Short communication
The differential vitality of intraradical mycorrhizal structures
and its implications
P.L. Staddon*, A.H. Fitter
Department of Biology, University of York, PO Box 373, York YO10 5YW, UK
Received 4 November 1999; received in revised form 23 March 2000; accepted 6 April 2000
Abstract
Preliminary assessment of the effect of storage on the vitality of mycorrhizal fungi inside plant roots gives insights into how fresh root material has to be when undertaking functional studies on plant±mycorrhizal fungus associations and also, perhaps more importantly, it highlights aspects of mycorrhizal inoculum potential. Here we report on the vitality of mycorrhizal structures in roots refrigerated at 58C over a 3 week period. The change in vitality followed the same pattern whether the roots were excised from the soil or not. The vitality of internal hyphae and arbuscules remained constant for 2 weeks and 6 days, respectively, but then declined. The vitality of vesicles showed no change over the 3 week period.q2001 Elsevier Science Ltd. All rights reserved.
Keywords: Arbuscular mycorrhizas; Arbuscules; Internal hyphae; Vesicles; Mycorrhizal vitality; NBT-succinate vital staining
Most plant species form mycorrhizas (Smith and Read, 1997), a symbiotic association between plant roots and fungi. The most common type of mycorrhiza is the arbus-cular mycorrhiza (AM), formed by 75% of plant species. In AM associations, the plant partner supplies carbohydrates to the fungus, which in turn provides the plant with mineral nutrients, especially phosphorus, as well as other possible bene®ts (Newsham et al., 1995). Mycorrhizal associations are most commonly quanti®ed by assessing the level of fungal colonisation of the roots of the host plant. A frequently used measure of the extent and structure of mycorrhizal colonisation is the percentage of root length colonised by internal hyphae, arbuscules and vesicles. However, a problem with such measurements is that it is not possible to determine whether the mycorrhizal fungal biomass is alive or dead. This is particularly critical for studies investigating functional aspects of the symbiosis. Vital staining techniques, such as the nitro blue tetrazolium chloride-succinate method (Schaffer and Peterson, 1993), allow this limitation to be overcome. A disadvantage with vital staining is that the material must be freshly collected for analysis, which can be problematic for large-scale ®eld experiments. Here we report a study with the objective of determining how long soil/root material can be stored at low
temperature (58C) before the vitality of the mycorrhizal fungi present inside plant roots is adversely affected. This study will also give insights into which intraradical mycor-rhizal structures might be responsible for the inoculum potential of root fragments (Friese and Allen, 1991), which are thought to act as inocula in disturbed soils (Abbott and Gazey, 1994) and are often a component of prepared inoculum (the storage of which has received surprisingly little attention) (Sylvia and Jarstfer, 1994).
Turves were collected from a hill grassland site (Sour-hope Research Station, Yetholm, Roxburghshire, UK) on 23 February 1999. The plant community was dominated by the grasses Agrostis capillaris,Festuca rubra,Nardus stricta, Anthoxanthum odoratum and Poa pratensis and closely matched the National Vegetation Classi®cation (NVC) community U4d. The turves were placed in a glasshouse at the University of York, UK. On 5 May, two random sets of four turves (5£5 cm2, 10 cm deep) were removed from the glasshouse ready for the storage experiment. The shoots were removed from all turves, therefore removing the carbon supply to the roots. One set (set B) was placed directly in a refrigerator at 58C in the dark, whilst the other (set A) was immediately used to extract roots. Three random samples were taken from each turf in set A, and one random sample from each of the four turves were kept together to form three sets of four replicates. The roots were cut into segments around 1 cm in length and placed into modi®ed test tubes ®tted with a muslin mesh at the base, which were Soil Biology & Biochemistry 33 (2001) 129±132
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* Corresponding author. Tel.: 144-1904-432883; fax: 1 44-1904-432860.
then placed in deionised water in a refrigerator (58C) for a speci®ed length of time (1, 6 and 8 days, a set of four repli-cates for each period) until the staining procedure. The other set of turves (set B) was refrigerated (58C) for 7 days in the dark before the roots were extracted, as for the previous set, and then kept at 58C for a further period (1, 7 and 14 days). These two treatments were chosen to determine whether the removal of the carbon supply to the roots by cutting the shoots at the soil surface or the excision and fragmentation of the roots was the most important cause of any change in mycorrhizal vitality. It may have been preferable to have a clearer overlap in the timings of the treatments, however
this design nevertheless allowed to test whether mycorrhizal vitality in the two sets followed a different pattern.
