Introduction. MRI is established modality for the diagnosis of ovarian malignancies. Advances in MRI technology, including DW imaging, could lead to the further increase in the sensitivity of MRI for the detection of peritoneal metastases. The aim of this study was to assess the accuracy of DW imaging for detection of peritoneal metastatic disease in patients suspected of having potentially early ovarian cancer and secondly to evaluate ADC values of peritoneal implants. Materials and Methods. The prospective study group consisted of 26 women with sonographic or/and CT diagnosis of suspected ovarian tumor. Based on the results of the above imaging, in none of them was extraovarian spread of disease or ascites recognized. All patients underwent MRI with DW imaging. Results. Overall, 18 extraovarian peritoneal lesions were found on DW images in 10 from 26 examined patients. All implants had diameter ≤10 mm. The presence of all lesions diagnosed by MRI was confirmed intraoperatively. Histopathologic findings in 17 proofs confirmed ovarian cancer. PPV was 94%. On all DW images  the mean signal intensities of peritoneal lesions were significantly higher than the mean signal intensities of normal adjacent tissue (p = 0.000001). About 3300 new cases of ovarian cancer are annually diagnosed in Poland of which 70% are advanced (stages III and IV) . CurrentThe treatment of patients with advanced-stage ovarian cancer is based on debulking surgery and adjuvant chemotherapy. The surgeon aims at achieving a maximal possible cytoreduction . NumerouWhen the disease has spread intra-abdominally, complete surgical tumor debulking is increasingly difficult. In patients with massive peritoneal spread, the method of choice is neoadjuvant chemotherapy, followed by interval surgery . The int b values) and various methods of calculation of apparent diffusion coefficient (ADC).Magnetic resonance imaging (MRI) is established modality for the diagnosis of ovarian malignancies. This technique may be also utilized to determine the extension of disease, including detection of peritoneal metastatic disease in these patients. Advances in MRI technology, including improvement of DW imaging technique, could lead to the further increase in the sensitivity of MRI for the detection of peritoneal metastases. Studies have recently investigated the efficacy of DW images to detect peritoneal implants in different pelvic and abdominal malignancies . The resThe aim of this study was twofold: firstly, to assess the accuracy of DW imaging for detection of peritoneal metastatic disease in patients suspected of having potentially early ovarian cancer and secondly to evaluate ADC values of peritoneal implants.The prospective study group consisted of 26 women aged from 34 to 67 years with transvaginal and transabdominal sonographic diagnosis of suspected ovarian tumor. All patients had elevated serum levels of CA 125 or CA 19-9. In eight patients additional computed tomography (CT) of abdomen and pelvis was performed. Based on the results of the above imaging, in none of them was extraovarian spread of disease or ascites recognized.All patients underwent MR imaging of the abdomen and pelvis at our institution. MR imaging examinations were performed in all 26 patients using a 1.5 T clinical whole-body MR system  with the Spine Matrix coil and combined two Body Matrix coils for larger coverage.MRI protocol for the detection of the abdominal and pelvic lesions contained turbo spin-echo (tse) T2-weighted images, fat-suppressed T2-weighted, T2-TIRM, DW EPI, and pre- and postcontrast dynamic 3D T1 GRE in transverse orientation. The details of the applied parameters of MR imaging are presented in 2.Axial DWI images were acquired using the same multislice EPI sequence for all patients: 34 × 6 mm slices  and 30 × 6 mm slices (pelvic part); 380 × 380 mm FoV; 128 × 96 matrix; TR = 3800 ms; TE = 73 ms; with diffusion weightings of 0, 50, 100, 150, 200, 400, 800, and 1200 s/mmIn all patients Gadobutrol  was administered, at a dose of 0.1 mL/kg bodyweight, immediately followed by a bolus of 20 mL of physiological saline (NaCl 0.9%).Regions of interest were outlined in Multimodality Workplace Station  by a genitourinary radiologist (with experience in pelvic MR imaging), who documented the number and location of peritoneal metastases on DW images and ADC maps. The number and location of metastatic peritoneal implants were confirmed intraoperatively and compared with MRI findings.b values DWI images by using the T2-weighted images for guidance. ROI included largest possible part of the lesion, avoiding partial volume effect, areas of necrosis, and artifacts. Then ROI was copied and pasted from DWI image to corresponding ADC map and the measurement on ADC map was recorded. ADC was measured twice for each lesion and these measurements were averaged. Separate ADC measurements were performed for adjacent, normal tissues .Subsequently, freehand regions of interest (ROI) were drawn on the ADC and all S = S0  ·  exp(−b  ·  ADC), where S is the signal intensity after application of the diffusion gradient and S0 is the signal intensity at b = 0 s/mm2. Eight b values were applied for ADC calculation.ADC values were calculated by monoexponential regression with the following formula: The number and location of metastatic peritoneal implants were confirmed intraoperatively and compared with MRI findings.The reference standard for the diagnosis was histopathologic proof obtained intraoperatively.b values images  between peritoneal implants and normal surrounding tissue were analyzed using an unpaired t-test; ROC curve analysis p < 0.05 indicated a statistically significant difference.All statistical analyses were performed using the STATISTICA ver.12 (Statsoft) software package. Comparisons of mean ADC and mean Overall, 18 extraovarian peritoneal lesions were found on DW images in 10 from 26 examined patients. The location of these lesions is presented in  b values of 0, 50, 100, 150, 200, 400, 800, and 1200 s/mm2) the mean signal intensities of peritoneal lesions were significantly higher than the mean signal intensities of normal adjacent tissue (p = 0.000001). Mean ADC values of peritoneal lesions were significantly lower than those of adjacent tissues (p = 0.0005)  and 85% (specificity) for ADC to 100% (sensitivity) and 100% (specificity) for ectively .Magnetic resonance imaging is regarded as an accurate technique for the detection and characterization of peritoneal spread of abdominal and pelvic malignancies. Advances in MRI technology, such as introduction of high-performance gradient systems, parallel imaging, or increased field homogeneity, resulted in improvement of quality of several MR techniques, including diffusion-weighted imaging. According to published reports this method could be also implemented for the detection of metastatic peritoneal disease in patients with malignant ovarian lesions .At present, CT is the method of choice in preoperative evaluation of ovarian cancer and has been proved an accurate technique for predicting the results of cytoreduction in bulky disease . In casep = 0.006) and ovarian mass (p = 0.015) [Sala et al. in 2012 described the group of 22 ovarian cancer patients in which the men ADC for peritoneal metastases was lower than that of omental (= 0.015) . b values  and the method for ADC calculation  have important implication for calculated ADC values of analyzed lesions. On the expense of prolonged imaging time, we implemented 8 b values  aiming to obtain more reliable ADC values than with the use of less b values. However, alternatively, we cannot exclude that monoexponential model applied in this study for ADC calculation could be less accurate than biexponential model [DW imaging, especially when quantitative analysis is performed, has several limitations. The acquisition as well as analysis of DW images is not standardized. The choice ofal model .One of the important limitations of this study is small number of patients. Therefore our results have to be regarded as preliminary in terms of sensitivity and specificity of DW imaging for the detection of small peritoneal implants and should be confirmed on larger group of patients.The results of this preliminary study confirmed that implementation of DW imaging has potential for the detection of small peritoneal metastatic implants, which is very important especially in patients with potentially early malignant ovarian masses. We presume that implementation of DW imaging of the abdomen and pelvis may provide important, supplementary information regarding extension of neoplastic disease and influence patient's treatment.


