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Quantitative assessment of the rhizoplane microflora by direct microscopy
QUANTITATIVE ASSESSMENT OF THE RHIZOPLANE MICROFLORA BY DIRECT MICROSCOPY A. D. ROVIKA.* E. I. N~WMAK Dcpartmcnt
of Botany.
H. J. BOWEN and R. CAMPBELL
The University.
Bristol BSX IUG. England
Summary- The hacterlal populations on roots of several grassland species were estimated by combining the tcchniqucs of direct counting of stained bacteria itt .siru with standard sampling techniques of plant ecology. The eight plant species examined had bacterial cover of the root surface within the range 4 10 per cent. In a more detailed experiment with two plant species, the method was sufficiently precise to demonstrate that a 7.7 per cent cover of coots of Loliur~l ~CVYVI~~J L. by bacteria was significantly different (P < @05) from a 6.3 per cent cover on Pla~ttrcgo /~IIIL.~U~~IL. The length of fungal mycelium per unit root surface area was estimated hq a modification of the lint intercept method. which was originally developed for mcasurmg root lengths. When hyphal diameters were also measured. fungal cover per cent and volume could hc estimated. Estimates of the numbers of rhiroplnne bacteria and fungi made hq the spread-plate counting method on a non-selective agar showed that the mean number of bacteria from Loliurn was greater (but not sipnificantly ditfcrent) than the counts on Pltr~~tcqc~:houe\er. Lo/k/n did have significantly more Gram-negative bacteria as estimated hq crystal violet agar than did Pltrr~rugo. The numbers of hactcria catimatcd bq direct microscopy were about IO-fold greater than estimates by plate counts. For II given period of time. direct microscopy gave a more prease estimate of total bacterial and fungal abundance than did plate counts hut the technique docs require continuous concentration by the ohscrvcr over long periods
Although direct microscopy has been used to dcmonstrate micro-organisms on the surfaces of roots (Rovira. 1956: Trolldcnicr. 1973). there arc few studies in which the technique has been used to quantitatively assess the microbial populations on roots. Waid (1957) used percent frequency of occurrence of fungal hyphae along roots as observed by direct microscopy to show fungal succession from the root apex while Thornton (1965) used r-tests to compare fungal populations on roots of different ages and species. Parkinson and Crouch (1969) measured hyphal lengths on roots of Pir~rs nigrtr var. laricio and found these to range from 9.2 to 14% mm:mm’. The only quantitative assessment of rhizoplane populations of bacteria by direct microscopy is that of Bowen and Theodorou (1973). who estimated the percentage cover by micro-organisms in rcprcsentative fields of stained roots of Pirnrs ditrtcc Don. The choice of “representative” fields to estimate rhizoplane populations does not enable a valid statistical analysis of population differences. whether due to plant species. soil conditions, root age or any other causes. In this paper we have applied standard sampling techniques of quantitative plant ecology on a micro-
* Permanent address: laide. South ,4ustralia
CSIRO.
