Blackwell Science Ltd
Longevity of mycorrhizal roots depends on branching
order and nutrient availability
Hooshang Majdi1, Eva Damm2 and Jan-Erik Nylund3
1Department of Ecology and Environmental Research, PO Box 7072, Sweden; 2Department of Forest Mycology and Pathology, PO Box 7026, Sweden; 3Department of Forest Management and Products, PO Box 7060, SE-750 07 Uppsala, Sweden
• The effects are reported of branching order and nitrogen fertilization on the lon-gevity of mycorrhizal short roots at a Norway spruce (Picea abies) stand in south-west Sweden.
• A 4-yr qualitative analysis was made, using minirhizotrons, of the emergence fre-quency and life span of individual mycorrhizal roots of three root orders (unbranched, main axes of branched and side branches of branched short roots, orders 1, 2 and 3, respectively).
• Longevity of mycorrhizal roots varied with branching order and soil depth, being greatest in unbranched (order 1) roots and those at the greatest soil depth (40– 85 cm). Nitrogen addition decreased the proportion of higher mycorrhizal root orders (a fivefold reduction in order 3 compared with controls), while increasing the median longevity. Seasonal effects were recorded; mycorrhizal roots produced in spring and summer remained unsuberized during the winter.
• Mycorrhizal root longevity depends on the branching order of mycorrhizal roots and nitrogen addition decreases branching density while increasing longevity. The survival of mycorrhizal roots, which remain vital and unsuberized over winter, enhances water and nutrient efficiency of Norway spruce.
Key words: Branching order, longevity, mycorrhizal roots, minirhizotrons, median longevity, nitrogen.
© New Phytologist (2001) 150: 195–202 Author for correspondence:
Hooshang Majdi Tel: +0046 18672428 Fax: +0046 18673430 Email: hooshang.Majdi@eom.slu.se Received: 27 July 2000 Accepted: 3 November 2000 Introduction
The dynamics of short root emergence and survival of ecto-mycorrhizal roots in forest trees has not been a very well under-stood area of tree and soil sciences until recently. Emergence and decline of fine roots occur simultaneously during the growing season, making estimates of production and mortality by sequential destructive methods impossible. Additionally, mycorrhizal roots with small diameter, that is < 0.3 mm, through which most of the mineral and water uptake takes place ( Jensén & Petersson, 1980; Yanai, 1994), are not detected using these methods (Hendrick & Pregitzer, 1993). The total surface area of roots varies as a function of number, size, and longevity (Fitter & Stickland, 1992). Like leaves, mycorrhizal roots are modular in nature, and their emergence and
disap-pearance may be very dynamic (Hooker et al., 1995; Fitter, 1996; Majdi & Nylund, 1996).
Tree seedlings grown in observation boxes, using artificial substrates (Finlay & Read, 1986) have been employed for ecto-mycorrhiza studies, but no data on their dynamics (production and longevity) and branching order have been presented, sup-posedly because of the artificiality of the systems and the lack of methods to follow individual roots. Previous mycorrhizal studies (Kårén et al., 1997) in the present study site have been focused on species diversity as observed through fruit bodies and by PCR-RFLP ( genetic fingerprinting ) of root tips.
Minirhizotrons have been used to study root processes from demographic perspective, and to quantify the rates of mycorrhizal short root production and longevity (Majdi & Nylund, 1996). However, studies on the pattern of mycorrhizal NPH065.fm Page 195 Tuesday, February 27, 2001 10:49 AM
root emergence in relation to branching order and nutrient availability are rare and the influence of mycorrhizal root order on life span has not been investigated earlier. The advent of the minirhizotron technique has opened up new opportun-ities in this field. In some previous studies (Majdi & Kangas, 1997), the dynamics of long roots (production, and longev-ity) have been studied, and the possibilities and limitations of this technique have been discussed (Majdi, 1996).