Vitis vinifera L. cv. Cabernet Franc, Shiraz, and Cabernet Sauvignon in the Sunraysia region of Victoria, Australia. Lighter pruning did not change the total carbohydrates concentration and composition in wood and roots within seasons in Cabernet Franc and Shiraz. However, the total carbohydrate pool (starch and soluble sugars) at the end of dormancy increased under lighter pruning, due to higher vine size, associated with retention and growth of old-wood (trunk and cordons). Water deficit negatively impacted trunk and leaf starch concentrations, over the day and within seasons in Cabernet Sauvignon. Soluble sugars concentrations in these tissues tended to be higher under limited water supply, possibly due to higher sugar mobilization as photosynthesis decreased. Trunk carbohydrate concentrations markedly varied within and between seasons, highlighting the importance of interactive factors such as crop load and climate on carbon status. The period between fruit-set and véraison was shown to be critical for its impact on the balance between carbon accretion and depletion, especially under water deficit. The lower leaf and trunk starch concentration under water deficit resulted in a decrease of yield components at harvest, while similar yields were reached for all pruning systems. The sugar allocated to berries at harvest remained remarkably stable for all practices and seasons, irrespective of vine yield and carbohydrate status in vegetative organs in Shiraz and Cabernet Sauvignon.Light pruning and deficit irrigation regimes are practices which are widely used in high yielding commercial vineyards in the warm climate regions of Australia. Little information is available on their impacts on carbohydrate dynamics in vegetative organs within and between seasons, and on the resulting plant capacity to maintain productivity and ripen fruits. This study was conducted to address this gap in knowledge over five vintages on  In high yielding vineyards grown in warm regions of Australia, there has been widespread adoption of lighter pruning techniques such as mechanical hedging and minimal pruning, together with deficit irrigation techniques to enhance red wine quality  reduced berry set and berry mass  is the most common deficit irrigation strategy implemented in warm climate grape-growing regions. This practice is aimed at optimizing the balance between grapevine vigor and potential production. The reduction of irrigation amount after berry set in RDI slows canopy development and decreases berry size  the effect of pruning regimes  on vine size parameters, partitioning of soluble sugar and starch in key vegetative tissues at winter dormancy by Cabernet Franc and on trunk starch concentrations during and between seasons in Shiraz, (b) the effect of irrigation treatment  on within-day, within-season and between-season changes in key physiological parameters and tissue carbohydrate concentrations and composition in Cabernet Sauvignon, and (c) vine capacity to maintain productivity and adequately ripen the fruits under the different pruning or irrigation treatments.Three experiments were conducted in commercial vineyards in the Sunraysia region of Victoria, Australia, to address the effects of pruning (Experiment 1 and 2) or water supply (Experiment 3) on vine carbohydrate status, and on yield components and berry sugar content.Experiment 1 (1986–87) was carried out at the CSIRO Division of Plant Industry Sunraysia site . Cabernet Franc, clone AC.72.8186, vines were planted in 1972 at a row and vine spacing of 3.0 × 2.4 m in two parallel rows and trained on a 1.3-m high, 0.4-m T-trellis. The soil was a gradational reddish, calcareous, mottled sandy clay loam. Irrigation by overhead sprays was over 9 ML/ha per annum . They were planted in 1994 with Shiraz (12 ha) grafted onto Schwarzmann (Experiment 2), and with own-rooted Cabernet Sauvignon (12 ha) (Experiment 3), at a density of 1366 vines per ha (3 m between row and 2.44 m within row). The soil was a Nookamka sandy loam  treatment was applied throughout the season to meet 100% of estimated ETc. On the Cabernet Sauvignon site (Experiment 3), STD was compared with a Prolonged Deficit (PD) treatment. The STD and PD irrigation treatments were allocated randomly to plots in a block design with 12 replicates. A more detailed description of the irrigation treatments design is provided by Cooley et al.  for Experiment 2 2004–2006)  and Expey et al. . The PD y et al. . It (PD)In Experiment 1, the size and the carbohydrate status of Cabernet Franc vines, managed under different pruning regimes, were determined at the end of winter dormancy, prior to budburst  and Cabernet Sauvignon (Experiment 3) at winter dormancy pre-budburst, fruit set, véraison, berry ripeness/harvest, and leaf fall. In addition, trunk reducing sugars and sucrose concentrations were assayed at all stages in Experiment 3. Trunk samples were taken approximately mid-way between the graft union and the training wire, using an increment borer. They were collected on six replicate vines for spur and mechanical pruning and four replicate vines for minimal pruning in Experiment 2, and on 12 replicates vines for each water treatment in Experiment 3  were assessed, using a CIRAS-1 Photosynthesis System  on basal leaves opposite to the bunches of 12 plants per irrigation treatment on 1 day in 2003 , during the extreme deficit period of the PD treatment. Water potential measurements (Ψl) were measured on the same leaves with a pressure chamber . These leaves were then snap frozen in liquid nitrogen, and stored at −70°C until they were assayed for starch concentrations. Leaf samples were ground in liquid nitrogen using a mortar and pestle. Starch analysis was conducted using the Megazyme starch analysis kit, as above for trunk samples , daily changes in photosynthesis (An), stomatal conductance (gs) and leaf temperature  concentrations (°Brix) were determined on the berry juice using a temperature compensating digital refractometer .P = 0.05).The effects of pruning or irrigation treatments by season or of season on plant size, leaf physiology, tissues carbohydrates, and productivity were assessed with a One-Way analysis of variance. Where significant differences were found, mean values were separated using Fisher's least significant difference (LSD) test  and Genstat 6 (Experiment 3) statistical packages , and with R (Experiment 2)—language and environment for statistical computing  but lower with spur pruning compared to minimal-spur or minimal-cane in old-wood and roots , but soluble solids were higher with spur pruning (25.7°Brix) than with either minimal pruning treatment (22.9–23°Brix), possibly due to desiccation Table . As a coP < 0.05) during season 2004–05 (14.4% dw) compared with season 2005–06 (15.4% dw). The lowest trunk starch concentrations were observed at véraison in 2005 , and at leaf fall in 2006 (14% dw)  compared with Experiment 1 at most phenological stages  and for all pruning treatments Figure . HoweverP < 0.05), partly due to lower berry weight (12% reduction). No seasonal difference in TSS was observed (mean TSS 24.6°Brix)  compared with 2006 (mean of 20 kg per vine) (P < 0.05) compared with STD , and they were again lower during the véraison period around 100 days after budburst in 2005  were observed pre-budburst during season 2003–04 between STD and PD treatments, suggesting no carry over effect from the stress incurred in season 2002–03. In 2004–05, trunk starch concentration was similar between STD and PD treatments  for STD than PD throughout the day, but not at predawn  period in 2003, which was pre-véraison or 119 days after budburst. Minimum and maximum air temperatures on this day were 13 and 32°C, respectively, and minimum and maximum VPD were 0.4 and 4 kPa, respectively. Leaf water potential was higher  in season 2004–05 (mean of 19.5 kg per vine) compared to season 2003–04 (mean of 15 kg per vine) and 2002–03 (mean of 13 kg per vine). Berry weight was similar in season 2002–03 and season 2004–05 but significantly lower in season 2003–04  and a smaller root system, thus highlighting the importance of these tissues for carbohydrate storage. Vine vigor was reduced in the minimal-cane treatment through lower pruning weight and node numbers Table . When grAlthough minimal-cane pruned vines of Cabernet Franc were 25% smaller than spur and minimal-spur vines, they only produced 10% less total berry sugar Table . In the Significant within season variations in trunk carbohydrate were observed on Cabernet Sauvignon, and to a lower extent on Shiraz Figures , 4. As rWater deficit reduced leaf starch concentration over the day and trunk starch concentrations over the season in Cabernet Sauvignon Figures , 6. ThesIn addition to the changes in starch and soluble sugar concentrations in vegetative tissues, water deficit also lowered berry size and vine yield, with maximal reductions of 18 and 21%, respectively Table . However−1, respectively in 2005  when compared at dormancy and throughout the season. Trunk carbohydrate status of Cabernet Sauvignon and Shiraz differed during season 2004–05. Vines of the varieties were similarly managed  at the same site. Trunk starch concentrations of Shiraz were very high at all stages of growth , compared to Cabernet Sauvignon, where maximum levels at dormancy were around 6% dw in 2004–05 and dropped to values lower than 2% dw at the commencement of véraison (Figures 5 Tables . When vi5 Tables , carbohyYield markedly varied between seasons in Shiraz and Cabernet Sauvignon under control irrigation Tables , 3. In cIn spite of the large varietal and seasonal differences in yield and carbohydrate concentrations in vegetative tissues, the capacity of plants to ripen berries was remarkably stable in our experiments. TSS in berries only varied between 24.0°Brix and 25.7°Brix for Shiraz and Cabernet Sauvignon, irrespective of years and pruning and irrigation treatments.The impacts of lighter pruning and deficit irrigation techniques on carbohydrate dynamics and vine production were addressed in this study for warm irrigated Australian vineyards. The total carbohydrate pool in grapevines was shown to be largely dependent on the size of the vine, mainly determined from old wood and roots system, rather than on the carbohydrate concentration in vegetative tissues. Starch was the major carbohydrate in all vegetative tissues. Soluble sugars also significantly contributed to the total carbohydrate pool in the above ground tissues, and to a lower extent in roots. Carbohydrate composition varied between tissue types, but was similar for all pruning treatments. Large varietal differences in carbohydrate pools were observed, although they were possibly confounded by crop load effects.Within seasons, the lowest concentrations of stored carbohydrates in woody trunks occurred between fruit set and véraison period, indicating that this period was critical for carbon balance. Water deficit tended to lower recovery of trunk carbohydrates post-véraison and worsen carbon starvation through its negative impact on starch concentrations. Deficit irrigation practices had the potential to impact on source activity, sink reserves, and yield in grapevines. These responses were subject to significant season to season variation which may be due to differences in the response of sink reserves to the water stress and crop load which may then impact on the source reserves. Alternatively, there may be interactions occurring between high ambient temperatures, high VPD and soil water deficit, which significantly affect the canopy's ability to produce carbohydrate and partition it according to sink demand.Despite the large variations in carbohydrate pool in vegetative tissues, and also the variation in yield components, berries accumulated sugars at a similar rate for all pruning systems, irrigations levels and seasons.