Divismn
of Soils. Ade-
scopic scale to estimate the populations of bacteria and fungi on the roots of several plant species. For two of the plant species we have compared these direct counts with the numbers estimated by the conventional dilution plate method. MATE:RIALS AND YIETHODS
The species selected for this study are plants common in permanent grassland on neutral to slightly acid soils in lowland Britain. The species were four grasses. Allfhosur7rhurll odo~turt~ L., C~r10s~ru.s cristatus L.. Holcus lunntu.s L. and LoII’tfntpr~rnnr L., and four dicotqledonous herbs. H~poclwrris ratikuta L.. Plu~tuylo IUK~YI/LI~L~ L., Rurmz IICP~OS~I L., and Trjfi,lium rrprns L. All were grown from seed collected locally, except for Tr$~liur~ which was the commercial variety S. 100. All eight species were included in experiment 1. the results of which were the basis for selecting Loliur,~ and Pl~~~tu(~ofor the more detailed experiment 2. The soil in which the plants were grown came from permanent grassland and had the following properties: mechanical analysis. 7’:” fine gravel (c2.0 mm), 8’>;, sand (0.2- 2 mm), 170o fine sand (0.06-0.2 mm), 46% silt (0.002 -0.06 mm) and 274(, clay ( <0.002 mm); pH 4.5 (in water): 6.2”,, organic matter (wet digestion). For experiment I the plants were grown in &cm free-draining pots standing in saucers. about four
212
A. D. ROVIKA.E. 1. Nr WMAN.H. J. BOWN
plants per pot. with four replicate pots per species. They were grown from August to October in a greenhouse without artificial light. For experiment 2 the plants were grown in 13-cm pots. two plants per pot and nine pots per species. They were grown from December to February with supplementary illumination over an IX-h da!. The soil was kept moist (about 45”,, water) by allowmg the free-draining pots to stand in saucers in which water was maintained at about I cm depth. After I? weeks the plants were graded according to sire and paired pots. one of 0x11 species, were used on each day.
To observe the rhizoplane micro-organisms in situ. it is necessary to wash most of the soil from the root surface. Experiments were conducted with Loliuttt to estimate the loss of root surface bacteria with the gentle washing necessary to clean the roots for direct microscopy. Roots which had been immersed briefly in sterile distilled water (SDW) to remove excess soil were transferred to a conical flask containing 50ml SDW and gently washed to the point at which they could be used for direct microscopy. These roots were transferred to a flask with lOOmI SDW and 5g glass beads (3 4 mm dia) and shaken vigorously for 15 min with a wrist action shaker before preparing dilutions and plate counts. The results showed that the gentle washing removed 3 x IO” bacteria.‘g fresh weight of root lvhile the 15 min shaking removed 90 x 10” bacteria;g fresh weight of root. This result was consistent for several roots and thus WC concluded that the washing required to produce roots for direct examination uould not remove more than S4 per cent of the rhiLoplane population as estimated by the plate count method. Direct examination of roots and plate counts of the suspensions during the shaking period revealed that 40 and 80 prr cent of the bacteria were removed from the roots after 5 and 10 min shaking respectively and that virtually no bacteria or fungi remained on the roots after 15 min. Maceration of the roots after I5 min shaking did not increase plate counts. These experiments indicated that a shaking time of 15 min on a Grifin Flask shaker (Griffin and George Ltd., Birmingham U.K.) with the roots immersed in 100 ml SDW in ;I 250 ml comcal flask containing glass beads is sufficient to remove the rhizoplane microflora. Sektiott o/‘tmrs. The roots which had been gently washed were trimmed to exclude those parts within I cm of the base of the pot. In experiment 1. one piece of root of about 10 cm, subjectively estimated to be of about average diameter, was chosen from each pot. Counts were made on a I-cm portion at each end of the selected root. In cxpcriment 2. the roots wcrc cut into lengths ofabout 2 cm. and spread out in ;I flat-bottomed dish. For the direct counting, twelve Z-cm root segments were then selected randomly. by taking the segments nearest to 12random points on the dish: no distinction was made between the main root ;IY~Sand finer sccondarj roots in this selection of roots.