In this paper, we present a qualitative analysis over a 4-yr period on ethology viz emergence and life span of mycorrhizal roots. Our main objective was to investigate the emergence frequency of individual mycorrhizal roots (< 0.3 mm in dia-meter) and their longevity in relation to branching order and nutrient availability in a Norway spruce stand in SW Sweden. Materials and Methods
The experimental area, Skogaby, is located in south-west Sweden (56°33′ N, 13°13′ E) and is 95 – 115 m above sea level. The site is a homogeneous, second-rotation Norway spruce stand planted in 1966 on a stony sandy loam developed in glacial till. Current wet deposition of nitrogen and sulphur at the site is about 18 and 10 kg ha−1 yr−1, respectively, and
throughfall deposition is 16 and 20 kg ha−1 yr−1, respectively
(K. Wiklund, pers. comm.). The soil is a Haplic podzol with clay content of 3 – 4% in the whole profile and a sand fraction (> 0.06 mm) of about 70 %. The effective base saturation prior to treatments in the humus layer was 30% and in mineral soil layers varied between 7% –12%. The pH (H2O) in the humus layer was 3.9 and in upper mineral soil layers 4.1. Further physical and chemical characteristics of the soil are described by Bergholm et al. (1995).
Field plots measuring 45 × 45 m were established in 1987 in a randomised experimental design with four blocks and six treatments (Bergholm et al., 1995). The field experiment started during the growing season of 1988.
In this study, we report data from 1991 to 1994 from the control (C) and the liquid fertilization treatment (addition of 100 kg N ha−1 and other nutrients yearly; IL). The ground
vegeta-tion is sparse, dominated by mosses, the grass (Deschampsia
flexuosa) and some bilberry (Vaccinium myrtillus).
Minirhizotrons were installed in 1990 and recording of roots started 1 yr after installation in order to maintain stabilization of the density of the root systems (cf. Joslin & Wolfe, 1999). Fifty minirhizotrons (100 cm long, 5 cm inside diameter and 6 cm outside diameter) were installed vertically to obtain accurate root intersections due to prevalent spruce fine root orientation. Tubes were labelled with an identification number and an index reference hole was made 5 cm from the top end to
standardize the orientation of the video camera at all sampling dates. Date, tube number and frame number were recorded on S-VHS videotape, using a microphone. We specifically studied four minirhizotrons from C and IL plots to investigate the pattern of mycorrhizal fine root emergence from individual long roots during 1991–94. The number of long roots in the present study was 70 and 500 associated individual mycorrhizal roots were followed during a 4-yr period. All minirhizotron tubes were recorded at monthly intervals beginning in August 1991. In total, 6000 frames (1.12 ×1.35 cm) were examined. When roots were observed in an image as white in colour for the first time they were considered as ‘new’ and if they remained white in colour at subsequent dates they were typi-fied as ‘white’. The new root remained white and turgid for a long time, but later it started to get an increasingly brown colour, due to incrustation of phenolics in the cortical cells, and accom-panied by increasing endoderm suberization (cf. Nylund, 1987). This transition is accompanied by decreased internal metabolism measured as carbohydrate turnover and nutrient uptake measured as P uptake (cf. Jensén & Petersson, 1980). Examining the videotapes, we were usually unanimous about the classification of a root as ‘dead’. A change in colour to black and loss of turgid of the apex were taken as criteria. Particularly long roots, but also order 1 and 2 mycorrhizal roots (see below, Data recording) could however, sometimes show renewed apical growth. Thus, a root classified as ‘dead’ may have a residual stellar activity, while the cortex cells are totally nonfunctional.
The images from videotapes were displayed on a colour monitor, and all new short roots associated with a long root were marked up on b/w printouts with information on emergence date, the mycorrhizal orders (see below), tube number and depth. The roots in each of the frames were at subsequent times organized in chronosequences to follow the development of individual short roots. As we followed all the recorded roots from all frames until they totally disappeared, we could measure the longevity of different mycorrhizal root orders. Kårén & Nylund (1997) investigated the mycorrhizal status of the study site and found a number of morphotypes in the humus layer. In our data all root tips recorded by minirhizotrons had a similar appearance. We found only one morphotype in the mineral soil, which was similar to that in the humus layer. However, judging from findings by Kårén & Nylund (1997) this unique ‘morphotype’ is likely a result of the colonization of several different species.
The long and mycorrhizal roots were classified (Fig. 1) according to the following definitions:
I) Long roots could normally be recognized from the short roots by appearance, growth speed, and lack of mycorrhiza.