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It was concluded that greater acquisition of mineral nutrients and higher yields of newer varieties were associated with greater total root length at maturity. The negative relationship between RLD and soil Nmin at harvest for all varieties suggests the importance of the spatial distribution of the root system for N uptake by maize.Larger, and deeper, root systems of new maize varieties, compared to older varieties, are thought to have enabled improved acquisition of soil resources and, consequently, greater grain yields. To compare the spatial distributions of the root systems of new and old maize varieties and their relationships with spatial variations in soil concentrations of available nitrogen (N), phosphorus (P) and potassium (K), two years of field experiments were performed using six Chinese maize varieties released in different eras. Vertical distributions of roots, and available N, P and K in the 0–60 cm soil profile were determined in excavated soil monoliths at silking and maturity. The results demonstrated that new maize varieties had larger root dry weight, higher grain yield and greater nutrient accumulation than older varieties. All varieties had similar total root length and vertical root distribution at silking, but newer varieties maintained greater total root length and had more roots in the 30–60 cm soil layers at maturity. The spatial variation of soil mineral N (N The development of new varieties, together with improved management practices, has played an important role in increasing grain yields of maize . New maiWidely extended lateral roots and branching angles are important for the acquisition of mineral nutrients. The optimal lateral root branching density in the maize root system depends on the relative availability of nitrate and P , 8–11. IIncreased nutrient uptake by plants can be achieved through the proliferation of roots in regions of the soil where nutrients are present, and thorough the acceleration of nutrient movement in the soil solution to the root surface , 17. Themin), Olsen-P and ammonium acetate (NH4OAc) extractable K.The objectives of the present work are (1) to compare the changes in root spatial distribution in the soil profile under field-grown maize released in different eras; and (2) to clarify the spatial variations of the concentrations of soil available N, P and K in different soil horizons and their relationship with root length distribution of maize grown under field conditions. To address these questions, field experiments were performed in two years using six Chinese maize varieties released in different eras. Vertical root distribution, expressed as RLD, in the 0–60 cm soil profile of excavated soil monoliths, was measured at silking and at maturity and compared with the spatial distribution of soil mineral N , China Agricultural University, Beijing, China. The soil is a typical Ustochrept with a silty loam texture, mesic soil temperature regime, and mixed mineralogy. The soil was sampled using an auger and analyzed before sowing in both years. The chemical properties of the 0–30 cm soil layer of the field sites in 2008 and 2009 were: CaCl2 extractable mineral N (Nmin) 8.0 and 7.7 mg kg−1, pH (H2O) 8.0 and 7.9, Olsen-P 7.6 and 7.1 mg kg−1, NH4OAc extractable K 86.3 and 97.6 mg kg−1, and organic matter 10.5 and 7.3 g kg−1, respectively. A few days before plowing and sowing, the field was irrigated sufficiently with a sprinkler in both years.The field experiments were performed in 2008 and 2009 at two adjacent sites at the Shangzhuang Experimental Station  and Jinhuanghou (JHH), two open-pollinated varieties released in the 1950s; Zhongdan 2 (ZD2) and Tangkang 5 (TK5), two hybrids released in the 1970s; Nongda 108 (ND108) and Zhengdan 958 (ZD958), two currently popular hybrids. They show early senescing, intermediate senescing and stay green traits, respectively. New varieties (ND108 and ZD958) had larger total leaf area and longer green leaf duration, took up more N, P and K, had higher grain yield and nutrient use efficiency than old varieties (BMY and JHH) . In 2008The maize varieties used in this study differed in the duration of their phenological stages. Plants were harvested at silking  and at physiological maturity  in 2009, when over 50% of the plants showed a visible black layer at the kernel base. In 2008, plants were only harvested at silking. At harvest, five consecutive shoots were cut at the stem base, and two whole root systems were excavated within a soil volume of 50 cm (length) × 28 cm (width) × 40 cm (depth). Soil was removed from roots by gently washing and the use of a banister brush. The harvested shoot and root samples were chopped to a fine consistency, dried to a constant weight at 60°C, and ground into fine powders. Samples of ground, dried plant tissue were used to determine the total N content by a modified Kjeldahl digestion method . The rem3) were dug one by one in a soil volume of 60 cm (length) × 30 (width) cm to a depth of 60 cm. The total number of soil cubes sampled under each plant was 108. The soil of each cube of a monolith was crushed, and all of the visible roots in each soil cube were removed by hand in the field and placed in individual plastic bags labeled with the spatial coordinates. These roots were washed free of soil after transfer to the laboratory and then frozen at -20°C until the measurement of root length as described by Peng et al. [To study the spatial distribution of roots in the soil profile, a monolith method was employed . Monolitg et al. . The rel−1 CaCl2 solutions and analyzed for soil Nmin (sum of NH4+-N and NO3—N) by continuous flow analysis  [−1 NaHCO3 for 0.5 h and then P concentration was analyzed colorimetrically according to the method described by Murphy and Riley [−1 NH4OAc by flame photometry according to the method described by Walker and Barber [After removing roots, the soil of each monolith cube was passed through a 3 mm sieve in the field. A portion of the sieved soil of each cube was then placed in a plastic bag labeled with the spatial coordinates and stored in the ice box. The bagged soil samples were extracted immediately after transfer to the laboratory with 0.01 mol LGermany) . The remnd Riley . The K cd Barber .min and RLD  in root length density, Nmin, Olsen-P and NH4OAc-K at a particular soil horizon  using the SAS package 9.1 . Kriging interpolation was performed using Surfer 7.0 software  in the contour maps Figs. ; S1 Fig. and RLD  were bes and RLD , in root and RLD , in soil horizon , in spec horizon , in the  horizon  at a parmaturity  were detNew maize varieties (ND108 and ZD958) had accumulated more dry weight (DW) and nutrients  in their shoots than older varieties at silking . DiffereThe distribution of roots of different maize varieties in a soil volume of 60 cm (length) × 30 cm (width) × 60 cm (depth) was investigated at silking in 2008 and at silking and maturity in 2009. These distributions are presented as projected two dimensional RLD across the width of a monolith ; S1 Fig.−1) than older ones at silking and maturity, showing a higher proportion of thicker roots in new varieties (Root DWs of new varieties (ND108 and ZD958) were larger than those of older varieties at silking and the relative difference in root DW between the new and older varieties increased after silking . In 2009arieties . Howeverarieties , especiaarieties . Positivarieties .The growth angles of shoot-borne roots were not determined directly in this study. However, to assess whether new varieties had steeper roots than older varieties, the proportion of the root length in each soil cube excavated along the middle of the row was determined for each soil horizon for plants at silking and at maturity . Similarmin) concentration  were grown was less than that in the soil when older varieties were grown at both silking and maturity  and least for NH4OAc extractable K (19.9%–30.7%)  . Despite%–30.7%) , there wtivation .Since the maize varieties used in this study differed in the duration of their phenological stages, they were harvested different times after sowing at silking and maturity. However, the larger shoot DW and grain yield of new varieties compared with old ones are because of not only a longer growth season in the new varieties, but also larger total leaf area and longer green leaf duration (stay-green) , 32, 33.Despite having a larger root DW ; S1 Fig.Although both new and older maize varieties had similar total root lengths at silking, new varieties accumulated more mineral nutrients than older varieties. The amounts of mineral nutrients taken up by plants with an ample supply are often determined by plant demand, rather than simply by the size of their root system , 40–45. At maturity, new maize varieties had greater total root length than older varieties . The resLateral roots constitute the majority of the total root length  and are The experiments reported here were conducted with the same plant density. It is known that the size of the maize root system is reduced with increasing plant density , 57. Furmin concentration in the top 0–20 cm horizons both at silking and maturity  had similar total root length, root vertical distribution in the 0–60 cm soil profile and root growth angles to older maize varieties at silking when grown under the experimental conditions described here. Thus, the greater nutrient accumulation and grain yield of new maize varieties was not predicated based on greater total root length than older varieties at silking. It is, perhaps, related to the regulation of nutrient uptake capacity of the root by plant demand. Nevertheless, since most of the soil resources are distributed in the topsoil, the proliferation of roots in the topsoil before silking and the maintenance of roots in the topsoil after silking ensure that new maize varieties take up sufficient soil resources to support their greater nutrient demand and higher grain yield than older varieties. Larger root DW in new maize varieties is also beneficial for lodging resistance at high plant density . AlthougS1 Fig4OAc extractable K concentration were shown in row from the top to bottom respectively, in a soil volume of 60 cm  × 60 cm (depth) × 30 cm (width). Each 10 ×10 cm2 area was the projection of three 10×10×10 cm3 soil cubes. Maize varieties were indicated at the top right side. Total root length per plant (m), soil mineral nitrogen content (g), soil Olsen-P content (g) and soil residual NH4OAc extractable K content (g) in the whole 60×30×60 cm3 soil volume were indicated at bottom right side in each panel, respectively. n = 3.Root length density, soil mineral nitrogen concentration, soil Olsen-P concentration and soil NH(TIF)Click here for additional data file.S2 FigP<0.05). Data are mean values from 3 replicates of each genotype. Error bars denote the standard deviation.Specific root length means the ratio of root length to root dry weight. Different letters above the bars within the same soil layer indicate significant differences among maize varieties ((TIF)Click here for additional data file.S3 Fig* and ** represent significance at 0.05 and 0.01 probability level, respectively.(TIF)Click here for additional data file.S4 Fig3 cube (P<0.05). Data are mean values from 3 replicates of each genotype. Error bars denote the standard deviation.Different letters above the bars indicate significant differences between maize varieties in a 1000 cm(TIF)Click here for additional data file.