and
R. CAMIWLI
Staitzitty uttd direct ttric~roscopq of’roots. The 1 or 2 cm root segments were stained with phenolic aniline blue for 3-6 min. The staining time, which varied for fine and coarse roots. was selected to stain bacteria and fungi dark blue against a light blue or clear background (Rovira, 1956). The segments were mounted in water. placed under a no. 1 cover slip glass and examined under high power ( x 675). The eyepiece graticule was divided to give squares of 18 x 18Ilm for counting. In experiment 1. fi\c fields were selected within each l-cm root segment. and counts were made in five squares in each field. In experiment 2 there were again five fields per segment. but onl) three squares per field. The fields were approximately regularly spaced along the root segment. but the obscrvcr was not looking down the microscope while he moved the slide; this avoided any possibility of selecting a particular arca. When the field was obscured by soil or root hairs. the slide was moved again. Only counts of the bacteria on the surface of the root itself were made. with no cstimates of bacteria on root hairs which were sparsely colonized. For further details on the arrangement of the fields and squares. see Newman and Bowen (1974). In experiment 2 the diameter of each root segment was measured. When a square contained 40 or fewer bacteria. clearly separated. the number of individuals was counted to the nearest 5. Where there were tnore than 40 bacteria per square or where tht: individual cells could not be clearly distinguished, the percentage of the square occupied was estimated on the scale I?. 25. 50. 75 or 100 per cent cover. In both experiments all the direct observation was pcrformcd by one person (H.J.B.). to reduce observer error in estimating cover percentage. For calculating the mean cover percent of all squares the numbers of individuals have been converted to cover on the assumption that the mean projccted area of one bacterium is 1/tm’. This figure is based on measurements of the sire of the bacteria on a number of root scpmcnts. By this conversion 40 bactcria per square give 12 per cent cover. In experiment 2. the length of fungal hyphae per unit area of root surface was estimated by a modification of the line intercept technique of Newman (1966). If hyphac are growing on a plane surface, the length of hyphae can be estimated by placing random lines on the surface and counting the number of times hyphae cross these lines. In the experiment. the random lines (each 36,~m long) were provided by two lines on the eyepiece graticule at right angles to each other. For every field, the eyepiece was twisted to give the lines a “random” direction before counting the number of hyphae crossing them. An estimate of L. the mean length of mycelium per unit area, is given by
L = n,l;. 7H M,hcre :L’ is the total number of intersections and H is the total length of the random lines. If II is in mm. L is in mm mm’.
Direct estimation of rhizopfane
To summarize the direct observations on each plant species: experiment l-bacteria, 4 pots x 2 root segments x 5 fields x 5 squares = 200 squares; experiment 2-bacteria, 9 pots x 12 segments x 5 fields x 3 squares = 1620 squares: fungi. 9 pots x 12 segments x 5 fields = 540 fields.
Fifteen clumps of 2cm length of roots were taken from the dish to give a sample of 0&0~6 g fresh weight and transferred to 100 ml SDW with 5 g glass beads in a 250ml conical Bask. Serial dilutions were prepared of the water in which the roots were shaken for 15 min as described earlier. From the appropriate di~utioi~s a drop of 176 ~1 was spread over one half of a Petri dish containing one of three agar media: each drop was duplicated on another plate. Spread plates were used rather than pour plates because of savings in time and materials with no measurable loss in accuracy of counts (Ridge and Rovira, unpublished results). “Total” bacteria were estimated with 0.37; Tryptic Sov Agar (TSA) prepared from Tryptic Soy Broth (Difco Code: 0.370-01) solidified with 1.57; agar; Dr. J. K. Martin (CSIRO Adelaide, personal communication) has shown that for soil and rhizosphere samples. counts with TSA were similar to those obtained with the YPS medium of Bunt and Rovira (1955); TSA has the advantage of being a standard laboratory medium of reproducible composition. Plates were counted after 7 days at 26°C to minimize losses due to spreading bacteria ; Zagallo and Katznelson (I 957) and Rovira and Brisbane (1967) showed that rhizosphere and rhizoplane bacteria have faster growth rates than most soil bacteria and would produce visible colonies in 7 days. Gram-negative bacteria were estimated by incorporating sterile crystal violet solution into TSA at a final
Table
2. Abundance
of bacteria
(numberjmg root dry weight Total bacteria gram-I~egative bacteria Fi~amentous fungi
concentration of 2 ppm immediately before pouring the plates. Comparisons which we had made between this TSA-CV medium and the crystal violet medium of Holding (1960) showed similar counts of Gram-negative bacteria. Plates were incubated at 26-C for 7 days. Filamentous fungi and yeasts were counted on 4’!,, malt agar (Oxoid CM 90) to which streptomycin at 30 ppm was added immediately before pouring plates. The filamentous fungi were counted after 2 days and the yeasts after 7 days at 26’C. In most samples the yeasts were more numerous than filamcntous fungi and were counted at a higher dilution.
