Measurement of development of endomycorrhizal mycelium using three different vital stains

New Phytol. (1990), 11, 297-302 Measurement of development of endomycorrhizal mycelium using three different vital stains BY C. H A M E L, H. F Y L E S AND D. L. SMITH Plant Science Department, Macdonald College of McGill University, Ste-Anne de Bellevue, Quebec, Canada, H9X ICO {Received 1 November 1989; accepted February 1990) SUMMARY The extraradical hyphae of Glomus intraradices Schenck & Smith associated with a mixture of alfalfa (Medicago sativa L. cv. Saranac) and bromegrass {Bromus inermis Leyss. cv. Tempo) were tested for metabolic activity by three enzyme staining procedures. Mycorrhizal plants were grown for, 9 or 12 weeks and the vital staining methods were evaluated and compared at each date. Succinate dehydrogenase activity was assessed by the reduction of nitro blue tetrazolium (nitro-bt), and NADH diaphorase activity by the reduction of indonitrotetrazolium (INT). Fluorescein diacetate (FDA) hydrolysis was used to assess the activity of esterases in the extraradical hyphae. Percentages of intraradical hyphae that were active in alfalfa and bromegrass were also determined after staining root samples with nitro-bt and chlorazol black E. The percentage of live intraradical infection declined with the maturing symbiosis. In contrast, no reduction with time in the percentage of extraradical hyphae that were metabolically active was shown by nitro-bt, suggesting a different turnover for extra- and intraradical hyphae. The FDA method gave the most precise estimates of enzyme activity (low SE) followed by the INT method, while the nitro-bt method was the least precise. Different percentages of extraradical hyphae that were metabolically active were obtained with the different methods. The nitro-bt assay deviated from the others as it did not show declining percentage of active extraradical hyphae as the symbiosis aged. Key words: Vital stains, vesicular-arbuscular mycorrhizas, enzyme staining procedures, extraradical and intraradical hyphal activity. method (Sutton & Sheppard, 1978), or optical observations (Abbott, Robson & Du Boer, 1984), but The colonization of plant roots by VA mycorrhizal these methods do not distinguish active from inactive fungi often changes the pattern of plant growth mycelia. Such a distinction is necessary as physio(sanders et al., 1977). Therefore, an assessment of logical processes require the presence of metabolithe state of root colonization of experimental plants cally active hyphae. For example, P transport into is made in practically all studies involving mycor- the fungus from the soil and from the fungus to the rhizas. Various methods have been used to quantify host are active transport processes (Sanders et al., VA mycorrhizal infection. Most of these methods 1977; Smith & Smith, 198) and, therefore, require measure the extent of intraradical development of active hyphae. Measurements of enzyme activity have been used the fungi (Giovanetti & Mosse, 1980; Biermann & Linderman, 1981 ; Trouvelot, Kough & Gianinazzi- to assess the viability of VA mycorrhizal mycelium Pearson, 198). However, the effect on plant growth (MacDonald & Lewis, 1978; Kough & Gianinazzibrought about by VA mycorrhizal colonization can Pearson, 198; Schubert et al., 1987; Sylvia, 1988). be better explained by the increased absorbing The reduction of tetrazolium salts by dehydrogenases surface created by the extraradical mycelium of the is a common histochemieal method (Pearse, 1972). fungi (Sanders et al., 1977; Bethlenfalvay, Pacovsky In such colorimetric assays, tetrazolium salts act as & Brown, 1982; Graham, Linderman & Menge, electron acceptors in enzyme-catalysed oxidation 1982). A few attempts to quantify these external reactions which reduce them to formazan. T h e hyphae have been made using a chitin assay reduction of tetrazolium salts was used to assess the (Pacovsky & Bethlenfalvay, 1982), a sand aggregation activity of mycorrhizal fungi (MacDonald & Lewis, INTRODUCTION 20 ANP 11