Staphylococcus aureus. The genetic diversity of the ess locus, which encodes the T7SS, and the functions of proteins encoded within it are poorly understood.Type VII protein secretion (T7SS) is a specialised system for excreting extracellular proteins across bacterial cell membranes and has been associated with virulence in S. aureus. The ess loci were found to comprise of four distinct modules based on gene content and relative conservation. Modules 1 and 4, comprising of the 5’ and 3’ modules of the ess locus, contained the most conserved clusters of genes across the species. Module 1 contained genes encoding the secreted protein EsxA, and the EsaAB and EssAB components of the T7SS machinery, and Module 4 contained two functionally uncharacterized conserved membrane proteins. Across the species four variants of Module 2 were identified containing the essC gene, each of which was associated with a specific group of downstream genes. The most diverse module of the ess locus was Module 3 comprising a highly variable arrangement of hypothetical proteins. RNA-Seq was performed on representatives of the four Module 2 variants and demonstrated strain-specific differences in the levels of transcription in the conserved Module 1 components and transcriptional linkage Module 2, and provided evidence of the expression of genes the variable regions of the ess loci.We used whole genome sequence data from 153 isolates representative of the diversity of the species to investigate the genetic variability of T7SS across ess locus of S. aureus exhibits modularity and organisational variation across the species and transcriptional variation. In silico analysis of ess loci encoded hypothetical proteins identified potential novel secreted substrates for the T7SS. The considerable variety in operon arrangement between otherwise closely related isolates provides strong evidence for recombination at this locus. Comparison of these recombination regions with each other, and with the genomes of other Staphylococcal species, failed to identify evidence of intra- and inter-species recombination, however the analysis identified a novel T7SS in another pathogenic staphylococci, Staphylococcus lugdunensis.The The online version of this article (doi:10.1186/s12864-016-2426-7) contains supplementary material, which is available to authorized users. Mycobacterium tuberculosis secreting two T-cell antigens (termed ESAT-6/EsxA and CFP-10/EsxB) via the T7SS pathway. There is significant evidence that this system is an important virulence factor in mycobacteria. Genes encoding ESAT-6 and CFP-10 form part of the region of difference 1 (RD1), a cluster of genes that is deleted from the genome of the M. bovis BCG vaccine strains [The secretion of virulence factors is an essential process for bacterial pathogenesis, and bacteria have evolved numerous systems through which proteins can be secreted into the environment or injected into host cells . The Typ strains . This deThe mycobacterial T7SS comprises a number of membrane proteins that form a large 1.5 MDa complex . StructuBacillus subtilis [Bacillus anthracis [Staphylococcus aureus [Homologs of the mycobacterial T7SS components EsxA, EsxB and EccC are also encoded by some firmicutes and secretion of EsxA and/or EsxB have been demonstrated in subtilis , 11, Bacnthracis  and Staps aureus , 14. Hows aureus . This has aureus .S. aureus is a human commensal bacterium and an opportunistic pathogen that can cause a broad range of clinical manifestations in humans, including the majority of skin and soft tissue infections and a substantial proportion of invasive infections such as endocarditis and osteomyelitis [ess (ESAT-6-like secretion system) locus and are highly up-regulated during long-term persistence in the cystic fibrosis airway, consistent with a role in persistent infection [S. aureus strains Newman, USA300 and RN6390 have shown that the ess locus codes for two secreted WXG100 family proteins (EsxA and EsxB) and two secreted proteins lacking the WXG100 motif (EsxC and EsxD). Three ess-encoded transmembrane proteins, EssA, EssB and EssC, are reportedly essential for protein secretion, but a potential function has been assigned to only one of these, the EccC-like ATPase EssC. The ubiquitin-like protein EsaB has been proposed to regulate Ess activity at either the post-transcriptional or post-translational level. Finally, the membrane protein EssD also encoded at this locus, has a non-essential role in protein secretion [myelitis –17. The nfection . Collectecretion , 19–21.S. aureus is a clonal bacterial species and is dominated by a number of successful lineages [S. aureus lineages there is variation in the extensive arsenal of immune evasion and virulence factors that modulate the host cell interaction under complex genetic regulation [ess locus has previously been described in a small number of S. aureus strains from multi-locus sequence type (MLST) clonal complex (CC) 8 , homologs of which were present in all but one of the sequences analysed. The gene arrangement of Module 1 was as described previously [esxA, esaA, essA, esaB and essB (ASASM86) contained a genomic deletion of 9166 bp that resulted in the loss of these genes.Module 1 contained the first five genes of the eviously , 13, 20,eviously . A singlessC gene encodes a membrane-bound protein with three C-terminal ATPase domains and is a key component of all T7SS. Homologs of this gene were identified in all of the S. aureus isolates investigated. The 5’ region of this protein coding sequence (CDS) (approximately 3220 bp of essC) was conserved among all isolates, however the 3’ region (approximately 1230 bp of essC) and downstream CDS fell into four distinct variants. We have termed these variants essC1-4  and was found in strains belonging to CCs 1, 5, 7, 8, 9, 25, 41, 51 and 88. These included the reference strains Newman [essC3 (41/153 isolates), which was found in CC30 and ST239, and included the TW20 [essC4 variant was only identified in isolates belonging to CC22 (including the EMRSA-15 reference isolate HO 5096 0412) [essC2 was identified in CC15 and ST398  [essC variants associated with each isolate is included in Additional file ess loci of NCTC 8325, S0385, MRSA252 and HO 5096 0412 reference genomes strains as representative of the four essC variants, essC1, essC2, essC3 and essC4 respectively.The s Newman , COL [27s Newman , N315 [2s Newman  and MW2 s Newman , which hs Newman , 20, 24.e S0385) . A summaessC gene. The X-ray structure of the C-terminal 550 amino acids of EssC from Geobacillus thermodenitrificans, a thermophilic member of the firmicutes phylum has been reported [G. thermodentrificans EssC. It is clear from Fig. S. aureus recognise different repertoires of secreted proteins due to the C-terminal domain variation.It was notable that the sequence divergence associated with this module at the 5’ end occurred within the reported  and encoessC1 have been previously demonstrated to be co-transcribed with the genes encoding the core secretion components [esxC, esxB and esxD encode secreted T7SS substrates [essD encodes a transmembrane protein and esaE a predicted soluble protein of unknown function [essC1 variant in this study had the same arrangement of these five genes, by contrast none of the isolates with the other three essC variants  possessed any of these genes. Instead, each essC variant . Consequently, each variant Ess system has at least two WXG family proteins: an EsxA protein (found in Module 1) that is highly sequence-conserved regardless of EssC subtype and a Module 2 protein, conserved with the same EssC subtype but very poorly conserved across strains with different EssC subtypes. Additional proteins sharing sequence similarity with the WXG100 family of secreted proteins were identified in the essC2 and essC3 variants , SAEMRSA15_02500 (essC4 variant) and SAPIG0304 (essC2 variant) shared motifs associated with the CATH superfamily 1.20.5.170 [essC2 variant shared a motif or some peptide sequence with a hypothetical protein in the essC3 variant cluster.Although functions for remaining CDSs in Module 2 could not be predicted, the conservation of protein motifs implies conserved function in this cluster. For example, 20.5.170 . This doSAOUHSC_00270; SAOUHSC_00271; SAOUHSC_00272; SAOUHSC_00255 ; and a cluster of four genes which are always found together in the following combination: SAEMRSA15_2510, SAEMRSA15_2520, SAEMRSA15_2530 and SAEMRSA15_2540.Immediately downstream from Module 2 was a complex arrangement of CDSs that included a variable number of predicted transmembrane proteins, interspersed with a diverse arrangement of genes coding for hypothetical proteins of approximately 166 amino acids in length with a domain of unknown function named DUF600. These genes form Module 3, as illustrated in Fig. essC variant and the combination of genes in this region. For example, all essC3 variants were associated with homologs of the genes SAOUHSC_00272 and SAOUHSC_00255. Some, but not all, essC3 variants were associated with the SAEMRSA15_2510 to SAEMRSA15_2540 cluster. None of the isolates with the essC3 variant were associated with the SAOUHSC_00271 gene. Different isolates within the same clonal complex appeared to possess the same combination of hypothetical transmembrane proteins encoded in Module 3. The exception was CC8, which had a wide variety of combinations. Some of the hypothetical transmembrane proteins were encoded in multiple copies in the same isolate. For example in the strain Newman, NWMN_0230 encoded a protein with almost identical amino acid sequence to NWMN_0237  [The combination of these genes found in each genome varied between isolates. All strains had at least one homolog of these genes; no isolates possessed homologs of them all. Our analysis suggested that there may a relationship between the essC1 variant gene clusters, there was variation in the 3’ region of essD, the gene immediately upstream of the first DUF600 gene. Each essD variant was associated with a different downstream DUF600 protein variant, which broadly clustered into clonal complexes.The number of DUF600 genes present in representative of the different clonal complexes: the minimum number identified was two genes in CC22 isolates and the maximum number was 13 in CC8 isolates. The number of DUF600 genes also varied between isolates within the same clonal complex. In the ess locus is unclear. Several features support an association with the T7SS, including: i) these genes were always on the same coding strand, following immediately from Modules 1 and 2 described above; ii) there was little intergenic space between coding regions, suggesting that these genes may be under the same transcriptional control as other elements of the T7SS; iii) some of the genes encoded in this region shared sequence identity with genes that flanked known secretion component genes within the ess locus. For example, SAOUHSC_00270 shared some sequence motifs with SAOUHSC_00254  and SAOUHSC_00271  with SAOUHSC_00255, genes that neighboured SAOUHSC_00256 – the gene immediately upstream of esxA. SAR0288, part of the cluster of genes immediately downstream of essC3 also shared 67 % amino acid sequence identity with SAOUHSC_00254.The relationship between this cluster of transmembrane and DUF600 proteins encoded at the SAOUHSC_00279 and SAOUHSC_00280 represented as Module 4 in Fig. ess locus: SAOUHSC_00279 contained a cystatin-like fold  which was also present in SAEMRSA_2520, while SAOUHSC_00280 harboured the DUF4064 domain  which was also present in SAEMRSA_2530.Downstream of the highly variable Module 3 is a region that contains two genes  and, more specifically, to look for evidence of the expression of the hypothetical components of the ess loci identified in our analyses. Examination of the mapped strand-specific cDNA sequence data confirmed the gene prediction models of the ess regions, and revealed variation in the abundance of transcript across the ess region  Kneuper et al. mapped the transcriptional start site upstream of esxA, and also identified a promoter in the esxA-esaA intergenic region [esxA and with the other components of the Module 1  present on separate transcripts; esxA is present on a moncistronic transcript, and esaA, essA, esaB, essB on a polycistronic transcript that also contains components of Module 2, including essC and downstream CDSs.RNA-Seq was used to examine the transcriptional profiles of the cination . This hieviously . The resc region . RNA-Seqess clusters revealed strain-specific differences in the levels of transcription , essD (P = 0.0)). Although some conserved genes did not show evidence of recombination (e.g. esxA) other genes that are encoded in the conserved part of the ess locus did reach statistical significance using this method (esaB (P = 0.017), essB (P < 0.0001)). These indicate that the variation between the same gene in different isolates was more likely to have occurred by recombination than convergent selection or the random accumulation of homoplastic SNPs. To illustrate this, Fig. essD genes of isolates with the essC1 subtype, an apparent hotspot of recombination events within the ess operon.We applied further recombination detection methods using the Phi test within the PhiPack package  on indivS. aureus with publicly available reference genomes for other species in the Staphylococcus genus was conducted to investigate the wider distribution and genetic arrangement of this locus. As shown in Fig. ess locus was present in Staphylococcus lugdunensis, with homologs of the conserved portion of the locus from esxA to essB (SLGD_01975 to SLGD_01971) and a gene similar to the essC4 variant (SLGD_01970). There was also a cluster of genes similar to those downstream of essC4, including homologs of SAEMRSA15_02470 to SAEMRSA_02500, SAEMRSA_02520 and SAEMRSA_02540 (SLGD_01964 to SLGD_01969). There were two genes with homology to those coding for DUF600 proteins (SLGD_01962 and SLGD_01963), but no obvious homologs of the two conserved genes found in Module 4 of the ess operon (SAOUHSC_00279 and SAOUHSC_00280).Comparative genomic analysis of Staphylococcus genus, none possessed the entire ess operon. However, Staphylococcus pasteuri contained one gene with sequence identity to the DUF600 gene (STP1_1226) and homologs of the cluster of four hypothetical transmembrane-protein coding genes, SAEMRSA_02510 to SAEMRSA_02540 (STP1_1228 to STP1_1231). Staphylococcus warneri also possessed homologous genes to this cluster (A284_00845 to A284_00860). None of the genes identify in these staphylococci exhibited high DNA identity to S. aureus homologs suggestive of interspecies recombination. A complete list of the species and isolates examined can be found in Additional file Of the other species examined within the  gene STP_1226 andess locus using S. aureus isolates from a broad range of clonal complexes from two distinct genome collections. We identified genes conserved across all isolates and characterised variants in the ess operon, including a number of further candidate substrate proteins that may be secreted by the S. aureus T7SS.In this study we have described, for the first time, the organisational variation in the ess locus, it was striking that the cluster of genes in Module 1, from esxA to essB, was conserved among almost all isolates, suggesting a strong survival benefit or functional constraint on this cluster. In-frame deletions of each of these genes have been shown to abolish secretion of T7SS substrate proteins, supporting the assertion that these genes encode core components of the T7SS [Despite the variation elsewhere in the the T7SS , 14, 19.essC gene. This membrane-bound protein is essential for secretion of all effector proteins in the T7SS [essC variant was associated with a unique cluster of downstream genes, suggesting that the EssC C-terminus may play a role in interacting with the proteins encoded immediately downstream from essC. One of the proteins encoded downstream of each EssC variant is from the WXG superfamily, and in the commonly studied S. aureus Newman, USA300 and RN6390 strains it is EsxB. Studies have shown that esxB is expressed at a very similar level to essC [essC variant interacts with the C-terminal domain of its cognate EssC protein to activate secretion. It is also possible that this sequence-variable EssC domain may recognise variant-specific substrate proteins or accessory factors. While the genetic basis of this variation and its functional consequence remains unclear, the arrangement of genes and their incongruence with MLST suggest that recombination has played a role in the evolution of this locus. Comparative genomic analysis of S. aureus has revealed that recombination in the core genome is relatively rare [A key finding in this study was the identification of four variants of the the T7SS , 20. Var to essC , and str to essC . We postely rare . It is tessC, remains unclear. The best characterised cluster was downstream from essC1, which has been studied in a small number of CC8 isolates and includes genes encoding the secreted proteins EsxB, EsxC and EsxD and the transmembrane protein EssD. These proteins appear to have a functional relationship, with reports that strains lacking each of these proteins affect the stability or expression of the others, while EsxB and EsxD have been shown to form heterodimers [ess variants.The function of many of the proteins in Module 2, downstream from rodimers . FurtheressC3 and essC2, implied some redundancy in their function. However, there were also marked differences between variants. For example, the essC2, essC3 and essC4 variants all contained a hypothetical protein with the WXG motif  that was much larger (440–556 aa) than the previously identified T7SS substrates associated with essC1 (for example EsxB and EsxD are 104 and 105 amino acids in length respectively). There was also no transmembrane protein of a comparable size to EssD encoded in the other variants.Although there is a limit to information that can be derived from the bioinformatics analysis of four variants of Module 2 identified in this study, the observation that different variants encoded proteins that shared sequence motifs, particularly the modules associated with S. aureus  containing the DUF600 motif [essD gene, which raised the question of whether other genes in these variants serve an equivalent function.The association between EssD and the DUF600 hypothetical proteins has been observed previously, with the C-terminus of some EssD sequences in 00 motif . We obseess clusters. A recent study by Baek and colleagues demonstrated that the greatest sequence variation between strains of the same S. aureus lineage (NCTC 8325) occurred between homologs of the DUF600 proteins [Previous evidence has suggested that there is very little variation within coding regions between isolates belonging to the same lineage . Howeverproteins . This iness operon, associated with the DUF600 proteins. Their function and relationship to the T7SS will require further investigation. The presence of these hypothetical transmembrane proteins without the remainder of the T7SS apparatus in the genomes of S. warneri and S. pasteuri may suggest they are not key components of this system. However it is unclear why these genes are located in the same region of the S. aureus genome, often flanked by DUF600 proteins.There are many hypothetical transmembrane proteins encoded within Module 3 in the S. lugdunensis, a medium-pathogenic coagulase-negative staphylococcus which shares a number of clinical features with S. aureus, including the ability to form abscesses [S. aureus, the T7SS could have a role in abscess development and therefore in pathogenicity. Investigation into a broader range of staphylococcal isolates may provide clues as to the evolution of the ess locus.The T7SS has been described in other Gram positive bacteria , 3, 6, 7bscesses . The mecesxA, are under complex regulatory control and vary between closely related isolates in the same clonal complex [ess operon, which is likely to have implications for gene regulation in the broader range of clonal complexes. Mutants in the T7SS have impaired ability to persist in the complex interplay between host and pathogen during colonisation and abscess formation [S. aureus isolates, all of which possess the essC1 operon arrangement. It is unclear what effect other arrangements will have on the pathogenicity of S. aureus, or its abscess-forming phenotype.Having described a range of hypothetical proteins that will require further molecular research to determine their role and function, our study provides further avenues for investigation. Recent studies have suggested that the expression of key genes of the T7SS, including  complex . In thisormation , 41. HowS. aureus whole genome sequence data utilized in this study was generated by the Wellcome Trust Sanger Institute (WTSI). The S. aureus isolates were obtained from human bacteremia cases at Addenbrooke’s Hospital, Cambridge, UK between 2006 and 2012. Further stored isolates from neighbouring hospitals in the East of England, collected between 1998 and 2011, were also included. From this broad collection, 103 isolates were chosen to represent as many different clonal complex (CC) and MLST types (ST) as possible, to provide a snapshot of the genetic diversity of disease causing isolates from this large scale UK collection. To augment the collection, a further 50 published and publicly available S. aureus sequences from the European Bioinformatics Institute were included in the analysis. A full list of isolates used in this study, along with accession numbers, is presented in Additional file Written informed consent from patients was not required as all bacterial isolates were collected, processed and stored as part of routine clinical care. The study protocol was approved by the National Research Ethics Service (reference 11/EE/0499), and by the Cambridge University Hospitals NHS Foundation Trust Research and Development Department (reference A092428).S. aureus  were incubated shaking at 200 rpm for 16 h at 37 °C. The overnight culture was used to inoculate 25 ml TSB media to an OD600 of 0.03. 