To determine the proportion of mycorrhizal fungus colo-nisation alive in the roots, roots were vital stained using a modi®cation of the nitro blue tetrazolium chloride (NBT)-succinate method (deposition of formazan being the viabi-lity indicator) and counterstained with acid fuchsin (Schaf-fer and Peterson, 1993). The roots were incubated in NBT-succinate solution for 19 h at room temperature in the dark. After rinsing in deionised water, the roots were placed in formol saline solution for ®xation for 5.5 h at room tempera-ture in the dark. After rinsing in deionised water, the roots were cleared in KOH, acidi®ed in HCl, stained in acid fuch-sin solution and destained in lactoglycerol following the procedure described in Staddon et al. (1998). The roots were mounted in lactoglycerol and viewed by compound
microscope (Nikon EFD-3 Optiphot-2) at 250£
magni®ca-tion using both epi¯uorescence (Merryweather and Fitter, 1991) and normal light to facilitate visualisation. Scoring followed McGonigle et al. (1990) looking at a minimum of 60 intersections. At each intercept, the presence (or absence) of internal hyphae, arbuscules and vesicles was noted, as was the presence of formazan deposits on fungal structures (indicating viability). Due to low numbers, scoring for vesi-cles was subsequently performed separately with a mini-mum of 100 intersections.
TheF-ratio method (Mead and Curnow, 1983; Sokal and Rohlf, 1995) was used to determine whether there was any signi®cant differences between the two sets of data, i.e. between the samples harvested immediately and those harvested after the turves were stored for 7 days in the fridge. TheF-ratio tests showed that, for all three variables (internal hyphae, arbuscules and vesicles), all the points from both data sets could be ®tted by a single regression line, i.e. the residual sum of squares was not signi®cantly reduced by ®tting two lines as opposed to one line. The F-ratios obtained when comparing the two sets of data for the vitality of internal hyphae, arbuscules and vesicles were F2;201:22; F2;202:53 and F2;19 1:70; respectively (in all casesp.0:1). The two sets of data were therefore
combined for further analysis. The fact that there was no signi®cant difference between these two treatments on mycorrhizal vitality implies that the cutting of the carbon supply to the roots must be the key factor affecting changes in mycorrhizal vitality over time (see below).
Total mycorrhizal colonisation remained constant over the 3 week period of storage at 58C: total length colonised by internal hyphae (IH-TLC), by arbuscules (A-TLC) and by vesicles (V-TLC) averaged 42, 15 and 4%, respectively (Fig. 1), the slopes of the ®tted regression lines as a function of time not differing from 0 (IH-TLC:p0:878;A-TLC:
p0:709; V-TLC: p0:327). Over the 3 week period,
vital mycorrhizal colonisation decreased for internal hyphae and arbuscules (Fig. 1a and b): vital length colonised by internal hyphae decreased from ca. 20 to 8% (regression: p0:0045) and vital length colonised by arbuscules from P.L. Staddon, A.H. Fitter / Soil Biology & Biochemistry 33 (2001) 129±132
130
10 to less than 1% (regression:p0:0001). However, there
was no signi®cant change in vesicular vital length colonised
(regression: p0:188), which averaged 2% (Fig. 1c),
although the very variable data for vesicles may conceal undetected effects.