Table I. Per cent cover and per cent frequency of b;tctcria on root surfaces ofeight species m experiment 1, determmed by direct observation 0”
species Hypochorris radicata L. Holcus lanucus L. Lolitirn perenne L. Anthomanthum odoratum L. Cynosurus cristatus L. Rumrs acetosa L. Tr$oirtliumrepens L. Piunty ~an~f~olafa L.
cover*
9.3 8.6 8.4 8.1 6.4 6.3 6-2 3.7 10.1 3.2
LSD
12.1 66.7 7.1 1090
‘I,, Frequency? 81 72 64 70 12 61 66
59 < OQ5 19.7
* Per cent cover is the percentage of root area covered by bacterial cells. t Per cent fequency is the percentage of squares containing one or more bacteria. : Results of analysis of variance: P = significance level of differences between species; LSD = least significant difference between species at P = 0.05.
and fungi on root surfaces of Lo/iurtt pr~trtr and Pkzrtta~~o iur~oftrrrr determined by direct observation and plate counts
Direct observation Fungal hyphae. length. mm:mm’ Bacteria. frequency per cent Bacteria, cover per cent Bacteria. number/mm3 root x 10” Plate counts (number/mg root fresh weight “Total” bacteria Gram-negative bacteria Filamentous fungi Yeasts
21.3
microflora
14.3 66.2 6.3 990
in experiment
24 66 I.3
x 10’) 107 48 0.35 26
70 26 032 ‘0.95
< 0.05 to.1
50 21 0.31 24
x 10”)
YedStS
* Statistical significance of ditkrence “r LSD value at P = 0.05.
737 330 2.3 IX.1 between
the species. by unpaired
_ _
t test; P > O-1 is shown as
368 157 1% 14.2
7.
214
A.
D. ROVIRA.E. I.
H. J. BOWEFand R. CAMPIWLI
NEWMAN,
RICSL’LTS
To compare the numbers of bacteria estimated by direct observation and by plate counts. the cover percent values were converted to numbers per unit root volume. To achieve this conversion, certain assumptions needed to be made, firstly that the average cell covered an area of 1 LLrn’and secondly, that the layer of bacteria was only one cell deep. As we found no significant correlation between root diameter and bacterial cover (see below). a mean surface to volume ratio for each species was used to convert bacterial numbers per unit area of root surface to numbers per unit volume. The mean root diameters were 0.24 mm for Lolium and 0.23 for P(ur~cc~~o.By assuming that 1 mm3 fresh root weighs I mg it IS possible to compare the numbers per mm3 root volume directly with the “total” bacteria per mg fresh weight as estimated by plate count. Table 2 shows that when the numbers of bacteria arc estimated in this way, the direct count is about 10 times the plate count. This discrepancy would be greater if the bacteria are sometimes more than one cell deep on the root and less if one assumed a larger average cell six.
