Improving vigour assessment of pine (Pinus nigra Arnold) seedlings before their use in reforestation

ABSTRACT

We investigated whether changes in the root system of pine seedlings induced by stress (lifting of bare-root seedlings from the nursery bed irrespective of dormancy; prolonged storage of bare-root seedlings in a cold room) could provide a measure of plant vigour. Physiological parameters, such as growth potential and root electrolyte leakage, and morphological parameters, such as root length and number of root tips, were calculated. Computerised image analysis was used to measure root growth, overall and based on root-diameter class (0–0.5 mm, 0.5–1.0 mm and 1.0–1.5 mm). The efficiency of vigour assessment was evaluated by correlating the data for each parameter with percentage seedling survival. Root growth potential was more efficient than root electrolyte leakage, but both parameters were affected by seedling age. Total root length was a more efficient indicator of plant vigour than root tip number, particularly when referred to roots of the same diameter class. A comparative analysis of physiological and morphological parameters referred to the root systems improves their relative effciency.     

Issues in using the WinRHIZO system to determine physical characteristics of plant fine roots

Image analysis systems have facilitated rapid measurements of fine root length (RL), diameter (RD), volume (RV), etc. The WinRHIZO system is unlike other image analysis systems in that it can detect, and make corrections for, areas of root overlap. It is designed to be capable of using both Regent’s non-statistical method (WinRHIZO method) and Tennant’s statistical method (line-intersect method), and can simultaneously output the root measurements by both methods when they are chosen at the same time. This study tested: (1), the efficacy of the overlap correction function in the WinRHIZO system; and (2), the consistency of fine root measurements between the WinRHIZO and Tennant methods with two sets of root measurement data from winter wheat (Triticum aestivium L.). The results showed that there were significant differences in RL, RD and RV between small root samples with and without stumps. The impact of root stumps outweighed the overlap correction efficacy in WinRHIZO. The values from the Tennant method are significantly different from those using the WinRHIZO method, although both results are statistically closely correlated. This indicated how critical it was to use without-stump root samples when using image analysis systems to measure RL, RD, RV, etc., and to keep in mind that a significant difference in root measurements may be methodologically related when comparing the results of various experiments from these two methods. Our research results bear important implications for the study of root ecology.     

Issues in using the WinRHIZO system to determine physical characteristics of plant fine roots☆

Image analysis systems have facilitated rapid measurements of fine root length (RL), diameter (RD), volume (RV), etc. The WinRHIZO system is unlike other image analysis systems in that it can detect, and make corrections for, areas of root overlap. It is designed to be capable of using both Regent’s non-statistical method (WinRHIZO method) and Tennant’s statistical method (line-intersect method), and can simultaneously output the root measurements by both methods when they are chosen at the same time. This study tested: (1), the efficacy of the overlap correction function in the WinRHIZO system; and (2), the consistency of fine root measurements between the WinRHIZO and Tennant methods with two sets of root measurement data from winter wheat (Triticum aestivium L.). The results showed that there were significant differences in RL, RD and RV between small root samples with and without stumps. The impact of root stumps outweighed the overlap correction efficacy in WinRHIZO. The values from the Tennant method are significantly different from those using the WinRHIZO method, although both results are statistically closely correlated. This indicated how critical it was to use without-stump root samples when using image analysis systems to measure RL, RD, RV, etc., and to keep in mind that a significant difference in root measurements may be methodologically related when comparing the results of various experiments from these two methods. Our research results bear important implications for the study of root ecology.     

Automated image analyses for separating plant roots from soil debris elutrated from soil cores

Historically, destructive root sampling has been labor intensive and requires manual separation of extraneous organic debris recovered along with the hydropneumatic elutriation method of separating plant roots from soils. Quantification of root system demographics by public domain National Institute of Health (NIH-Image) and Root Image Processing Laboratory (RIPL) image processing algorithms has eliminated much of the labor-intensive manual separation. This was accomplished by determining the best length to diameter ratio for each object during image analyses. Objects with a length to diameter ratio less than a given threshold are considered non-root materials and are rejected automatically by computer algorithms. Iterative analyses of length to diameter ratios showed that a 15:1 ratio was best for separating images of maize (Zea mays L.) roots from associated organic debris. Using this threshold ratio for a set of 24 soil cores, a highly significant correlation (r² = 0.89) was obtained between computer image processed total root length per core and actual root length. A linear relationship (r² = 0.80) was observed between root lengths determined by NIH-Image analyses and lengths determined independently by the RIPL imaging system, using the same maize root + debris samples. This correlation demonstrates that computer image processing provides opportunities for comparing root length parameters between different laboratories for samples containing debris.     