II) Mycorrhizal roots could easily be distinguished from nonmycorrhizal ones, the former having woolly, turgid tips, the latter bearing root hairs and usually being pointed and being extremely rare.
III) Mycorrhizal short roots were classified according to branching as: order (1) unbranched, all those always stayed unbranched; order (2) main axis of branched and; order (3) side branches of branched roots.
Statistical analysis of the numbers of mycorrhizal roots in different dates and treatments was carried out using a two-way ANOVA design (P < 0.05) with emergence dates and treatment as main factors. Pairwaise comparisons between means were made using the Sidak t-test (P < 0.05). The Kaplan-Meier product-limit estimator curve (Kaplan & Meier, 1958) was used to describe the surviving proportion of mycorrhizal roots as a function of their age. Paired comparisons were made between treatments using a log rank test (Mantel & Haenszel, 1959, P < 0.001). The log rank test compares the order in which the roots die in groups and not in the medians. Results
Formation of long and short roots: seasonal patterns
Fig. 2(a,b) shows the relationship between the number of long roots and their associated mycorrhizal roots produced in C and IL plots. Pooling orders and date the total number of
mycorrhizal roots was significantly (P < 0.05) higher in IL plots relative to control plots. The average ratio of mycorrhizal root : long root, pooling birth dates, was in control plots 4 and IL plots 3. Pooling root orders, the emergence date of long roots had a significant effect on the emergence of their corresponding mycorrhizal roots especially in June, July and August 1992 (Fig. 2a,b).
Effect of mycorrhizal order and depth on life span
The mycorrhizal root orders had different longevity patterns during the study period. Longevity of mycorrhizal root order 3 was significantly (Log-rank test, P < 0.001) lower than other mycorrhizal orders (1 and 2) and those in order 2 showed higher longevity compared to order 3 (Fig. 3a). There was no significant different between the longevity of orders 1 and 2 (Fig. 3a). The median longevity of orders 1, 2 and 3 were 750, 980 and 400 d, respectively. Mycorrhizal roots at 0 – 20 cm depth had a shorter median longevity than those at 20 – 40 and 40 – 85 cm. The median longevity of mycorrhizal roots at 0 – 20, 20 – 40 and 40 – 85 cm depth was 600, 764 and 1000 d, respectively (Fig. 3b).
Effect of the treatments
The IL treatment had a significant effect on the relative frequency of the root orders. In the control plots with relatively low nutrient
Fig. 1Root system of Norway spruce showing mycorrhizal orders 1, 2 and 3 in relation to the long root.
levels, branched (order 2 and 3) mycorrhizal roots pre-dominated (Fig. 4a). Addition of water, nitrogen and other nutrients by the IL treatment resulted in most short roots being unbranched (order 1).
The degree of root branching (order 3 : order2-ratio) in C and IL plots at different depths showed different patterns (Fig. 4b). The nutrient availability is higher in the upper soil layer both in control and IL plots (Bergholm, unpublished
data). The branching density increased with increasing depth in treatment plots and at 0 – 20 cm the IL treatment decreased the branching density five times compared to control and more than 50% at greater depths (Fig. 4b).
Root longevity (pooling depths and orders) in IL and con-trol plots was significantly (P < 0.05) different (Fig. 5). The median life span of mycorrhizal roots in control and IL plots was 600 and 675 d, respectively.
Fig. 2 Means (n = 3) of production of long and mycorrhizal roots from the 1991–94 in control plots (a) and in IL plots from the 1991–93 (b).
Abbreviations: C, control; IL, irrigation liquid fertilisation. Numbers above bars indicate mycorrhizal root : long root ratios. NPH065.fm Page 198 Tuesday, February 27, 2001 10:49 AM
Previous results from the study site showed that long roots in general have a long life span even with high nitrogen input (Majdi & Kangas, 1997), and that root production was highest in late summer. The emergence of mycorrhizal roots in relation to long root production has a seasonal variability as long root production is low from the beginning of the growing season and emergence of mycorrhizal roots from these roots is high (Fig. 2a,b).
In contrast to data obtained from sequential cores, the results from the present study show that minirhizotron technique can be used to study even the dynamics of mycorrhizal roots since the emergence and disappearance of mycorrhizal roots can be measured simultaneously. However, minirhizotron studies are time consuming especially when root longevity is high. The present results cover mainly mineral soil layers as LFH-layer only occupied 3 frames (1.12 ×1.35 cm). Yet 50%
of fine roots are located in mineral soil layers (cf. Majdi & Persson, 1995).