298 C. Hamel, H. Fyles and D. L. Smith 1978; Kough, Gianinazzi-Pearson & Gianinazzi, 1987; Sylvia, 1988). Fluorescein diacetate (FDA) is taken up in cells by active transport where it is hydrolysed by esterases (Rotman & Papermaster, 19). Fluorescein, the product of FDA hydrolysis accumulates, since it is not eliminated from the cell as fast as its ester enters. Schubert and coworkers (1987) used the fluorescent dye FDA (Ingham & Klein, 1984) to assess the viability of mycorrhizal mycelium. We evaluated and compared three vital staining procedures during the development of endomycorrhizal mycelium. Two procedures involved the reduction of tetrazolium salts, nitro blue tetrazolium (nitro-bt) and indonitrotetrazolium (INT), and assay succinate dehydrogenase and NADH diaphorases, respectively. The third method involved the hydrolysis of FDA by esterases. MATERIALS AND METHODS Materials and growth conditions Extraradical hyphae were extracted from the soil of potted mycorrhizal alfalfa {Medicago sativa L. cv. Saranac) and bromegrass {Bromus inermis Leyss. cv. Tempo) plants. Seeds were surface sterilized in 70 % ethanol for one min and germinated in sand. An excess of Rhizobium meliloti (Nitragin Co.) inoculum was sprinkled over alfalfa seeds. Ten days later, seedlings were transplanted into 00 ml pots. Two alfalfa and two bromegrass plants were placed in each pot. Leek {Allium porum L.) roots (1 g fresh mass) colonized by Glomus intraradices Schenck & Smith were used as fungal inoculum and placed cm below the seedlings. T h e control pots received 1 g of autoclaved fungal inoculum and 10 ml of filtered (Whatman No. 1) washings of the fungal inoculum. Plants were grown in mixture, in an autoclaved Turface:sand (1:1, v/v) substrate for 12 weeks and were fed with a modified \ strength Hoagland's solution. Twelve inoculated pots and four noninoculated control pots were harvested after, 9 or 12 weeks from seeding. Harvested plant roots were washed, cut into segments (1 cm) and homogenized. A subsample was stained with chlorazol black F (Brundrett, Piche & Peterson, 1984) for determination by the grid intersect method of the percentage of total (active and inactive) root colonization (Giovanetti & Mosse, 1980). A second subsample was stained with nitrobt to measure the percentage of root containing active mycelium. In both methods, at least two hundred intersections were considered in determining the percentage of root colonization. agitated with a stream of pressurized water to detach hyphae adhering to the roots. The hyphae and root fragments floating out of the soil were collected on a 37 [im sieve. The recovered material was suspended in 2 ml of aqueous sucrose solution (0%) for 2 min. The supernatant was collected, resuspended in 2 ml ofthe same sucrose solution and centrifuged for 30 s at 100 rpm (Schubert et al., 1987). T h e supernatant was collected, rinsed with water and filtered on Whatman No. 1 paper filter. No hyphae were recovered from non-va mycorrhizal soil, while over 1 g of material was extracted from VA mycorrhizal soil. Such extraction has an advantage over soil samplings (Ingham & Klein, 1984a) because it gives quantities of clean hyphae which are easy to assay. Staining procedures Sampling. Three random samples (one for each method) were taken at harvest from the hyphae collected from each experimental pot, and assayed immediately. Nitro-BT reduction assay. Hyphae were incubated for 12 h at room temperature in a staining solution of 0 mm Tris buffer (ph 7-4) containing 0- mm MgClg, l O m M K C N, 1 mg ml"^ nitro blue tetrazolium, and 0-2 M sodium succinate (Kough et al., 1987). The stained hyphae were rinsed with water, mounted in polyvinyl alcohol ([ Ch2CH(OH) ] ) (Omar, Bolland & Heather, 1979) and examined under dark field microscopy for evaluation of hyphal viability. T h e percentage of live hyphae was determined by the grid intersect method (Giovanetti & Mosse, 1980). Twelve randomly selected microscope fields were examined per pot. Roots were also incubated for 12 h in the nitro-bt staining solution and cleared in 20 % (w/v) boiling aqueous chloral hydrate (CCL3CH(OH)2) for 10 min. The percentage of roots containing metabolically active VA mycorrhizal fungal hyphae was determined by the grid intersect method (Giovanetti & Mosse, 1980). Hyphae were observed through a grid placed in the microscope eyepiece. INT reduction assay. Hyphae were stained for 12 h at room temperature in a solution containing equal parts of I N T (1 mg ml"'), NADH (3 mg ml"^) and 0-2 M Tris buffer, at ph 7 4 (Sylvia, 1988). The stained hyphae were rinsed in water, mounted and examined for the percentage of viable hyphae as described previously. Twelve randomly selected microscope fields per pot were examined under transmitted light. FDA hydrolysis assay. Hyphae were stained for min in a solution prepared by dissolving mg The soil was placed in a large shallow pan and FDA in 1 ml acetone, then adding 0-1 M Tris bufter, Extraction of mycelium from the soil