2 ml of OD600 1.0 cells were harvested by centrifugation at 13000 g for 2 min and processed for RNA extraction using the Ambion Ribo-PureTM Bacteria kit as per the manufacturers’ guidance. Three biological and three technical replicates were performed for each strain. The RNA quality and concentration was assessed by Agilent BionalyserTM, NanodropTM and visually on a 1 % agarose gel containing 1 % gel red (Biotium) and visualised by gel doc imaging system (Bio-Rad).Strains of For RNA-Seq, total RNA was reverse transcribed using SuperScript III reverse transcriptase (Invitrogen). Actinomycin D  was added to the reaction to avoid spurious second-strand cDNA synthesis . cDNA wahttp://www.vicbioinformatics.com/software.velvetoptimiser.shtml) script to optimize the kmer length. Automatic annotation of the Accessory genome contigs was carried out using PROKKA [de novo assemblies using BlastN [Assemblies were created with Velvet v1.2.09  using thg PROKKA . Detaileg BlastN , and wasg BlastN .S. aureus strain in the T7SS literature) [In silico PCR using these primers was performed on the de novo assemblies for all isolates using a Python script, to determine the presence or absence of each gene in each isolate. In brief, the script used approximate regular expressions to identify all best matches to each primer sequence in each genome, allowing up to 3 mismatches per match. Where forward and reverse primers were in the correct orientation relative to each other and produced a product of approximately the correct length (defined as <10 kb), products were extracted as fasta files. If a gene could not be identified in an isolate using this method, tblastx comparisons [ess locus. In silico primers were developed for all novel genes identified and the in silico PCR process repeated until all genes in the ess locus had been identified. All primers used in this analysis are presented in Additional file Individual T7SS genes were identified from the publicly available Newman strain reference genome (the most commonly studied erature) . Primersparisons  were genparisons  to confiwww.ncbi.nlm.nih.gov/blast/), and Pfam [http://pfam.xfam.org/), InterPro [http://www.ebi.ac.uk/interpro/). Maximum likelihood phylogenetic trees were generated for whole genome sequences of the 103 isolates sequenced using RAxML [ess locus were generated using PhyML 3.1 [Bioinformatic searches on all genes and putative gene products were conducted using the web-resources NCBI BLASTn )  in the different strains and remove any extra source of bias in the data, we performed an extra normalization, using the expression value of a housekeeping gene, which is rpoD in our case. More specifically, we scaled the fragments per kilobase of transcript per million (FPKM) mapped reads values of the housekeeping gene across the four strains  and we resized the FPKM values of all the genes based on the hs.To compare the expression levels of the genes of interest (http://www.ebi.ac.uk/ena) under the study numbers ERP000871, ERP001009, and ERP001320; individual accession numbers are given in Additional file All sequences from this study have been submitted to the European Nucleotide Archive (ENA;


Limonium vulgare using 454-pyrosequencing. We hypothesize that the selective force exerted by plants rather than soil would regulate the dynamics of the root-associated bacterial assembly along the chronosequence. Our results showed that the soil and rhizosphere bacterial communities were phylogenetically more diverse than those in the endosphere. Moreover, the diversity of the rhizosphere microbiome followed the increased complexity of the abiotic and biotic factors during succession while remaining constant in the other microbiomes. Multivariate analyses showed that the rhizosphere and soil-associated communities clustered by successional stages, whereas the endosphere communities were dispersed. Interestingly, the endosphere microbiome showed higher turnover, while the bulk and rhizosphere soil microbiomes became more similar at the end of the succession. Overall, we showed that soil characteristics exerted an overriding influence on the rhizosphere microbiome, although plant effect led to a clear diversity pattern along the succession. Conversely, the endosphere microbiome was barely affected by any of the environmental measurements and very distinct from other communities.Proper quantification of the relative influence of soil and plant host on the root-associated microbiome can only be achieved by studying its distribution along an environmental gradient. Here, we used an undisturbed salt marsh chronosequence to study the bacterial communities associated with the soil, rhizosphere and the root endopshere of  Assessments of microbial diversity have revealed soils—the habitats where plants and microbes live together and build highly diverse interactions—as being among the most biologically diverse on Earth . However, this selective force is rather different at different locations, which has been justified by differences in agricultural practices  phylogenetic diversity to decrease as we intensified the association with the plant—higher in bulk soil and lower in the endosphere—regardless of the soil successional stage. Moreover, we expect (ii) diversity to be constant within the rhizosphere and endosphere across different successional stages but variable in the bulk soil. In addition, we predict (iii) the structure of the plant associated bacterial communities to cluster according to the degree of connection with plant rather than soil successional stages. Within each community, however, we expected (iv) the distribution of the bacterial patterns to cluster according to three main successional—initial, middle and late stages—following the soil and vegetation development along the chronosequence  or inside (endosphere) the roots, as well as in the bulk soil, in different soil types, along a salt marsh primary succession gradient , and spans more than 100 years of primary succession . Each sample was placed in a sterile plastic bag, sealed and transported to the laboratory within 24 h. Spades were sterilized with 70% ethanol between different sampling plots and plants. From each composite sample we sampled bulk soil, rhizosphere, and endosphere (see below).Sampling was performed twice (May and August) on all of the triplicate plots in the five stages to investigate the differences in season. As mentioned above, in order to quantify the relative influence of soil type and plant species on plant associated bacterial community, The plants were separated carefully from the adhering soil without damaging the roots by gentle shaking. All bulk (non-rhizosphere) soil samples were sieved (2 mm mesh size) and stored at −20°C for DNA extraction and 4°C for physicochemical measurements. Ten grams of roots with tightly adhering soil (rhizosphere soil) were transferred to an Erlenmeyer flask containing 180 mL of sterile sodium pyrophosphate (0.1%). After 30 min of shaking at 200 rpm at room temperature, 0.5 mL of the suspension with rhizosphere soil was used for DNA extraction or stored at −20°C.Plant roots (about 8 g) were thoroughly washed with running tap water, trimmed to remove adhering soil and dead tissues, and surface sterilized by immersion into 1.5% NaClO solution (3 min), 70% ethanol (3 min) and sterile distilled water (3 × 3 min). Sterility checks were performed by tissue-blotting surface-sterilized plant samples on R2A plates, and checking the plates after 2–7 days incubation at 28°C. Samples without bacterial growth were considered successfully sterilized and used for further study. The surface-sterilized root parts (5 g) were sliced with a sterile scalpel and immersed into 15 mL NaCl solution (0.9%). After shaking incubation for 1 h at 28°C, the suspension with root pieces was shaken using a horizontal vortex instrument . Large plant and fungal cells were removed with 5-μm filters  were subjected to total DNA extraction using 0.5 gram of soil and 0.5 mL of the suspension with rhizosphere soil, respectively, using the MoBio PowerSoil DNA extraction kit . We followed the instruction manual, except for the addition of glass beads  to the MicroBead tube and three cycles of bead beating  for 60 s. A total of 26 composite endophytic cell pellet samples  were excluded because of very small pellets) were subjected to total DNA extraction using the MoBio UltraClean Microbial DNA Isolation Kit . We followed the instruction manual, except for heating the preparations at 65°C for 10 min with occasional bump vortexing for a few seconds every 2–3 min. Extracted DNA was further quantified using the PicoGreen dsDNA assay  within the wavelength range of 485–535 nm.−1 FastStart High Fidelity (FSHF) Taq DNA Polymerase, 2.5 μL 10 × FSHF Reaction buffer without MgCl2, 2.3 μL 25 mM MgCl2 stock solution, 0.5 μL 10 mM PCR nucleotide mix, 0.25 μL 20 mg mL−1 bovine serum albumin (BSA) , and 0.5 μL each of 10 μM primer and 5 ng DNA template. The thermal cycler protocol was 95°C for 5 min, 30 cycles of 95°C for 40 s, 56°C for 45 s, 72°C for 40 s and a final 10-min extension at 72°C. The primer set 515f/1061r targeting regions V4–V6 of the 16S rRNA bacterial gene was used, which can provide sufficient resolution for the precise taxonomic classification of microbial sequences  agarose gel, and the gel containing the extract bands was excised and purified using the QIAquick Gel Extraction kit , and then quantified using PicoGreen dsDNA assay . Amplicons from all samples were pooled in equimolar concentrations into four composite samples containing 25, 25, 27, and 29 samples, respectively, and sequenced at Beckman Coulter Genomics  on a Roche GS-FLX 454 automated pyrosequencer running Titanium chemistry.http://metagenomics.anl.gov/).Before analyses, raw sequencing data generated from the 454-sequencing runs were demultiplexed and processed using the Quantitative Insights into Microbial Ecology (QIIME) toolkit . To test the significance of the dissimilarity among different sources and different successional stages in the weighted unifrac distance matrix, we used permutational multivariate analysis of variance (PerMANOVA)  with linear discriminant analysis (lda), resulting in the best prediction of group identities of the sites  were identified using One-way analysis of variance (ANOVA), followed by Tukey HSD pairwise group comparisons in the R environment  , salinity , and soil water content (SWC). The soil pH, however, was highest at the 5-year stage (mean pH 8.51 ± 0.07) and decreased over succession (mean pH 7.51 ± 0.16 at the 105-year stage). With the development of the succession and sedimentation caused by the tidal regime, the soil physicochemical conditions and the level of silt and clay particles on the salt marsh with progressively increasing elevation were found to improve along the chronosequence, reaching a peak at the 65-year stage (except for nitrate levels). In addition, the salinity level also