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.     

Synthesis of Jatropha curcas oil-based biodiesel in a pulsed loop reactor

Jatropha curcas oil (JCO) has a high content of free fatty acids and has been used extensively as a feedstock in biodiesel production. In the present study, the transesterification reaction of JCO to Jatropha curcas methyl ester (biodiesel) was performed in a continuous pulsed loop reactor under atmospheric conditions. The JCO was pre-treated prior to the reaction to reduce the free fatty acid content to below 1% (w/w). The operating parameters of the loop reactor were optimised based on the conversion of the JCO to Jatropha curcas biodiesel and included reaction temperature, molar ratio of oil to MeOH, reaction time and oscillation frequency. The findings show that the highest reaction conversion of 99.7% (w/w) was achieved using KOH catalyst and 98.8% conversion was obtained using NaOCH₃ catalyst. The optimal operating conditions were a molar ratio of 6:1, an oscillation frequency of 6 Hz, temperature of 60 °C, feedstock FFA content of 0.5% (w/w) and only 10 min of reaction time. As a commercial commodity, the physical properties of biodiesel were analysed, and they compared well with the characteristics of fossil-based diesel fuel.     

Predicting Jatropha curcas seed-oil content, oil composition and protein content using near-infrared spectroscopy—A quick and non-destructive method

The potential of near-infrared reflectance spectroscopy (NIRS) to estimate the oil content, fatty acid composition, and protein content of Jatropha curcas seeds was studied. Seventy-four intact kernels from various sources were scanned by NIRS. All samples were analyzed for oil content (hexane extractions), fatty acid composition (gas chromatography), and protein content (Kjeldahl). Calibration equations were developed for oil content, individual fatty acids (oleic C18:1, linoleic C18:2, stearic C18:0 and palmitic C16:0), and protein content. The performance of the calibration equations was evaluated through external and cross-validation. The results showed that NIRS was a reliable, accurate and nondestructive technique to estimate oil and protein contents, as well as oleic and linoleic fatty acid concentrations in J. curcas kernels; NIRS provides a rapid, simple, and cost-effective alternative method for screening intact J. curcas kernels.     

Value added waste of Jatropha curcas residue: Optimization of protease production in solid state fermentation by Taguchi DOE methodology

The oil extraction of Jatropha curcas created the large amount of the by-product from its seeds. An application of solid-state fermentation (SSF) was considered to be of value to these raw materials. This study investigated the potential of a utilization of deoiled J. curcas seed cake as substrate for protease productions by Aspergillus oryzae. While various parameters for SSF was conventionally individually optimized, five parameters were simultaneously examined based on Taguchi method. The effect of three different levels of five factors, including moisture content of substrate, inoculums size, incubation temperature, type of porous substrate and incubation time were examined. The optimum conditions for the protease production by A. oryzae obtained from this experiment were 45% moisture content of substrate, 10% inoculums size, 30 °C incubation temperature, deoiled J. curcas seed cake mixed with cassava bagasse ratio 4:1 as porous substrate at 84 h of incubation time. By adjusting the conditions to these optimum levels, the protease production increased up to 4.6 times as many as the protease yield from the non-optimizing experiment. The use of statistical approach, Taguchi method, provided a satisfactory outcome in defining the optimum conditions for protease production by A. oryzae. Further, the utilization of deoiled J. curcas seed cake as substrate for SSF was proven as the suitable practice for this agricultural waste, in order to develop for an industrial use.     