Using a fine root extraction device to quantify small diameter corn roots (≥0.025 mm) in field soils

The study of fine roots growing under field conditions is limited by the techniques currently available for separating these roots from soil. This study had two objectives: to measure the total root length of field grown corn (Zea mays L.) by root diameter class, and to develop an inexpensive and efficient root washing device that would effectively capture all of the roots in a field soil sample. An inexpensive Fine Root Extraction Device (FRED) was constructed from readily available materials and was successful at extracting all roots, including very fine diameter roots (0.025 mm), from field soil samples. Greater than 99.7% of marked roots introduced to the FRED were recaptured by the device. Soil samples from three depths, and on three dates, from field grown corn were placed in the FRED. We found that more than 56% of total root length occurred in roots whose diameters were smaller than 0.175 mm, and more than 35% of root length occurred in roots smaller than 0.125 mm in diameter. Corn roots of the diameters described here have not been reported in field soils prior to this study. Root researchers who fail to measure these very fine roots will significantly underestimate root length density. Widespread use of the FRED should improve our understanding of root distribution in field soils.     

Two independent estimations of stand-level root respiration on clonal Eucalyptus stands in Congo: up scaling of direct measurements on roots versus the trenched-plot technique

Root respiration at the level of a forest stand, an important component of ecosystem carbon balance, has been estimated in the past using various methods, most of them being indirect and relying on soil respiration measurements. On a 3-yr-old Eucalyptus stand in Congo-Brazzaville, a method involving the upscaling of direct measurements made on roots in situ, was compared with an independent approach using soil respiration measurements conducted on control and trenched plots (i.e. without living roots). The first estimation was based on the knowledge of root-diameter distribution and on a relationship between root diameter and specific respiration rates. The direct technique involving the upscaling of direct measurements on roots resulted in an estimation of 1.53 micromol m(-2) s(-1), c. 50% higher than the mean estimation obtained with the indirect technique (1.05 micromol m(-2) s(-1)). Monte-Carlo simulations showed that the results carried high uncertainty, but this uncertainty was no higher for the direct method than for the trenched-plot method. The reduction of the uncertainties on upscaled results requires more extensive knowledge of temperature sensitivity and more confidence and precision on the respiration rates and biomasses of fine roots.     

Genotype and Planting Density Effects on Rooting Traits and Yield in Cotton （Gossypium hirsutum L,）

Root density distribution of plants is a major Indicator of competition between plants and determines resource capture from the solh This experiment was conducted in 2005 at Anyang, located in the Yellow River region, Henan Province, China. Three cotton （Gossyplum hlrsutum L.） cultivars were chosen： hybrid Btcultlvar CRI46, conventional Btcultlvars CRI44 and CRI45. Six planting densities were designed, ranging from 1.5 to 12.0 plants/m^2. Root parameters such as surface area, diameter and length were analyzed by using the DT-SCAN Image analysis method. The root length density （RLD）, root average diameter and root area Index （RAI）, root surface area per unit land area, were studied. The results showed that RLD and RAI differed between genotypes; hybrid CRI46 had significantly higher （P 〈0.05） RLD and RAI values than conventlonal cultlvars, especially under low planting densities, less than 3.0 plants/m^2. The root area index （RAI） of hybrid CRI46 was 61% higher than of CRI44 and CRI45 at the flowering stage. The RLD and RAI were also significantly different （P = 0.000） between planting densities. The depth distribution of RAI showed that at Increasing planting densities RAI was Increasingly distributed in the soil layers below 50 cm. The RAI of hybrid CRI46 was for all planting densities, obviously higher than other cultivars during the flowering and boll stages. It was concluded that the hybrid had a strong advantage in root maintenance preventing premature senescence of roots. The root diameter of hybrid CRI46 had a genetically higher root diameter at planting densities lower than 6.0 plants/m^2. Good associations were found between yield and RAI In different stages. The optimum planting density ranged from 4.50 plants/m^2 to 6.75 plants/m^2 for conventional cultlvars and around 4.0-5.0 plants/m^2 for hybrids.     