The vitality of internal mycorrhizal hyphae remained relatively constant, around 40% until 14 days, then declined to 16% by 21 days, whereas the vitality of arbuscules was 60% until 6 days then declined sharply between 6 and 8 days to 30% reaching nearly 0% by 21 days. (Fig. 2a). Over the 3 week period, the overall slopes of the regressions as a function of time were21.12 p0:0165and22.94
p,0:0001 for internal hyphae and arbuscules,
respec-tively. These slopes differed signi®cantly by a factor of 2.6 (F-ratio method for testing difference in slopes (Sokal and Rohlf, 1995), p,0:05) implying that the vitality of
arbuscules decreased substantially faster than that of inter-nal hyphae. Contrary to interinter-nal hyphae and arbuscules, the vitality of vesicles showed no change over time (slope of regression not signi®cantly different from 0: p0:998)
(Fig. 2b). Vesicular vitality averaged 48% over the duration of the experiment. The large variability in vesicle numbers and vitality (Figs. 1c and 2b) was likely due to the general patchy distribution of vesicles.
These results highlight the need for the immediate vital
staining of roots once harvested, or at the most they may be refrigerated for only a few days. There was a signi®cant difference in the response of the vitality of internal hyphae and arbuscules as a function of storage time: the vitality of internal hyphae took over 2 weeks to decrease by 50%, whereas only 1 week was taken for the vitality of arbuscules to decrease similarly. As for the vitality of vesicles, there was no change over time. Assuming that the main effect of stopping newly ®xed carbon arriving in the roots is an inhi-bition of formation of new fungal structures, this gives some measure of the longevity of mycorrhizal fungal structures inside roots. Arbuscules are known to have a relatively short life-span, the onset of senescence beginning from 4 days after arbuscule formation (Read, 1991), and vesicles are known to act as storage structures for many mycorrhizal fungi (Mosse, 1973).
This rapid decline in vitality of internal hyphae and arbus-cules also raises the question why inoculum consisting of root fragments (albeit dried ones) stored for several months at 58C remains effective (Staddon, personal observation). One possibility is that such inocula invariably contain spores, which act as the initial source of mycorrhizal infec-tivity. However, another possibility is that the root frag-ments contain a small number of viable structures, which are relatively infective. From the data presented here, the only candidate for this would be the vesicle as only the vesicles (which were recorded in very small numbers) showed no evidence of any change in vitality (averaging nearly 50%) over the 3 week period at 58C. The potential for intraradical vesicles to act as propagules has previously been demonstrated by Biermann and Linderman (1983). Vesicles may therefore play a far greater role in mycorrhizal inoculum infectivity than previously suggested (e.g. Tommerup and Abbott, 1981). Smith and Read (1997) state that ªvesicles, like spores, store large amounts of lipid and contain many nuclei, which together with their thick walls suggest a function either as propagules or to support the regrowth of intercellular hyphaeº, although as they imply there is very little published on the inoculum potential of vesicles (only half a paragraph on vesicles in their 13 page section on ªSources of Inoculumº). This is not to say that vesicles are important for mycorrhizal fungal survival under all conditions. For example, in a study on cotton agroecosystems, McGee et al. (1997) showed that plants inoculated with root fragments were not colonised by mycorrhizal fungi and they noted that there were no vesicles in those root pieces.
If the nutrient exchange organs of arbuscular-mycorrhizal fungi are as susceptible to low temperature and death of their host as this study implies, how do mycorrhizas persist in annual dominated temperate ecosystems? In cases where some perennials are present, it can be easily envisaged that those perennials act as a refuge for the mycorrhizal fungi. But what of ecosystems with no perennial component and substantial physical disturbance, such as agricultural ones for instance? Do the mycorrhizal fungi survive winter solely
P.L. Staddon, A.H. Fitter / Soil Biology & Biochemistry 33 (2001) 129±132 131
in a dormant state (see Helgason et al., 1998)? Is such a state unique to spores (Miller, 1987) or can other mycorrhizal structures, such as vesicles, also be dormant (over a period of several months)? According to Miller and co-workers (Addy et al., 1994, 1997; McGonigle and Miller, 1999) extraradical mycorrhizal hyphae may remain infective over winter, although it is dif®cult to distinguish this from the role of spores, which have been shown to be the prime structures for winter (and/or disturbance) survival along with mycorrhizal fungi inside root fragments (Friese and Allen, 1991). As Abbott and Gazey (1994) point out, very little is known about the basic dynamics of mycorrhizal fungal populations.
Acknowledgements
This research forms part of the Soil Biodiversity NERC Thematic Programme. We thank two anonymous referees for comments on this manuscript.
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