Antifungal activity of Vitex agnus-castus extract against Pythium ultimum in tomato

Vitex agnus-castus methanolic extract showed strong antifungal activity against Pythium ultimum in tomato under both in vitro and in vivo conditions. The 0.2% extract delayed the mycelial growth of the fungus and showed significant antifungal activity against P. ultimum on tomato seedlings with an efficacy comparable to that of the synthetic fungicide. To determine the involvement both of plant extract and pathogenic fungus in PR gene induction, tomato plants were treated with V. agnus-castus extract and/or inoculated with P. ultimum. The expression of four PR genes (PR-1, PR-2, PR-5, PR-6) was monitored at five time points within 48 h of the extract treatment and fungal inoculation. The PR-1 and PR-4 genes were activated directly by V. agnus-castus extract up to 12 h after treatments; at 24 h, the direct activation by plant extract disappeared and a synergistic inducing effect of extract and pathogen applied simultaneously on the plant was observed. The PR-6 gene was not activated directly by the V. agnus-castus extract but only when applied together with the pathogen; activation of the PR-6 gene occurred 24 h after treatments and the gene expression increased at 48 h. There was no activation of PR-5 gene by the plant extract. The ability of V. agnus-castus extract to enhance plant defence responses upon pathogen inoculation might be further investigated. The activation of various PR genes suggests that induction of defence responses by V. agnus-castus extract in tomato may be regulated by more than one signalling pathway.     

In vitro regeneration from petiole explants of non-toxic Jatropha curcas

Jatropha curcas, a multipurpose shrub has acquired significant economic potential as biodiesel plant. The seeds or pressed cake is toxic due to the presence of toxic substances and is not useful as food/fodder despite having the best protein composition. A simple, efficient, and reproducible method for plant regeneration through direct organogenesis from petiole explants of non-toxic J. curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (57.61%), and number of shoot buds (4.98) per explant were obtained when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 μM TDZ. The Induced shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM 6-benzyl aminopurine (BA), and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation and subsequent elongation was achieved on MS medium supplemented with 2.25 μM BA and 8.5 μM IAA. The elongated shoots could be rooted on half-strength MS medium with 15 μM IBA, 11.4 μM IAA and 5.5 μM NAA with more than 90% survival rate.     

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.     

Marker gene excision in transgenic Brassica napus via Agrobacterium-mediated Cre/lox transient expression system

Oilseed rape (Brassica napus L.) is one of the most important oil crops in the world. However, study on marker-free transgene of B. napus for bio-safety purpose is limited in this allotetraploid crop. In order to advance marker gene excision research, a newly designed Cre/lox system combining crossing and auto-excision strategy was introduced into B. napus. The system consists of 2 sets of independent vectors including pC35Spro::T7RP carrying T7 RNA polymerase and pCT7pro::Cre carrying T7 promoter respectively. After hybridization of 2 according types of transgenic B. napus, marker gene would be removed as T7 RNA polymerase facilitate T7 promoter to promote Cre gene expression. Totally 52 and 46 positive T₀ transgenic lines of these 2 vectors were obtained after identification by PCR and test trip. T₁ plants from 3 T₀ positive pC35Spro::T7RP lines and 2 T₀ positive pC35Spro::T7RP lines were identified as single copy according to segregation ratio and were chosen for crossing. However, expression of CP4 EPSPS (glyphosate resistance gene) and OXY (bromoxynil resistance gene) were not found in F₁ progeny, which proved that the excision was not complete. The possible reasons for our limited success were investigated and detailed analyses were performed. Although this system is not applicable for generating transgenic B. napus free from selectable marker gene, it provided valuable experience and clue for further improvement of this technique. Many other advantages and further improvement will be progressed in future work.     

Cloning,Expression, and Purification of a GDSL-like Lipase/Acylhydrolase from a Native Lipase-Producing Bacterium,Lactobacillus fermentum

