors on the process of retting in hemp.