Significance of temperature and precipitation for maize root distribution in the field

Measurements of maize (Zea mays L.) root distribution with depth in the soil for nine years in a 11-year period revealed significantly different distribution patterns. Weather variations were expected to be related to the amount of roots found in each of the five 15-cm soil layers. The objective of this study was to attempt to explain root distribution in the field on the basis of precipitation and temperature data for the nine growing seasons. Growing degree days (GDD), accumulated in daily increments from planting to silking, were used to describe temperature effects. Correlations were calculated for weekly time increments of GDD versus root length densities at silking in all soil layers. Root length density below 30 cm was correlated (P=0.05) with GDD for two weeks following planting, whereas no relation was found between GDD and root length density in the topsoil. Amount of precipitation was accumulated in weekly increments from silking to planting and correlated with root length density in the soil layers at silking. This procedure evaluated the relation between precipitation and root growth during the vegetative growth period. Root length density in the 0 to 15 cm layer was found to be related significantly (P=0.05) to precipitation. The period 3 weeks prior silking gave the highest correlation coefficient (r=0.79). Journal Paper no. 10,629. Purdue Univ. Agric. Exp. Stn., W. Lafayette, IN 47907. Contribution from the Dep. of Agronomy. The research was supported in part by BARD, United States-Israel Binational Agricultural Research and Development Fund, and Deutsche Forschungsgemeinschaft.     

短期氮磷配施对刨花楠细根形态及其土壤微生物的影响

以1年生刨花楠幼苗为研究对象,通过不同的氮磷配施实验,采用扫描根系法和磷脂脂肪酸法,研究不同氮磷配施处理对刨花楠幼苗1—4级细根根序形态特征及其土壤微生物的影响。结果表明:(1)4种氮磷配施处理均显著增加了刨花楠1—2级根的比根长和比根面积(P0.05),降低了3—4级根的比根面积(P0.05);(2)通过不同梯度的氮磷配施,1—2级细根的根组织密度呈下降态势,而3—4级根的组织密度则显著增加(P0.05),体现低级根与高级根之间的权衡;(3)4种氮磷配施处理都显著降低刨花楠1—4级细根的平均直径(P0.05);(4)随着氮磷比的增加,微生物总量及细菌、真菌与放线菌数量等均呈现先增加后降低的趋势,并均在N∶P为10∶1时达到最大;(5)氮磷配施条件下,细菌、真菌等与1—2级细根的比根长和比根面积呈显著正相关,而与4级根的比根长和比根面积则呈显著负相关,革兰氏阳性菌、真菌等与3—4级根的组织密度存在显著正相关,而与1—2级根的组织密度无显著相关性。各级根序的平均直径均与土壤微生物无显著相关性。研究结果表明,短期氮磷配施以N∶P为10∶1的效果最好,其最有利于提高刨花楠苗木细根的养分吸收能力与养分吸收效率,苗木通过调整细根形态来适应氮沉降,其地下生物群落如土壤微生物及其与细根的关系也发生变化,进而影响地下生态系统碳氮循环和养分流动。     

Linking root respiration to chemistry and morphology across species

Root respiration is a critical physiological trait involved in root resource acquisition strategies, yet it is less represented in root trait syndrome. Here we compiled a large dataset of root respiration associated with root chemical and morphological traits from 245 plant species. Our results demonstrated that root respiration correlated positively with root nitrogen concentration (RNC) and negatively with root tissue density (RTD) across and within woody and non‐woody species. However, the relationships between root respiration and specific root length (SRL) and root diameter (RD) were weak or even insignificant. Such root respiration–traits relationships were not completely in line with predictions by the root economics spectrum (RES). Furthermore, the principal component analysis showed that root trait syndrome was multidimensional. Root respiration was associated more strongly with the RNC‐RTD axis (the classical RES) than with the orthogonal SRL‐RD axis for woody species, but not for non‐woody species. Collectively, the linkages of root physiological, chemical, and morphological traits provide a better understanding of root trait covariation and root resource acquisition strategies.     