Background: Lipase enzymes are of great importance in various industries. Currently, extensive efforts have been focused on exploring new lipase producer microorganism as well as genetic and protein engineering of available lipases to improve their functional features. Methods: For screening lipase-producing lactobacilli, isolated strains were inoculated onto tributyrin agar plates. Molecular identification of lipase-producing Lactobacilli was performed by sequencing the 16Sr DNA gene, and a phylogenetic tree was constructed. The LAF_RS05195 gene, encoding lipase protein in L. fermentum isolates, was identified using specific primers, amplified by PCR (918 bp) and cloned into the pET28a (+) vector. The recombinant proteins were expressed 2, 4, 6, and 12 hours after induction with IPTG and assessed using the SDS-PAGE. Enzymatic activity of the purified recombinant protein was measured at 410 nm in the presence of ρ-NPA and ρ-NPP. Results:Among five identified native lipase-producing isolates, one isolate showed 98% similarity with Enterococcus species. The other four isolates indicated 98% similarity to L. fermentum. After purification steps with Ni-NTA column, a single protein band of about 34 kDa was detected on SDS- PAGE gel. The enzymatic activity of purified recombinant protein alongside ρ-NPA and ρ-NPP was measured to be 0.6 U/ml and 0.2 U/ml, respectively. Conclusion:In the present research, a novel lipase/esterase from L. fermentum was cloned and expressed. The novel lipase/esterase has the merit to be further studied due to its substrate specificity. Key Words: Escherichia coli, Gene expression, Lactobacillus, Lipase, Phylogeny     

Jatropha curcas seed cake as substrate for production of xylanase and cellulase by Aspergillus niger FGSCA733 in solid-state fermentation

Jatropha curcas seed-cake was evaluated for use as a solid state fermentation substrate for production of cellulolytic and xylanolytic enzymes by Aspergillus niger. Supplementation of the seedcake with 10% thatch grass (Hyperrhaenia sp.) resulted in a fivefold increase in xylanase production. Ammonium chloride supplementation increased production of xylanase by 13%. Under the same conditions, cellulase production was not influenced by supplementation with grass or the nitrogen sources used. Maximum xylanase was produced at 25 °C whilst cellulase was maximally produced at 40 °C. Highest xylanase activity was obtained when the cultures had an initial pH of 3 whereas cellulase was maximally produced at an initial pH of 5. Under optimised conditions, 6087 U and 3974 U of xylanase and cellulase respectively were obtained per gram of substrate. Zymograms of crude enzyme extracts showed six active bands ranging from 20 kDa to 43 kDa for cellulase and a 31 kDa active band for xylanase.     

小桐子ＡＣＯ１基因的克隆与表达分析

     为探讨ACO1基因在小桐子抗逆中的作用,本研究基于小桐子最新注释的基因组数据库,首次克隆了小桐子ACO1基因的全长编码框序列,命名为JcACO1,并对其功能结构域、系统进化、基因结构及器官和低温表达特性进行了分析。结果表明,克隆的JcACO1基因全长为1 022bp,编码319个氨基酸,预测分子量为36.0kDa,等电点5.5。序列比对表明,小桐子JcACO1在序列中部保守性较高,具有1个Pcbc superfamily保守结构域。进化树分析显示,小桐子JcACO1基因与毛果杨、橡胶树、木薯的同源性较高。qRT-PCR表达分析表明,小桐子幼苗JcACO1基因存在器官表达特异性,在叶与茎中表达量较高,而在根中表达量较低。另外,JcACO1基因在三种器官中都受低温诱导,低温胁迫24h时,基因在根与茎中表达量最大。说明JcACO1基因表达量升高,以响应小桐子的抗冷胁迫。     

Assessment of genetic stability in micropropagules of Jatropha curcas genotypes by RAPD and AFLP analysis

