Seed treatment technology: An attractive delivery system for controlling root parasitic weed Striga with mycoherbicide

Coating sorghum seeds with Fusarium oxysporum (Foxy 2) for control of the root parasitic weed Striga, appears to be an attractive option for minimizing the inoculum amount, establishing the biocontrol agent in the potential infection zone of the host plants, and offering a simple, easy and economical delivery system. Our investigations resulted in the selection of appropriate seed coating materials and a suitable type and form of fungal inoculum. The coating materials tested were arabic gum (AG10%, 20%, 40%), carboxymethylcellulose (CMC1%, 2%) and pectin (LS 440, LM-5 CS) 1%, while the fungal inoculum included microconidia and fresh and dried chlamydospores produced using different substrates. Foxy 2 survived the seed treatment processing and showed excellent viability on seeds for at least 8 months of storage after coating. In general, the performance of 40% arabic gum in combination with dried chlamydospores was the best among the other types of inoculum and coating material tested. Regardless of the type and form of inoculum and coating materials tested, Foxy 2 was able to colonize all roots, even root tips and hairs of the host (sorghum), thereby meeting important criteria of a promising candidate for controlling Striga when applied as a seed treatment. The efficacy of treated sorghum seed with Foxy 2 using different coating materials in reducing S. hermonthica infestation was evaluated in pot and root chamber trials. Foxy 2 markedly reduced Striga emergence and dry weight and increased the percentage of the diseased emerged Striga shoots. However, the efficacy of seed coating varied according to the type and form of fungal inoculum as well as coating material. Coating sorghum seed with dried chlamydospore inoculum homogenized into 20% arabic gum (as adhesive) showed the highest efficacy of 81 and 77% (i.e., percent reduction in healthy emerged Striga shoots compared to the control treatment) against Striga using either sterilized or non-sterilized soil, respectively. In root chamber bioassays, the application of Foxy 2 in combination with AG40% significantly caused disease in 77% of the germinated Striga seeds and in all tubercles after 25 days of sowing. These findings provide an optimized coating protocol as an attractive delivery system for bioherbicides for root parasitic weeds.     

Resolution of the structural isomers of partially methylesterified oligogalacturonides by polysaccharide analysis using carbohydrate gel electrophoresis

Pectins differing in their degree and pattern of methylesterification are important in diverse aspects of plant physiology and also in many industrial applications. Determination of methylesterification fine structure and knowledge of enzyme specificities in modification and fragmentation of pectin are key to understanding the relationship between structure and function. The development of methodologies for the detection, separation and sequencing of different partially methylesterified oligogalacturonides (Me-OGAs) is consequently very important. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) has been shown to be powerful for the quantitative resolution of species different in degree of polymerization (DP) and/or degree of methylesterification (DM). Mass spectrometry (MS) has, to date, been the only tool with which to obtain isomeric information. However, it is not quantitative, and the presence of isobaric species makes the interpretation of the fragmentation patterns complicated. Here, we present evidence that Me-OGAs with the same DP and DM but different patterns of methylesterification (structural isomers) can easily be separated and quantified using PACE.     

Jasmonate‐dependent modifications of the pectin matrix during potato development function as a defense mechanism targeted by Dickeya dadantii virulence factors

The plant cell wall constitutes an essential protection barrier against pathogen attack. In addition, cell‐wall disruption leads to accumulation of jasmonates (JAs), which are key signaling molecules for activation of plant inducible defense responses. However, whether JAs in return modulate the cell‐wall composition to reinforce this defensive barrier remains unknown. The enzyme 13–allene oxide synthase (13–AOS) catalyzes the first committed step towards biosynthesis of JAs. In potato (Solanum tuberosum), there are two putative St13–AOS genes, which we show here to be differentially induced upon wounding. We also determine that both genes complement an Arabidopsis aos null mutant, indicating that they encode functional 13–AOS enzymes. Indeed, transgenic potato plants lacking both St13–AOS genes (CoAOS1/2 lines) exhibited a significant reduction of JAs, a concomitant decrease in wound‐responsive gene activation, and an increased severity of soft rot disease symptoms caused by Dickeya dadantii. Intriguingly, a hypovirulent D. dadantii pel strain lacking the five major pectate lyases, which causes limited tissue maceration on wild‐type plants, regained infectivity in CoAOS1/2 plants. In line with this, we found differences in pectin methyl esterase activity and cell‐wall pectin composition between wild‐type and CoAOS1/2 plants. Importantly, wild‐type plants had pectins with a lower degree of methyl esterification, which are the substrates of the pectate lyases mutated in the pel strain. These results suggest that, during development of potato plants, JAs mediate modification of the pectin matrix to form a defensive barrier that is counteracted by pectinolytic virulence factors from D. dadantii.     