星毛委陵菜根系构型对草原退化的生态适应

对轻度、中度、重度和极度退化的草原群落中星毛委陵菜(Potentilla acaulis)根系构型参数及相应的土壤水分、容重和硬度等指标进行了分析, 以研究星毛委陵菜根系构型对草原退化的生态适应性。结果表明: 1)在以大针茅(Stipa grandis)为建群种的典型草原中, 随着退化程度的加剧, 星毛委陵菜在群落中的作用逐渐增强, 其根幅、根深、一级垂向根数、分蘖子株数和水平分蘖根长度显著增加; 2)根表面积、二级侧根长度、总根长和根分叉数4个根系构型参数是解释星毛委陵菜根系构型对草原退化生态适应的首选指标, 解释力依次减小, 累计贡献率为92.34%; 3)直径2 mm以下的根系对单株系星毛委陵菜的根表面积和总根长影响显著; 4)阔腰倒锥体三维根系构型是星毛委陵菜适应草原退化并使之成为建群种的优势构型。     

Patterns of variability in the diameter of lateral roots in the banana root system

The relative importance of root system structure, plant carbon status and soil environment in the determination of lateral root diameter remains unclear, and was investigated in this study. Banana (Musa acuminata) plants were grown at various moderate levels of soil compaction in two distinct experiments, in a field experiment (FE) and in a glasshouse experiment (GE). Radiant flux density was 5 times lower in GE. The distribution of root diameter was measured for several root branching orders. Root diameters ranged between 0.09 and 0.52 mm for secondary roots and between 0.06 and 0.27 mm for tertiary roots. A relationship was found between the diameter of the parent bearing root and the median diameter of its laterals, which appears to be valid for a wide range of species. Mean lateral root diameter increased with distance to the base of the root and decreased with branching density [number of lateral roots per unit length of bearing root (cm(-1))]. Typical symptoms of low light availability were observed in GE. In this case, lateral root diameter variability was reduced. Although primary root growth was affected by soil compaction, no effects on lateral root diameter were observed.     

Root distribution and water uptake patterns of corn under surface and subsurface drip irrigation

Information on root distribution and uptake patterns is useful to better understand crop responses to irrigation and fertigation, especially with the limited wetted soil volumes which develop under drip irrigation. Plant water uptake patterns play an important role in the success of drip irrigation system design and management. Here the root systems of corn were characterized by their length density (RLD) and root water uptake (RWU). Comparisons were made between the spatial patterns of corn RWU and RLD under surface and subsurface drip irrigation in a silt loam soil, considering a drip line on a crop row and between crop rows. Water uptake distribution was measured with an array of TDR probes at high spatial and temporal resolution. Root length density was measured by sampling soil cores on a grid centered on crop row. Roots were separated and an estimation of root geometrical attributes was made using two different image analysis programs. Comparisons of these programs yielded nearly identical estimates of RLD. The spatial patterns of RWU and RLD distributions, respectively normalized to the total uptake and root length, were generally similar only for drip line on a crop row, but with some local variations between the two measures. Both RLD and RWU were adequately fitted with parametric models based on semi-lognormal and normal Gaussian bivariate density functions (Coelho and Or, 1996; Soil Sci. Soc. Am. J. 60, 1039–1049).     