Jatropha curcas (Euphorbiaceae), a drought resistant non edible oil yielding plant, has acquired significant importance as an alternative renewable energy source. Low and inconsistent yields found in field plantations prompted for identification of high yielding clones and their large scale multiplication by vegetative propagation to obtain true to type plants. In the current investigation plantlets of J. curcas generated by axillary bud proliferation (micropropagation) using nodal segments obtained from selected high yielding genotypes were assessed for their genetic stability using Randomly Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) analyses. For RAPD analysis, 21 out of 52 arbitrary decamer primers screened gave clear reproducible bands. In the micropropagated plantlets obtained from the 2nd sub-culture, 4 out of a total of 177 bands scored were polymorphic, but in the 8th and 16th sub-cultures (culture cycle) no polymorphisms were detected. AFLP analysis revealed 0.63%, 0% and 0% polymorphism in the 2nd, 8th and 16th generations, respectively. When different genotypes, viz. IC 56557 16, IC 56557 34 and IC 56557 13, were assessed by AFLP, 0%, 0.31% and 0.47% polymorphisms were found, respectively, indicating a difference in genetic stability among the different genotypes. To the best of our knowledge this is the first report on assessment of genetic stability of micropropagated plantlets in J. curcas and suggests that axillary shoot proliferation can safely be used as an efficient micropropagation method for mass propagation of J. curcas.     

Isolation of entomopathogenic Bacillus from a biodynamic olive farm and their pathogenicity to lepidopteran and coleopteran insect pests

The occurrence of Bacillus entomopathogenic bacteria on a Tunisian biodynamic farm was determined by examining 75 samples from olive tree (Olea europaea L.) habitats. A total of 40 Bacillus isolates were characterized according to their phenotypic, physiological and biochemical parameters. Isolates of the species Bacillus subtilis, Bacillus mycoides, Brevibacillus brevis, Paenibacillus polymyxa, Bacillus licheniformis, Bacillus sp. (1), Bacillus sp. (2) and a standard strain Btk HD-1 were used separately in feeding bioassays on fresh artificial diet against larvae of lepidopterans Prays oleae (Bernard) and Palpita unionalis (Hübner) and coleopterans Hylesinus oleiperda (F.) and Phloeotribus scarabaeoides (Bernard), which are olive tree pests. Larvae were successfully reared on an artificial diet with 25 g powdered olive tree leaves. Compared to the control data, only Btk and the isolates of B. licheniformis, P. polymyxa and B. brevis were entomopathogenic. Larval mortality assessed 7 days post-treatment showed high mortality rates with Btk to lepidopteran larvae (86.6% for P. oleae and 80.9% for P. unionalis) and low mortality against coleopteran pests. B. brevis isolates showed high mortality rates against P. oleae (up to 67.9%). B. licheniformis isolates caused up to 59.2% larval mortality for P. oleae and 43.6% for P. unionalis. Highest coleopteran mortality was achieved by P. polymyxa isolates (up to 55%). According to the 16S rDNA results, isolates of each of the three entomopathogenic strains were similar. Proteins in the strain supernatants were toxic to P. oleae larvae with LC50 values of 10.0 (B. brevis), 12.5 (B. licheniformis) and 37.6 μg/ml (P. polymyxa). Also, P. polymyxa showed an LC50 of 12.4 mg/l against P. scarabaeoides. Our results suggest that entomopathogenic Bacillus present locally in the biodynamic farm could be used in biological control programmes of olive tree pests.     

Antifungal activity of prenylated flavonoids isolated from Tephrosia apollinea L. against four phytopathogenic fungi

Four prenylated flavonoids, isoglabratephrin, (+)-glabratephrin, tephroapollin-F and lanceolatin-A were isolated from Tephrosia apollinea L. growing in Egypt. The structures of compounds have been elucidated using physical and spectroscopic methods including (UV, IR, ¹H NMR, ¹³C NMR, DEPT, 2D ¹H–¹H COSY, HSQC, HMBC and NOESY). The isolated flavonoids showed considerable antifungal activity against four phytopathogenic fungi, namely Alternaria alternata, Helminthosporium sp., Colletotrichum acutatum and Pestalotiopsis sp. in a dose-dependent manner using the agar well-diffusion bioassay. They differ significantly in their activity with tephroapollin-F was the most effective. In a test using a concentration of 4 mg/ml of tephroapollin-F, strong fungicidal activities (32.8–58.3%) were produced against the test fungi, where C. acutatum, Helminthosporium sp. and Pestalotiopsis sp. showed greater susceptibility, while A. alternata was the least susceptible. Using the same concentration, the two flavonoids isoglabratephrin and (+)-glabratephrin showed moderate activities with % inhibition of fungal growth were ranged between (16.1–37.8) against A. alternata, Helminthosporium sp. and Pestalotiopsis sp., while showed a strong antifungal activity against C. acutatum (% growth inhibition were 46.4 and 42.9, respectively). In all treatments, the flavonoid lanceolatin-A exhibited weak to moderate activities. Using lower concentrations of the test flavonoids (2 and 1 mg/ml), weak to moderate antifungal activities were observed against all of the test fungal strains. In all cases and regardless of the flavonoid tested, C. acutatum was the most susceptible, while A. alternata was the least. The study recommends the use of the test compounds as rational fungicides of natural origin.     