Table 1 shows the percentage cover and per cent frequcncy of bacteria on the root surfaces of the plants used in experiment 1. There were significant differences hctwoen species in frequency. The differences in cover did not reach the conventional 5 per cent significance level. but since the species with higher cover tended also to have higher frequency, and since frequency did differ significantly. results suggest that there were real differcnccs in cover between species. Table 2 shows the results of experiment 7. By direct observation. there was no significant difference between Loliurf7 and Pl~tago in frequency of bacteria. The same was true in experiment I. where Loliun~ was lower in frequency than expected from its cover. In experiment 2 bacterial cover differed significantly bctwcen the spccics. Lolir~ having the higher value. as in experiment I. If the data from the two experiments arc combined and treated as if there had been a single cxpcriment with I! replicates, the difference in cover bctwccn f,o/iurr~ and P/LIIITLIMO was significant at P < WE. The length of fungal hyphae did not differ significantly hctween species. The plate counts showed dif-frrences bctwecn species for total bacteria. Gramnegative bacteria and yeasts. but only for the Gramncgativc bacteria was the differcncc statistically significant (at P < 005). Measurements of hyphal diameter were made on nthcr roots of the two species grown in the same soil and gave a mean diameter of 2.7 I’m for Lolim and 2.4 ~mi for P/c~tal/o. Using the length data in Table 2. this indicates th;;t the fungi had about Y,, cover on both species, about half the bacterial values. Rccalculation of the data of Parkinson and Crouch (1969) based on the assumption that the average diameter of fungal h!phac ~vas 2.5 llrn showed from 2.3 to 3.7 per cent of the roots covered by mycelia. The volume of fungal hyphae would bc about 7 x IO5 pm”.‘mm’ root surfi~ce. If the bacteria form a layer 1/lrn thick. their volume would be about 7 x 10”~tm3~mmL.
Table 3. Coefficients
Direct observation Bacteria (cover per cent) Fungi (length per mm’ root) Plate counts (number mg fresh Torul bacteria
weight
of
Rclutioc
&iewy
cfdiwcl
oh.s~wvrtio~~ cmd pht
UMN.\
The efficiency of the methods in detecting differences in microbial populations on roots of different plant species can be assessed from the least significant diffcrcnccs (LSD) in Tables I and 2. A more direct comprison can be made bj, calculating the coefticient of bariation (standard deviation t mean) as shown in Table 3. For plate counts, the calculations arc based on numbers per mg fresh weight: results calculated on a dry weight basis gave similar values. The values are consistently lower for direct observation than for plate counts. indicating a grcuter precision for direct counting in determining mean values and diffcrcnces between means. Against this, one must set the greater working time taken for the direct observations. namclq ;I total of about 45 h as against about 30 h for plate counts for the nine replicates. These times include all
variation (experiment 2)
0. I 60
@‘X8
0,X? 0.26 I
0.123
of root) 0,607
0,372
Gram-negative bxteriu Fllamentous fungi
0.492
0.706
Yeast?
I.044
0.757
0536
0.7
I2
0.653
Direct estimation of rhizoplanc microflora operations, spch as staining the roots for direct observation, and preparation of media. but do not include time for arithmetic calculations. Thus the plate counts took only about two-thirds the time, but on the other hand their mean coefficient of variation is nearly three times as great. Since to increase the precision j-fold (i.e. reduce the standard error to one-third) requires about a 9-fold increase of replication, a very great increase in the time spent on the plate counts would have been necessary to achieve the precision of direct observation.