A method to separate plant roots from soil and analyze root surface area

Analysis of the effects of soil management practices on crop production requires knowledge of these effects on plant roots. Much time is required to wash plant roots from soil and separate the living plant roots from organic debris and previous years’ roots. We developed a root washer that can accommodate relatively large soil samples for washing. The root washer has a rotary design and will accommodate up to 24 samples (100 mm diam. by 240 mm long) at one time. We used a flat-bed scanner to digitize an image of the roots from each sample and used a grid system with commercially-available image analysis software to analyze each sample for root surface area. Sensitivity analysis and subsequent comparisons of ‘dirty’ samples containing the roots and all the organic debris contained in the sample and ‘clean’ samples where the organic debris was manually removed from each sample showed that up to 15% of the projected image could be coveredwith debris without affecting accuracy and precision of root surface area measurements. Samples containing a large amount of debris may need to be partitioned into more than one scanning tray to allow accurate measurements of the root surface area. Sample processing time was reduced from 20 h, when hand separation of roots from debris was used, to about 0.5 h, when analyzing the image from an uncleaned sample. The method minimizes the need for preprocessing steps such as dying the roots to get better image contrast for image analysis. Some information, such as root length, root diameter classes and root weights, is not obtained when using this technique. Root length measurements, if needed, could be made by hand on the digital images. Root weight measurement would require sample cleaning and the advantage of less processing time per sample with this method would be lost. The significance of the tradeoff between information not obtained using this technique and the ability to process a greater number of samples with the time and personnel resources available must be determined by the individual researcher and research objectives.     

Distinguishing between metabolically active and inactive roots by combined staining with 2,3,5-triphenyltetrazolium chloride and image colour analysis

The objective of the present work was to develop a method to distinguish between metabolically inactive and active parts of plant roots. White clover (Trifolium repens L.) roots were stained with 2,3,5-triphenyltetrazolium chloride (TTC) followed by root colour classification with an interactive scanner-based image analysis programme (WinRHIZO). Roots inactivated by boiling were unstained and pale brown, whereas fresh samples with predominantly metabolically active roots turned dark red, red or pale red after staining. A small amount of very young, presumable active roots (0.8% of total active root length) failed to stain red with TTC. The colour analysis of inactive and active roots was based on four colour classes for boiled roots and seven classes for fresh roots, respectively, as defined upon visual examination of images. Pixel colours falling outside the defined classes were allocated to the nearest defined class – an option that increased objectivity and stability and reduced the required number of colour classes. For the fresh white clover roots, 75–86% of the total root length was determined as active, while 3–7% of the boiled roots fell into the same category. The percentage of total root length measured by WinRHIZO that was identified as metabolically active was linearly correlated with the percentage of fresh roots in mixtures of fresh and boiled roots (R²=0.99). Colour classes chosen à priori from one experiment could be used to distinguish fairly satisfactorily between active and inactive roots of another white clover cultivar grown under other conditions, but failed to classify activity in ryegrass (Lolium multiflorum Lam.) root samples. In the latter case, colour classes needed to be re-defined in order to produce reliable data. Our work shows that WinRHIZOs colour identification sub-module provides a new promising tool to classify root activity as identified after staining with TTC, but colour classes must be carefully evaluated on every new occasion.     

Effects of Soil Structure on Pea (Pisum sativum L.) Root Development According to Sowing Date and Cultivar

Spring peas are known to be very sensitive to compaction, particularly when sowing takes place soon after winter. Winter peas, which are sown in autumn, should present an opportunity to sow the crop in better soil structural conditions than for spring peas, because of more favourable moisture conditions at that time. As environmental conditions have a big influence on root systems, it is important to determine the effects of soil structure on pea root systems for different cultivars and sowing dates. A spring pea cultivar and a winter pea cultivar were both sown at two dates (one in autumn and one in spring) on soils with different plough-layer structures (compacted and uncompacted) at two sites in 2002 and one site in 2003. Soil structure was characterised by bulk density and the percentage of highly compacted zones in the ploughed layer. Root distribution maps were produced every month, from February to maturity. Root development was described in terms of general root dynamics, root elongation rate (RER) in the subsoil, final maximum root depth (Dmax) and root distribution at maturity. Root depth dynamics depended on compaction and its interaction with climatic conditions. The effects of compaction on RER in the subsoil depended on the experimental conditions. Dmax was reduced by 0.10 m by compaction. Compaction also reduced root distribution between 10 and 40% in the ploughed layer only. Pea cultivars differed in sensitivity to soil compaction, with a direct effect on the final depth explored by roots. These results are discussed in terms of their relevance to water and nutrient uptake.     