Measurement of development of endomycorrhizal myeelium at ph 7-4, to give a final concentration of 0 fig ml - 1 (Schubert et al., 1987). The stained hyphae were immediately observed with a Carl Zeiss Jenalumnar fluorescence microscope equipped with an exciting filter (B 229) and a barrier filter (G 247). Hyphae from each field of observation were photographed using a daylight film (Ektachrome 200 ASA), first in epifluorescent light and then in transmitted light. The percentage of live hyphae was determined using the grid intersect method. The photographic slides were projected on a screen on which a grid of lines was marked. Eight fields were counted at the first harvest but for the two following harvests, the observation of six fields was considered sufficient. 299 Table 1. Time-course of the percentage of soil hyphae that are metabolically active as estimated by three vital staining methods Assay ( o active hyphae) 1laivcsi (weeks) INT Nitro-BT FDA Means 9 12 9-3 a 2-ab 7-8b 3-2 -b -b 4 3 a 8-8 -a 1-2a 38-7b -1 3-9-8 3-9-0 Means Values are means of 12 replicates. Means followed by the same letter and in the same column are not significantly difterent (P < 0-0) by a protected L.S.D. test. Statistics Standard errors were calculated after pooling the variances from 2 to 20 fields for the tetrazolium assays, and up to 8 fields (depending on the harvest) for the FDA method. The pooled variances of each number of fields observed were averaged over the replicates and the harvests. Standard errors from each method were calculated for each number of fields observed. 80 7000403020100 RESULTS Weeks from seeding Percentage of active extraradical hyphae The percentages of soil hyphae that were active, obtained by two of the three methods, changed as the VA mycorrhizal association matured. Fungal activity decreaed significantly with increasing age when evaluated by the I N T and FDA methods, but remained constant according to the nitro-bt method (Table 1). Although results given by the FDA and the I N T methods presented the same trend across harvest dates, the latter gave higher percentages (P < 0-0) of active soil hyphae than the fluorescence method within dates, especially at week 12 (Fig. 1). The estimates of fungal activity given by the nitrobt method gave lower percent estimates of metabolically active hyphae than the other assays at the first two harvests but indicated an increase in hyphal activity at the final harvest. Standard error The FDA method had the lowest standard error for any given number of observations, and the nitro-bt method the highest (Fig. 2). Therefore, to obtain the same precision, more fields must be observed with the nitro-bt or the I N T methods than with the FDA method (Table 2). Note, however, that about 30% more intersections per microscope field were counted after FDA staining than after staining with either the I N T or the nitro-bt stains. This higher intersection number per field resulted from the utilization of difterent grids and difterent micro- Figure 1. Time-course of the percentage of soil hyphae that are metabolically active, as estimated by three vital staining methods. Values are means of 12 replicates. Bars, within weeks, with the same letter are not different at P < 0-0 by a protected L.S.D. test. 0, Nitro-BT;, I N T ; H, FDA. Q) CD -a c CD 3 4 7 8 9 10 11 12 13 14 1 1 17 18 19 20 Number of observations Figure 2. Standard error of three methods of assessing percentage activity of soil hyphae with increasing number of observations., Nitro-BT; A, I N T ; O, FDA. scopic magnifications obtained with and without a camera. These difterences between the FDA and the colorimetric methods lead to greater accuracy with the FDA method than the two colorimetric assays, at the same number of observed microscope fields. 20-2

300 C. Hamel, H. Fyles and D. L. Smith Root colonization Total (active and inactive) hyphal colonization of roots, as determined with chlorazol black E staining, increased as the symbiosis matured (Table 3). The nitro-bt dye showed less or no increase with time in the percentage of roots colonized by live hyphae Table 2. Number of observed fields required to achieve a specific standard error for each method Standard error 3 4 Methods INT 12 8 (observed Nitro-BT 19 10 fields) FDA Numbers are from standard error curves, derived from the means of the harvests. 