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.     

小麦 BES1基因家族的比较基因组学分析

BES1基因是植物体内一类重要的转录因子。本研究利用基因组测序数据,在小麦基因组中鉴定到20个 BES1基因家族成员,它们分布在14条染色体上。为进一步研究 BES1基因在小麦以及其他禾本科作物（水稻、玉米、高梁、谷子和大麦）中的功能和进化关系,对小麦等6种禾本科作物以及十字花科模式植物拟南芥的 BES1基因家族进行系统发育关系、结构特性和共线性关系分析,结果发现,系统发育分析将该家族成员分为Group A、Group B、Group C和Group D四类,大部分小麦 BES1基因集中在Group A;大部分小麦 BES1基因家族成员有2个外显子,最多有10个外显子;小麦与水稻 BES1基因之间存在更多的共线性关系。此外,在小麦根和穗中还鉴定到3个具有较高表达水平的 BES1基因（ TaBES1-3A-2、 TaBES1-3B-2和 TaBES1-3D-2）,表明 BES1基因在小麦生长发育过程中发挥着重要作用。     

Breeding dominant genic male sterility restorer line of Brassica napus L.

To facilate breeding process of Brassica napus, a microspore culture and molecular marker-assisted screening combined system were proposed in this research. At early flowering stage, F₁ offspring of hybridized combination HY15A ​× ​HF06 was used as donor for microspore culture to analyze effects of colchicine concentration on embryogenic and diploid rates of microspore. Treatment with 50 ​mg/L colchicine resulted in embryogenic rate of 3.56 embryos/bud, which was substantially higher than control (0.78 embryos/bud). A total of 1,387 embryos and 862 single plants were obtained after induction culture. Ploidy detection was performed for the regenerated plants by flow cytometry. Diploid rates of microspores treated with 50 ​mg/L and 70 ​mg/L colchicine were 17.2% and 21.0% respectively, which was significantly higher than control (10.5%). Totally 108 single plants that doubled successfully were randomly selected and screened using molecular marker BE10. Approximately 54 of 108 plants generated a 305 bp amplification product, whereas the other 54 plants showed a 398 bp band, thereby satisfying 1:1 separation ratio (x²_0.05 ​= ​0.0093). These coincided with field identification results. Findings of this study indicated that homozygous breeding material could be obtained by microspore culture in a short time, thereby remarkably accelerate breeding.     

Biocontrol of rice blast by the phenaminomethylacetic acid producer of Bacillus methylotrophicus strain BC79