Boron bridging of rhamnogalacturonan‐II,monitored by gel electrophoresis,occurs during polysaccharide synthesis and secretion but not post‐secretion

The cell‐wall pectic domain rhamnogalacturonan‐II (RG‐II) is cross‐linked via borate diester bridges, which influence the expansion, thickness and porosity of the wall. Previously, little was known about the mechanism or subcellular site of this cross‐linking. Using polyacrylamide gel electrophoresis (PAGE) to separate monomeric from dimeric (boron‐bridged) RG‐II, we confirmed that Pb²⁺ promotes H₃BO₃‐dependent dimerisation in vitro. H₃BO₃ concentrations as high as 50 mm did not prevent cross‐linking. For in‐vivo experiments, we successfully cultured ‘Paul's Scarlet’ rose (Rosa sp.) cells in boron‐free medium: their wall‐bound pectin contained monomeric RG‐II domains but no detectable dimers. Thus pectins containing RG‐II domains can be held in the wall other than via boron bridges. Re‐addition of H₃BO₃ to 3.3 μm triggered a gradual appearance of RG‐II dimer over 24 h but without detectable loss of existing monomers, suggesting that only newly synthesised RG‐II was amenable to boron bridging. In agreement with this, Rosa cultures whose polysaccharide biosynthetic machinery had been compromised (by carbon starvation, respiratory inhibitors, anaerobiosis, freezing or boiling) lost the ability to generate RG‐II dimers. We conclude that RG‐II normally becomes boron‐bridged during synthesis or secretion but not post‐secretion. Supporting this conclusion, exogenous [³H]RG‐II was neither dimerised in the medium nor cross‐linked to existing wall‐associated RG‐II domains when added to Rosa cultures. In conclusion, in cultured Rosa cells RG‐II domains have a brief window of opportunity for boron‐bridging intraprotoplasmically or during secretion, but secretion into the apoplast is a point of no return beyond which additional boron‐bridging does not readily occur.     

Arabidopsis PECTIN METHYLESTERASE17 is co-expressed with and processed by SBT3.5, a subtilisin-like serine protease

Background and Aims

In Arabidopsis thaliana, the degree of methylesterification (DM) of homogalacturonans (HGs), the main pectic constituent of the cell wall, can be modified by pectin methylesterases (PMEs). In all organisms, two types of protein structure have been reported for PMEs: group 1 and group 2. In group 2 PMEs, the active part (PME domain, Pfam01095) is preceded by an N-terminal extension (PRO part), which shows similarities to PME inhibitors (PMEI domain, Pfam04043). This PRO part mediates retention of unprocessed group 2 PMEs in the Golgi apparatus, thus regulating PME activity through a post-translational mechanism. This study investigated the roles of a subtilisin-type serine protease (SBT) in the processing of a PME isoform.

Methods

Using a combination of functional genomics, biochemistry and proteomic approaches, the role of a specific SBT in the processing of a group 2 PME was assessed together with its consequences for plant development.

Key Results

A group 2 PME, AtPME17 (At2g45220), was identified, which was highly co-expressed, both spatially and temporally, with AtSBT3.5 (At1g32940), a subtilisin-type serine protease (subtilase, SBT), during root development. PME activity was modified in roots of knockout mutants for both proteins with consequent effects on root growth. This suggested a role for SBT3.5 in the processing of PME17 in planta. Using transient expression in Nicotiana benthamiana, it was indeed shown that SBT3.5 can process PME17 at a specific single processing motif, releasing a mature isoform in the apoplasm.

Conclusions

By revealing the potential role of SBT3.5 in the processing of PME17, this study brings new evidence of the complexity of the regulation of PMEs in plants, and highlights the need for identifying specific PME–SBT pairs.     

一株具有脱胶功能大麻内生真菌的分离和鉴定

采用组织块法对大麻(Cannabis sativa)根、茎、叶等组织中的内生真菌进行分离,利用平板透明圈法筛选具有脱胶功能的菌株,对获得的脱胶菌菌株进行形态学鉴定和分子生物学鉴定。结果表明:(1)从大麻根、茎、叶的组织部位共分离得到内生真菌16株,茎部分离到9株真菌,叶部5株,根部2株。(2)编号为DM6的内生真菌具有较强的果胶分解能力,其透明圈直径为2.49cm。(3)形态学鉴定发现,内生真菌DM6不能产生孢子,菌丝较为粗壮、分支较少、有明显的隔;分子学鉴定表明,内生真菌DM6与Phoma aliena(KC311486)序列的相似性最高,为99%,并且在系统发育树上位于同一分支上。因而内生真菌DM6可以鉴定为茎点霉属一种(Phomasp.)。     