8 3 (Eig. 3). As a result, the proportion of the root colonized by active (living) hyphae tended to peak after 9 weeks and declined at week 12, especially in bromegrass roots. The proportion of the root colonized by active hyphae was calculated by dividing the percentage of total root colonization by the percentage of roots colonized by active hyphae. DISCUSSION Our results show that not all root infection was active and confirm previous findings (Kough & Gianinazzi- Pearson, 198; Kough et al., 1987; Schubert et al., 1987; Sylvia, 1988). The percentage of root infection that was active declined as the symbiosis matured. This declining activity of intraradical fungi contrasts with the observed constancy of extraradical fungal activity as determined with the same stain, nitro-bt. Kough & Gianinazzi-Pearson (198) also found differences in metabolic activity between external 0 (a) 0 (b) c o CD N 'E _oo 40 30 20 1 -.1 i" u " o CC 20 10-10 - Weeks n 1 12 12 Weeks Figure 3. Percent root colonization as estimated with nitro-bt ( ) or chlorazol black E ( ). {a) Colonization of alfalfa roots; () colonization of bromegrass roots. Values are means of 12 replications. Bars indicate standard deviations. Table 3. Percent root colonization, as estimated with nitro-bt or chlorazol black E Alfalfa Bromegrass Weeks Staining Chlb-E methods Nitro-BT Proportion of colonization - that is active (%) Staining imethods Chlb-E Nitro-BT Proportion of colonization - that is active \ /o) 9 12 8-Oc 37-2b 4 3 a -7b 2-a 24-1 a 7-Oa 70-3 a 4-1 b ll-4b 19-0 a 22-3 a 9- a 7-8a 8-3 a 87-7 a 41-4b 37-b Numbers represent the means of 12 replications. Means followed by the same letter and in the same column are not significantly different {P < 0-0) by a protected L.S.D. test.

Measurement of development of endomycorrhizal mycelium and internal VA mycorrhizal mycelia, according to assays for phosphatase and succinate dehydrogenase, although they found no difference using an assay for lipase activity. These findings suggest that the intraradical and extraradical mycelia of VA fungi have different lifespans. It is likely that the extraradical hyphae are consumed by soil organisms and replaced by new ones, giving the semblancfe of greater longevity. The turnover of extraradical hyphae is probably greater than that of intraradical mycelia which are protected by the roots. In most studies, the importance of endomycorrhizal fungi to plants is evaluated through the measurement of the percentage of roots colonized by the endophyte. As shown here, much of the intraradical mycelium is not metabolically active. Therefore, the usual staining method for evaluation of the microsymbiont could be misleading; for example, the percentage of root colonized by both live and dead hyphae may or may not be related to the effects of the symbiosis at a given time in the season or to a particular development stage of the plant. Histochemical tests of viability, such as the enzymatic assays used in this study, can be useful in determining the amount of active hyphae present in the roots of mycorrhizally infected plants. However, care must be taken when selecting the assay system. Our results emphasize that assays of different enzymes may give conflicting results. The exact causes of those differences observed here are uncertain. The three staining methods differ in many aspects. Most notably, they are based on different mechanisms. The nitro-bt staining solution provides an electron source (sodium succinate), and blocks the flow of electrons to the respiratory chain with KCN, thereby allowing an easier reduction of the tetrazolium salt. Magnesium ions, which enhance enzymatic activity (Pearse, 198), are also provided. This solution then removes limitations to the activity of succinate dehydrogenase, and the hyphae turn blue where succinate dehydrogenase is present. On the other hand, the I N T staining solution provides only reducing power (NADH) to endogenous NADH diaphorases and does not contain activity-maximizing materials. The FDA solution contains only a substrate, FDA, for esterases and provides neither a source of electrons nor activity-maximizing materials. However, FDA staining has been correlated to O^ utilization and COg evolution in soil fungi (Ingham & Klein, 1984), parameters which directly relate to general metabolic activity. FDA staining should therefore reflect the general vitality of mycorrhizal mycelium. The three staining methods also differ in the enzymes that they assay. Hyphae containing functional NADH diaphorases turn red under I N T staining while those containing active esterases fluoresce. The nitro-bt assay is specific for succinate dehydrogenase while the I N T and FDA methods are 301 less enzyme specific. The constancy of enzymatic activity indicated by the nitro-bt method, compared with the decline indicated by the other methods, suggests that the nitro-bt method assays only the presence of potentially active succinate dehydrogenase, while the other two methods test for more general metabolic activity. Different vital staining methods may give different estimates of hyphal activity in soil and, therefore, the choice of an assay and the interpretation of its results must be made with care. 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