Strain BC79, isolated from primeval forest soil in Qinling, Mountains, China, was identified as Bacillus methylotrophicus based on morphological, biochemical, physiological and chemotaxonomic analyses as well as phylogenetic 16S rDNA sequencing data. This strain was able to suppress mycelial growth and conidial germination of numerous plant pathogenic fungi in dual cultures on solid media. For exploring potential biocontrol activity, we assessed fermentation conditions for studying B. meth1ylotrophicus BC79. The active substance of BC79, phenaminomethylacetic acid, was extracted by TLC and HPLC, and identified as the strongest inhibitory substance described in B. methylotrophicus. Experiments in a greenhouse showed that application of BC79 culture filtrates 24 h before inoculation of Magnaporthe oryzae, the causal agent of rice blast, had 89.87% biocontrol efficiency. B. methylotrophicus BC79 colonized rice plant tissues and at 10 days after filtrate application, its population in leaves (1.65 × 10⁸ CFU/g) was much larger than in stems (6.78 × 10⁷ CFU/g) or roots (3.56 × 10⁷ CFU/g). Field trials indicated that BC79 culture filtrate (4000 g/667 m²) showed the highest efficiency for M. oryzae, with 84.8% biocontrol effect, followed by of 15% phenaminomethylacetic acid extract (75.5%) and 20% tricyclazole (76.1%). Seedling and post-transplant stages were the best periods to apply BC79 for control of rice blast. The B. methylotrophicus BC79 strain hence has enormous potential as an agricultural agent for biocontrol of rice blast.     

Effects of partial rootzone drying on yield,yield components,and irrigation water use efficiency of canola (<Emphasis Type="Italic">Brassica</Emphasis>?<Emphasis Type="Italic">napus</Emphasis> L.)

Effect of PRD (partial rootzone drying) on yield and yield components of canola (Brassica napus L.) was investigated in greenhouse conditions. The treatments were: T₁, full watering of both sides of roots; T₂, alternate irrigation on both sides; T₃, half of irrigation water in T₁ was given to one side; T₄, same as T₃ but without plate; T₅, same as T₂ but without plate. In T₁, T₂, and T₃ treatments, the boxes were evenly separated into two compartments with thin plates. The results showed that grain yield of T₁ to T₅ treatments was 18.11, 16.38, 12.44, 9.29, and 8.66 g plant⁻¹. T₂ treatment increased plant height by 46.9% and 1000-seed weight by 17.8%, but reduced lateral branches by 16.7% and number of pods by 24%, over T₁ treatment. T₂ treatment was the most efficient (irrigation water use efficiency = 0.679 kg m⁻³) and treatment T₅ was the least efficient (0.359 kg m⁻³). The difference between irrigation water use efficiency (IWUE) of T₂ and T₅, and T₃ and T₄ treatments, was significant (p < 0.05). Therefore, halving the amount of applied irrigation water and applying this water alternatively on both sides of the root zone will produce the highest IWUE. This study showed that PRD irrigation management has high influence on rooting system of canola. This phenomenon could affect nutrients uptake and consequently all aspects of plant growth and development.     

Characterization of fungicide resistant isolates of Phaeoacremonium aleophilum infecting grapevines in Spain

Fungicide resistance in Phaeoacremonium aleophilum, one of the most frequent fungal pathogens associated with grapevine trunk diseases, was investigated and found to exist in some isolates of the pathogen against a commercial formulation, Escudo^®. The effect of this compound and its two active substances, carbendazim and flusilazole, was first evaluated on the mycelial growth of P. aleophilum. Escudo^®-resistant isolates were estimated at a frequency of 24% in Spanish vineyards. Then, the two active substances were used individually to test their effect on mycelia growth of twelve single-spore cultures originating from six Escudo^®-resistant isolates. Flusilazole (DMI-triazole) did not inhibit mycelia growth of any single-spore cultures of P. aleophilum. Carbendazim (benzimidazole) used alone allowed the growth of the same single-spore cultures that were also resistant to Escudo^®. AFLP characterization of sensitive and resistant single-spore cultures showed genetic diversity within P. aleophilum isolates but no AFLP markers were associated with resistance. New primers set (L2/R1) were designed to partially amplify the exon 6 of the beta-tubulin gene of P. aleophilum. Two different point mutations resulted in glycine (GGC) or lysine (AAA) replacing the glutamic acid (GAG) at codon 198 of the beta-tubulin gene in some of the resistant single-spore cultures studied. Resistant single-spore cultures of P. aleophilum were shown to have different aggressiveness levels as sensitive single-spore cultures by inoculation of wood segments of Vitis vinifera in the presence and absence of fungicide.