Cell Wall,Cytoskeleton, and Cell Expansion in Higher Plants

To accommodate two seemingly contradictory biological roles in plant physiology, providing both the rigid structural support of plant cells and the adjustable elasticity needed for cell expansion, the composition of the plant cell wall has evolved to become an intricate network of cellulosic, hemicellulosic, and pectic polysaccharides and protein. Due to its complexity, many aspects of the cell wall influence plant cell expansion, and many new and insightful observations and technologies are forthcoming. The biosynthesis of cell wall polymers and the roles of the variety of proteins involved in polysaccharide synthesis continue to be characterized. The interactions within the cell wall polymer network and the modification of these interactions provide insight into how the plant cell wall provides its dual function. The complex cell wall architecture is controlled and organized in part by the dynamic intracellular cytoskeleton and by diverse trafficking pathways of the cell wall polymers and cell wall-related machinery. Meanwhile, the cell wall is continually influenced by hormonal and integrity sensing stimuli that are perceived by the cell. These many processes cooperate to construct, maintain, and manipulate the intricate plant cell wall--an essential structure for the sustaining of the plant stature, growth, and life.     

Mucilage secretion: an adaptive mechanism to reduce seed removal by soil erosion?

Diaspores of many plant species inhabiting open vegetation in semi‐arid environments secrete mucilage after wetting (myxospermy) that glues the diaspores to the ground and prevents movement when the mucilage dries. In the present study, we test whether mucilage secretion can be considered as a selective response to soil erosion in plant species inhabiting semi‐arid environments. We relate the amount and type of mucilage secretion by seeds of Helianthemum violaceum and Fumana ericifolia (Cistaceae) to the number of raindrop impacts needed to remove these seeds after gluing them with their own mucilage to the ground and also the time that these seeds resist water run‐off without detaching. We also compare the amount of seed mucilage production by plants growing in habitats without erosion and plants affected by severe erosion by fitting mixed effect models. Our results show an important phenotypic variation in the amount of mucilage secretion in both species, although it is suggested that the effect of mucilage secretion in the rate of seed removal by erosion is species‐ and mechanism‐dependent. For F. ericifolia, the amount of mucilage secreted by the seeds is directly proportional to their resistance to raindrop impacts and is positively related to the intensity of the erosive processes that the plants experience. Nevertheless, all the seeds resist the force of run‐off during 60 min, irrespective of the amount of mucilage they produce. In H. violaceum, mucilage secretion per se, and not the amount of mucilage produced by the seeds, has an effect on the rate of seed removal by erosive processes. Furthermore, cellulosic fibrils were found only in the mucilage of F. ericifolia but not in H. violaceum. Overall, our results only partially support the hypothesis that a selective response to soil erosion exists. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 241–251.     

Growth and protopectinase production of Geotrichum klebahnii in batch and continuous cultures with synthetic media

Geotrichum klebahnii ATCC 42397 produces a protopectinase (PPase-SE) with polygalacturonase (PGase) activity. The microorganism was aerobically cultivated in synthetic media. Glucose, fructose and xylose yielded the highest enzyme levels (10–11 PGase units ml⁻¹). Galacturonic acid repressed enzyme production and no growth was obtained with disaccharides and pectin. Specific enzyme activity obtained in an O₂-limited culture was similar to that found in nonlimited ones. A growth yield (Y _x/s) of 0.49 g of cell dry weight per gram of glucose consumed was obtained in a typical batch bioreactor culture. Enzyme production was growth associated, and no major products other than biomass and CO₂ were detected. The volumetric enzyme activity reached a maximum around D=0.3–0.4 h⁻¹ in glucose-limited continuous cultures. However, it varied strongly (together with microorganism morphology) even after retention times ≥8 at any D tested (0.035–0.44 h⁻¹) though the rest of the culture variables remained fairly constant. No correlation between morphology and enzyme activity could be obtained. Enzyme production was poor in urea- and vitamin-limited continuous cultures. In all cases, biomass and CO₂ accounted for ≅100% of carbon recovery though Y _x/s values were different. Journal of Industrial Microbiology & Biotechnology (2000) 25, 260–265. Received 20 April 2000/ Accepted in revised form 15 September 2000     

枇杷冷藏过程中果肉木质化与细胞壁物质变化的关系

“大红袍”和“解放钟”枇杷果实在 1℃下贮藏时 ,细胞壁物质代谢异常 ,果肉硬度持续升高而出汁率逐渐降低 ,果胶酯酶 (PE)和多聚半乳糖醛酸酶 (PG)活性和水溶性果胶含量下降 ,原果胶含量、苯丙氨酸解氨酶(PAL)活性及木质素和纤维素含量不断增加。约经 3周贮藏后 ,果实出现果皮难剥、果肉质地变硬、粗糙少汁的异常劣变现象。在 12℃下贮藏的枇杷果实 ,细胞壁物质代谢正常 ,果肉硬度增加少 ,PE和PG活性及水溶性果胶含量较高 ,无原果胶增加现象 ,PAL活性呈下降趋势 ,木质素和纤维素含量变化不大 ,果实不出现木质化败坏。这些结果表明冷藏枇杷的木质化败坏可能是一种低温失调现象