increased over time during succession, due to an accumulative effect.A detailed description of the soil data is given in Table To investigate the diversities of the bacterial communities associated with bulk and rhizosphere soil and endosphere along the chronosequence, the pyrosequencing data were rarefied to the depth of 760 reads per sample after being binned into 46,972 different OTUs, of which 48.76% of the sequences  were singletons. Similar α-diversity values were shown for bulk soil and rhizosphere, which were significantly higher than those observed for endosphere Figure , partialR2 = 0.29, P < 0.001), successional stage  and sampling time  with 20.84% of the total variation explained by CAP1, and there were a significant seasoning effect  and a more important effect of successional stage .The clustering of bacterial communities from the bulk soil, rhizosphere and endosphere at different successional stages, performed with CAP analyses by using sources as the constraint. The first two constrained axes explained 28.97% of the total variation, with a significant effect of source  and reflecting the influence of successional phase as well as sampling time. Interestingly, the clustering effect within each phase became stronger after the initial stage, reaching the highest level at the middle stage , the difference level between each other was not as strong as that for bulk soil and rhizosphere. In addition, the dispersion level of the community structure within each successional phase was found to increase toward the late succession  along the succession for bulk soil (from both samplings) and rhizosphere (from the first sampling) associated bacterial communities—indicating a more stable community structure as succession proceeded—whereas for the endosphere, the differences between communities in initial stages was lower than at late stages of succession , by using successional stage as the constraint Figure . Bulk so Figures . On the n Figure . Indeed,By partitioning the variation in the community dissimilarities of bulk soil, rhizosphere, and endosphere, we found that soil characteristic, including the selected subset of soil physicochemical parameters  and the soil texture indicators , explained much higher partition of the variation in community dissimilarities for the microbiome associated with bulk soil (38.26%) and rhizosphere (38.4%), compared with successional stages and seasonal changes Figure . SimilarAll together, these results partially corroborate our hypothesis that bacterial community structure is determined by plant rather than successional stage. This was especially true for endophytes, where the effect of successional stages was completely absent. As expected, clustering according to successional stage was detected for bulk and rhizosphere bacterial communities. Intriguingly, endophytes experienced higher turnover in community composition at later stages of succession, contradicting our prediction that plant selection should lead to more similar communities.P < 0.05) compared with the bulk soil and rhizosphere. The rhizosphere was found to accumulate significantly higher Chloroflexi and Planctomycetes (P < 0.05) than the bulk soil and endosphereat the middle stages . Among the six main genera of Proteobacteria , which was the most abundant genus, belonging to the family Oceanospirillaceae.Within the most predominant phylum, Proteobacteria, the classes Alpha- and Gammaproteobacteria were more abundant than Beta- and Deltaproteobacteria Figure . Among ta Figure , the endP < 0.05). At the class level of the phyla Proteobacteria, Deltaproteobacteria increased significantly across succession in the bulk soil and rhizosphere (P < 0.001). Conversely, Alphaprotobacteria in the bulk soil and rhizosphere were found to show decreasing patterns (P < 0.05). For Gammaproteobacteria, hump-shaped and inverse-hump-shaped patterns were found in the bulk soil and rhizosphere, respectively, reaching the peak at the 15-year stage (32.05 ± 5.42%) and the bottom at the 35-year stage (21.82 ± 4.07%). Despite being the most dominant genus, no significant pattern in relative abundance was shown for Marinomonas along the chronosequence (Figure Choloflexi and Gemmatimonadetes progressively increased along the chronosquence within the bulk soil and rhizosphere Figure . In the e Figure . In addiL. vulgare, a salt mash plant that is present along most of the successional gradient.In this study we directly examined the relative contribution of soil and plant effect on the diversity and structure of bacterial communities varying in their degree of association with plants, by making use of a gradient of soil development in a salt marsh primary succession. We specifically investigated the drivers of root-associated bacterial communities related to the rhizosphere and endosphere of By providing a comprehensive overview of the phylogenetic diversity of root associated bacterial community along the salt marsh chronosequence, we showed that bacterial communities associated with bulk and rhizosphere soil were equally diverse whereas those associated with the endosphere were significantly less rich, which has also been reported recently (e.g., Saleem et al., In opposition to the soil or rhizosphere microbiome, endophytic bacterial communities are often simple, encompassing up to hundreds of different bacterial types (Hardoim et al., We next focused on understanding the root-associated bacterial community turnover across successional stages, by assessing of the shifts bacterial β-diversity among bulk soil, rhizosphere and endosphere. The distinctive community structure established in each source corroborated our prediction that the bacterial communities would cluster according to the degree of connection to the plant Figure . Thus, bThe clustering patterns according to successional stage was shown for bacterial communities within bulk soil and rhizosphere when those were analyzed separately Figures , confirmL. vulgare abundance, which takes place at the intermediate stage of succession, indicates the most appropriate successional stage for this species. In that case, plants could be experiencing higher competition for nutrients and more demanding conditions due to the increase in salt concentration, consequently influencing the plant control over their microbiome, especially for endophytes. Although, we cannot pinpoint the specific factor leading to the higher turnover in endophytic communities at the end of the succession, the opposite trend observed for the communities associated with bulk soil and rhizosphere makes the selection pressure exerted by plants even more spectacular.The endophytic bacterial communities, however, showed much higher dispersion level within each successional phase Figure . In addiL. vulgare over the succession, whereas Gammaproteobacteria decreased, mostly due to a decrease in the genus Pseudomonas. The communities associated with endophytes were largely enriched in Proteobacteria, which was the predominant phylum across all samples. Within this phylum, the significant accumulation of the genus Marinomonas within the endosphere corroborated once more that the plant selective force plays a substantial role in regulating the endophytic bacterial community composition (Figure Marinomonashas been found among many different marine environments (Sanchez-Amat and Solano, Marinomonas suggest it could exert beneficial traits on the plant hosts, therefore leading to its dominance in endosphere. Contrary to Marinomonas, the genus Pseudomonas and Cellvibrio, could be considered as opportunistic endophytes as their relative abundances peaked only at the early stage of succession.Finally, by delving into the taxonomic composition of root-associated bacterial assemblages along environmental gradients of the succession, we could detect a few patterns. For instance, Chlorofexi, Plantomycetes, and deltaproteobacteria increased in relative abundance in the rhizosphere of n Figure . The genOverall, this study offers for the first time an overview of the variation of root-associated bacterial composition along a salt marsh chronosequence. Importantly, by focusing on non-domesticated plants in a natural ecosystem, we could show that the importance of soil in driving bacterial community composition is dependent on the plant compartments, being influential only in the rhizosphere but not on the endosphere. The distinctive dominant bacterial composition within endosphere validates the existence of the plant selective force on the root-associated microbes, resulting in the distinctive taxonomy composition and diversity in the rhizosphere and the internal root tissues.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gastrointestinal (GI) manifestations are common in autistic children. Polyunsaturated fatty acids (PUFAs) and carnitine are anti-inflammatory molecules and their deficiency may result in GI inflammation. The relationship between the increased frequency of GI manifestations and reduced levels of PUFAs and carnitine was not previously investigated in autistic patients. This study was the first to investigate plasma levels of PUFAs and serum carnitine in relation to GI manifestations in autistic children.Plasma levels of PUFAs  and serum carnitine were measured in 100 autistic children and 100 healthy-matched children.Reduced levels of serum carnitine and plasma DHA, AA, linolenic and linoleic acids were found in 66%, 62%, 60%, 43% and 38%, respectively of autistic children. On the other hand, 54% of autistic patients had elevated ω6/ω3 ratio. Autistic patients with GI manifestations (48%) had significantly decreased levels of serum carnitine and plasma DHA than patients without such manifestations. In addition, autistic patients with GI manifestations had significantly increased percentage of reduced serum carnitine (91.7%) and plasma DHA levels (87.5%) than patients without such manifestations , .Reduced levels of plasma DHA and serum carnitine levels may be associated with the GI problems in some autistic patients. However, this is an initial report, studies are recommended to invesigate whether reduced levels of carnitine and DHA are a mere association or have a pathogenic role in GI problems in autistic patients. Some cases of autism may be associated with several organic conditions including disturbance of brain energy metabolism which depends on adequate supply of polyunsaturated fatty acids (PUFAs) and carnitine and normal mitochondrial function ,2. FattyThe optimal range of the ratio of omega6/omega3 (AA/DHA) varies from 1/1 to 4/1. High ω6/ω3 ratio promotes the pathogenesis of many diseases. Whereas low ω6/ω3 ratio exerts a suppressive effect. A ratio of ω6/ω3 of 4/1 is the optimal ratio of brain mediated function . The rolGastrointestinal (GI) disturbances such as abdominal pain, chronic diarrhea, constipation, vomiting, gastroesophageal reflux, and intestinal infections.are commonly reported in children with autism, and may contribute to behavioral impairment ,10. RepoThe relationship between the increased