The root order within the branched hierarchy of the roots (orders 1, 2 and 3) was clearly an important determinant of life span. The median longevity of root order 3 was much lower than other mycorrhizal root orders (Fig. 3a). In contrast Reid et al. (1993), reported the longer life span for higher order of nonmycorrhizal roots of kiwifruit.
The principal reason why roots of order 3 had a shorter life span lies in the fact that mature order 2 roots carry them, and thus they emerge later but die simultaneously with their carriers. However, the difference may also be caused by the distance to the long root, which reduces access to carbon from the long root, and also by the greater activity in terms of water and nutrient uptake and respiration (Pregitzer et al., 1997). The root orders 1 and 2 remained unsuberized and vital during the winter. These short roots ought to be important for water and nutrient retention during the winter since they
Fig. 3(a) Longevity of different mycorrhizal
root orders across depths and treatments during 1991–94. (b) Longevity of mycorrhizal short roots at three depths across treatments during 1991–94.
occur at depths where the soil is not normally frozen, but also in early spring when trees start growing and new root produc-tion has not started.
In agreement with earlier root studies (Majdi & Kangas, 1997) the median longevity of mycorrhizal roots (across orders) decreased with depth (Fig. 3b). This effect may be related to soil temperature and water availability (Eissenstat & Yanai, 1997). The percentage of mycorrhizal root orders showed
different patterns in control and IL treatments (Fig. 4a). Addi-tion of nitrogen decreased the number of mycorrhizal root order 3 proportionally compared to other orders while in control plots with relatively less access to nitrogen and water the branched density of mycorrhizal roots (orders 2 and 3) was increased.
Our findings show that in relatively nutrient poor soils, root systems exploit the soil by increased branching. Nitrogen
Fig. 4 (a) The percentage of individual
myco-rrhizal orders within control and IL plots. Abbreviations: C, control; IL, irrigation liquid fertilisation. (b) The ratio of mycorrhizal orders (order 3 : order 2) at three depth and control and IL plots. Abbreviations: C, control; IL, irrigation liquid fertilisation. NPH065.fm Page 200 Tuesday, February 27, 2001 10:49 AM
availability decreased the number of mycorrhizal roots (order 3) and enhanced the proportion of unbranched mycorrhizal roots (Fig. 4a). In culture experiments, Read (1991) reported that the ectomycorrhiza formation can be regarded as ecolog-ically advantageous, facilitating the exploitation of nutrient-poor environments. Other results on effects of nitrogen addition (ammonium sulphate) indicate that neither mycor-rhizal colonization nor relative amount of fungal biomass decrease (Kårén & Nylund, 1997).
The similar pattern of longevity in IL and control plots supports other observations (Kårén & Nylund, 1997) that the high nitrogen input (100 kg ha−1) does not endanger the overall
mycorrhizal colonization. In addition Wiklund et al. (1995), investigated sporocarp production of ectomycorrhizal fungi at the study site and found that, sporocarp production was decreased by nitrogen addition. In spite of nitrogen addition at Skogay site, the overall impression of the stand is good health, vigorous growth, and well developed species rich ecto-mycorrhiza. Our previous studies (Kårén & Nylund, 1997), focused on humus-layer mycorrhiza, showed nearly 100% mycorrhiza colonisation of roots in plots treated by nitrogen. However, investigations by Kårén & Nylund (1996) showed that sporocarp surveys correlate poorly with community structure of mycorrhizal colonisation.
We conclude that the pattern of mycorrhizal root longevity depends on the branching order of mycorrhizal roots, while addition of N decreases the proportion of branched mycor-rhizal roots. We conclude also that nitrogen addition does not reduce the longevity of mycorrhizal short roots and, con-sequently, carbon consumption for root growth and construc-tion is likely reduced.
We would like to express our gratitude to Ulf Johansson and Rolf Reutlert for technical and computing assistance. Roger Finlay
is acknowledged for his comments on the manuscript. The Carl Tryggers Foundation and Karl Erik Önnesjö Foundation for Scientific Research and Development financed this work. References
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