Response of plant roots to elevated atmospheric carbon dioxide

Plant root response to atmospheric CO₂ enrichment can be great. Results from this controlled environment investigation demonstrate substantial effects on root system architecture, micromorphology and physiology. The most pronounced effects were an increase in root length (110%) and root dry weight (143%). Root diameter, stele diameter, cortex width, root/shoot and root weight ratios all increased; root numbers did not increase. The long-term implications for belowground processes could be enormous.     

亚热带典型树种根毛特征及其与共生真菌的关系

根毛和共生真菌增加了吸收面积,提高了植物获取磷等土壤资源的能力。由于野外原位观测根表微观结构较为困难,吸收细根、根毛、共生真菌如何相互作用并适应土壤资源供应,缺乏相应的数据和理论。该研究以受磷限制的亚热带森林为对象,选取了21种典型树种,定量了根毛存在情况、属性变异,分析了根毛形态特征与共生真菌侵染率、吸收细根功能属性之间的关系,探讨了根表结构对低磷土壤的响应和适应格局。结果表明:1)在亚热带森林根毛不是普遍存在的, 21个树种中仅发现7个树种存有根毛, 4个为丛枝菌根(AM)树种, 3个为外生菌根(ECM)树种。其中,马尾松(Pinus massoniana)根毛出现率最高,为86%;2)菌根类型是理解根-根毛-共生真菌关系的关键,AM树种根毛密度与共生真菌侵染率正相关,但ECM树种根毛直径与共生真菌侵染率负相关; 3) AM树种根毛长度和根毛直径、ECM树种根毛出现率与土壤有效磷含量呈负相关关系。该研究揭示了不同菌根类型树种根毛-共生真菌-根属性的格局及相互作用,为精细理解养分获取策略奠定了基础。     

IJ_Rhizo: an open-source software to measure scanned images of root samples

Background and aims

This paper provides an overview of the measuring capabilities of IJ_Rhizo, an ImageJ macro that measures scanned images of washed root samples. IJ_Rhizo is open-source, platform-independent and offers a simple graphic user interface (GUI) for a main audience of non-programmer scientists. Being open-source based, it is also fully modifiable to accommodate the specific needs of the more computer-literate users. A comparison of IJ_Rhizo’s performance with that of the widely used commercial package WinRHIZO? is discussed.

Methods

We compared IJ_Rhizo’s performance with that of the commercial package WinRHIZO? using two sets of images, one comprising test-line images, the second consisting of images of root samples collected in the field. IJ_Rhizo and WinRHIZO? estimates were compared by means of correlation and regression analysis.

Results

IJ_Rhizo “Kimura” and WinRHIZO? “Tennant” were the length estimates that were best linearly correlated with each other. Correlation between average root diameter estimates was weaker, due to the sensitivity of this parameter to thresholding and filtering of image background noise.

Conclusions

Overall, IJ_Rhizo offers new opportunities for researchers who cannot afford the cost of commercial software packages to carry out automated measurement of scanned images of root samples, without sacrificing accuracy.     

A procedure for determining average root length density in row crops by single-site augering

A simplified procedure has been formulated and tested for determining average root length density (RLD) by auger sampling at a single site in wheat, corn and mustard. It involves the determination of horizontal root distribution in the representative half of the unit soil strip (distance from base of plant to mid-point in the rows) by excavating small monolith segments in the top soil layer. Average RLD is computed by dividing the integral of polynomial function fitted to the horizontal root distribution (in the unit soil strip) with its length. The average RLD, thus, obtained is interpolated on the curve between root length density and horizontal distance from the plant base (d) in the representative half of the unit soil strip. Root length density determined by centering 5 cm diameter auger at the interpolated d gave minimum deviation from the average RLD of that layer compared to the other possible single site sampling schemes with same-sized auger. These results indicate that for row crops, the best centre for single-site augering is about one-third of distance from the plant base to mid-way between the two rows.