frequency of GI manifestations and reduced levels of PUFAs and carnitine was not previously investigated in autistic patients. This study was the first to investigate plasma levels of PUFAs and serum carnitine in relation to GI manifestations in autistic children. This may add new biological indices and therapeutic implications for autism.This cross-sectional study was conducted on 100 children with autism. They were recruited from the Autism Research and Treatment Center, Faculty of Medicine, King Saud University, Riyadh, Saudi Arabia. Patients were fulfilling the criteria of the diagnosis of autism according to the 4th edition of the Diagnostic and Statistical Manual of Mental Disorders . The autThe control group comprised 100 age- and sex- matched apparently healthy children They included 78 males and 22 females. They were the healthy older siblings of the healthy infants who attend the Well Baby Clinic, King Khalid University Hospital, Faculty of Medicine, King Saud University, Riyadh, Saudi Arabia for routine following-up of their growth parameters. The control children were not related to the children with autism, and demonstrated no clinical findings suggestive of immunological, GI or neuropsychiatric disorders. Their ages ranged between 3 and 10 years (mean ± SD = 5.96 ± 2 years).None of the studied subjects was vegetarian or was on a restricted diet such as casein and guten-free diet. All studied subjects had normal body weight (body mass index (BMI) was between the fifth and less than the 85th percentiles based on age and sex). Also, none of the studied subjects had abnormal results of stool analysis.The local Ethical Committee of the Faculty of Medicine, King Saud University, Riyadh, Saudi Arabia, approved this study. In addition, an informed written consent of participation in the study was signed by the parents or the legal guardians of the studied subjects.This was based on clinical history taking from caregivers, clinical examination and neuropsychiatric assessment. Gastrointestinal symptoms were assessed with the parent-report version of the Questionnaire on Pediatric Gastrointestinal Symptoms by an experienced pediatric gastroenterologist according to the Questionnaire on Pediatric Gastrointestinal Symptoms - Rome III Version used by previous studies that assessed gastrointestinal dysfunction in autism . In addiThe degree of the disease severity was assessed by using the Childhood Autism Rating Scale (CARS)  which raBlood samples were collected in the morning following at least 10 hour period of fasting. Blood samples were equally subdivided into two clean tubes, the first tube was dry and the blood was left to clot, then the serum was separated and stored at −20°C until assay of carnitine. The second tube was containing fluoride EDTA as an anticoagulant, then centrifugation of the samples was done immediately and plasma was separated and stored in sterile aliquoutes at −80°C until assay of PUFAs.Serum carnitine was measured by using enzymatic ultraviolet test from Roche Diagnostics Gmbh, Cat. No. 1′ 242008, Germany . To incrThe principle was the injection of separated fatty acids from the plasma into the gas chromatography instrument. Separation of fatty acids from plasma was done by addition of 3 ml of 3 g/L solution of nonanoic acid in 100 μL of acetyl chloride slowly with magnetic strirring for 45 min at room temperature. Three ml of 6% potassium carbonate solution was added in water with stiring while cooling the mixture in an ice bath. After that, 300 μL of hexan and vortex were added and the sample was cooled at 4°c for 30 minutes. Then, centrifugation was done for 10 minutes. Finally, 100 μ of the upper layer was removed and 1.1 μL was subjected to Gas Chromatography H5890  . To incrU-test was used for comparison of data. Chi-square test was used for comparison between qualitative variables of the studied groups. Spearman’s rho correlation coefficient “r” was used to determine the relationship between different variables. For all tests, a probability (P) of less than 0.05 was considered significant. As data distribution was non-parametric, patients were considered to have decreased serum carnitine, plasma DHA, linolenic, arachidonic and linoleic acids if their levels were below the 5th percentile of the control values . On the other hand, patients were considered to have elevated ω6/ω3 ratio if their levels were above the 95th percentile of the control values (3.84).The results were analyzed by the commercially available software package . The data was non-parametric and was presented as median and interquartile range (IQR), which is the difference between the 75th and 25th percentiles. Mann–Whitney Autistic patients had significantly lower levels of plasma PUFAs and serum carnitine than healthy children. On the other hand, ω6/w3 ratio (AA/DHA) was significantly higher in autistic patients than healthy children Table . Low serThere was a significant positive association between the reduced serum levels of carnitine and the elevated ω6/w3 ratio in autistic children as the percentage of elevated ω6/w3 was significantly higher in autistic patients with reduced serum levels of carnitine (94.7%) than patients with normal serum carnitine levels (48.4%)), P < 0.001.Plasma levels of PUFAs and serum levels of carnitine had no significant correlations with CARS and the age of autistic children (P > 0.05). Also, plasma levels of PUFAs did not correlated significantly with serum carnitine levels (P > 0.05).Gastrointestinal manifestations were found in 48 autistic children (48%). They include recurrent unexplained attacks of epigastric pain and chromic constipation in 20 patients, chronic constipation and bloating in 22 patients and recurrent attacks of diarrhea, with normal results of stool analysis, in 6 patients. These symptoms were reported by the parents of autistic children. They were recurrent, severe and the patients were attending the clinic because of these agonizing symptoms. None of the autistic children had abnormal results of stool analysis.Autistic patients with GI manifestations had significantly lower serum carnitine and plasma DHA than patients without such manifestations. On the other hand, ω6/w3 ratio was significantly higher in autistic patients with GI problems than patients without such manifestations Table .Table 2Autistic patients with GI manifestations had significantly higher percentage of reduced serum carnitine (91.7%) and plasma DHA levels (87.5%) than patients without such manifestations , . On the other hand, autistic patients with GI manifestations had significantly higher percentage of elevated ω6/w3 ratio (62.5%) than patients without such manifestations (15.4%), P < 0.001.In the current study, autistic patients had significantly lower levels of plasma PUFAs than healthy children. On the other hand, ω6/w3 ratio (AA/DHA) was significantly higher in autistic patients than healthy children. One preliminary study found that average total levels of omega-3 (n-3 PUFAs) in the autistic children were about 20% lower than mentally retarded children used as controls. Levels of DHA were 23% lower. These deficiencies resulted in a significantly higher ratio of n-6 to n-3 in 25% of autistic children ,21,22. A. Some studies of human infants suggest that dietary DHA may play a role in cognitive development and in some neurodevelopmental disorders like autism [The mechanisms by which DHA can impact on neuronal functions involve the modulation of membrane biophysical properties, the regulation of neurotransmitter release (such as dopamine and serotonin), the synthesis of oxygenated biologically active derivatives, and the nuclear receptor mediated transcription of lipid responsive genes . Some ste autism . Placeboe autism  conductee autism .T. Carnitine homeostasis in humans is maintained by a modest rate of endogenous synthesis, absorption from dietary sources, and efficient tubular reabsorption by the kidney [The current study revealed lower levels of serum carnitine in autistic patients than healthy children. A previous study also reported carnitine deficiency in a group of autistic children. Acetyl carnitine is needed for mitochondrial transfere of PUFAs and synaptic transmission of multiple neurotransmitters which affect attention, antagonize deterioration of ability to learn and improve long term memory. Carnitine has been proposed to be a conditionally essential nutrient, and even termed vitamin Be kidney . Carnitie kidney . A delete kidney . Mitochoe kidney ,32.Anhedonia is a condition in which the capacity of experiencing pleasure is totally or partially lost and it was reported in various psychiatric disorders . SelectiIn the present work, GI manifestations  were found in 48%.of autistic patients. The reported prevalence of GI abnormalities in autism is high . A previHow GI factors are related to autism is not yet clear. Many patients with autism have a history of previous antibiotic exposure, GI symptoms, abnormal food cravings, and unique intestinal bacterial populations, which have been proposed to relate to variable symptom severity . Recent In this work, autistic patients with GI manifestations had significantly lower serum carnitine and plasma DHA than patients without such manifestations. These results may demonstrate the possible association between GI manifestations and the reduced levels of serum carnitine and plasma DHA in autistic patients. The relationship between GI manifestations and plasma levels of PUFAs and serum carnitine in autistic children was not previously investigated.+ lymphocytes with increased production of basal and anti-CD3-stimulated pro-inflammatory cytokines in immune cells [All varieties of (n-3) acids  are essential components in mammalian metabolism, whether they act as anti-inflammatory molecules, immune-modulating agents, and the main components in guaranteeing cell membrane stability . PUFAs ane cells . L-carnine cells .This work may denote a possible association between GI manifestations and the reduced levels of serum carnitine and plasma DHA in autistic patients as a result of the decrease of their anti-inflammatory effects. It is not evident if GI manifestations are mere association or consequence to reduced PUFAs and carnitine levels in these patients. Further investigations are recommended, with a larger subject population, to determine the possible pathogenic role of the deficiency of PUFAs and carnitine levels in the occurrence of GI manifestations in autistic patients.Some autistic children have reduced levels of plasma PUFAs and serum carnitine which may be associated to GI manifestations in these patients. However, these data should be treated with caution until further investigations are performed, with a larger subject population, to determine whether the occurrence of GI manifestations is a mere association or a consequence to reduced plasma PUFAs and serum carnitine levels in autistic patients. Studies looking at a potential role of PUFAs and carnitine in treatment of autism and amelioration of GI problems are recommended.