The cover of bacteria over the surface of the roots of the eight species ranged from 4 to IO per cent; the fungal cover on Loliurn and P/~r~rtr~+~was about 3 per cent. With other species or other growing conditions the values might be different. but it is interesting to note that with Pillars rtrdic~rr Don. Bowen and Theodorou (1973) showed that the cover by bacteria of root segments four days old ranged from I.2 to IS.7 per cent (mean 5.1). while on one segment aged 90 days the cover reached 36.6 per cent. Bowcn cstimatcd by the plate count method the numbers of bactcria’mm’ which had colonized a formerly sterile root in three days and calculated from a mean cell size 3 x I llrn that the bacteria would cover some 15 per cent of the root (Bowen and Rovira. 1973). In view of the man) reports based on plate counts of the rhizosphere of marked stimulation of bacteria by plant roots. it was surprising to find such a relati\cly small proportion of the root surface occupied by bacteria and fungi. Calculations transforming the rhlroplane counts to per cent of root surface occupied by bacteria will help understand the ecology of bacterra and fungi on roots. The estimated IO-fold difference between direct counts and plate counts depends upon the ussumptions that the bacterial colonies are onI4 one cell deep and that each cell covers I Ltrn’. Jones and Mollison (1948) concluded that a major reason for the discrepancy between total counts and plate counts of soil samples was that many bacteria occurred as clumps of cells. each of which would give rise to only one colony on agar while being counted as several bacteria under the microscope. As many bacteria on roots occur in micro-colonies surrounded by mucilage of either plant or microbial origin. the clumping of cells could account for much of the discrcpancq. Numbers of rhiToplane micro-organisms are usually expressed on a root weight basis rather than per unit surface area and it would be of interest to know whether the populations of bacteria arc related to surface area or to volume among roots of varying diameter. The mean cover on each of the 108 root segments and the diameter of the segment for Loliur,~ and PluiltUgo in experiment 2 wcrc used to calculate the correlation between root diameter and bacterial cover. The correlation coeflicicnts were: Loli~nl -0.05. Phtugo -049. For fungal length per mm’ the correlation
115
coefficients were: Lolium -0.07. Pla~tu (I) it I-cq~~ircs wntinuous concentration h> the obscncr wcr long periods: (2) estimation of cover per cent is sub,jcctivc and within each experiment. or at lcast within cnch ropllcate block, all estimating should be done b> the same person: where colonization of the surface is sparse. conversion of counts of bacteria to cover invol\cs an assumption about mean bacterial sire: (3) any convcrsion of cover to numbers. weight or volume of bacteria requires assumptions of cell size ;~nd whether cells occur as a single or multiple layer on the root surliicc. The measurement of length of fungal mycelium on roots by the line intercept method does not suITer the same disadvantages as those outlined for bacteria. Firstly, hyphae are more easily seen than hactcria: secondly. the observer can accurately and easily record the number of times hyphae cross the lines. without having to make any subjective cstimatcs: and thirdly. if mean diameter of hqphac is determined from measurements made during observation, then ;ITC;I covered or volume can be cstimatcd. Although no significant difference in fungal length (mm mm’) on the roots of Lo/i~r~l and Pltr~~rtr,~oWIS detected. the prccision was quite high (L.S.D. = 3.6) and it v.ould have needed the examination of about tn ice the number of fields to cstabtish ahether the t 2.1 and 14.1 mm hyphae/mm’ for the two species were significantly d~fferent. This would not have been unduly time-consuning if the experiment had been designed to study ontb the relative fungal populations and not ken concerned primarily with the bacteria. .~~h,~o~~/ct/~~,~j~~,~~~.\ This rexarch was supported hv a grant from the Nutural Environment Research C’ounc~ilwhich sponsored Dr. A. 11. Rovirn :ls ii VisItins Research F~llob and Mr. H. .I. Bow~n ~1s:l Rcsuarch Assist:int.
216
A. D. ROVIICA.E. I. NI:WMA’V.H. J. Bowr u and R. C~WI%I I I HEFERENCES
BOWEN G. D. and ROVIRA A. D. (1973) Are modelling approaches useful in rhizosphere biology? In Mo&v,~ Methods in the StudY qfMicrohiul Ecoloq~,. (Th. Rosswall. Ed.). Bull. Ecol. Res. Conm. (Stockkh) 17, 443-450. BOWEN G. D. and.THEoix)Kor: C. (I 973) Growth of ectomlcorrhizal fungi around seeds and roots. In Ec~o,n~~corrllix~~--Tkeir Ecolq~~ md Physioloyy. (G. C. Marks and T. T. Kozlowski. Eds.) pp. 107~150. Academic Press. London. BUNT J. S. an’d ROVIKA A. D. (I 955) Microbiological studies of some sub-antarctic soils. J. Soil Sci.. 6. 121~ 12X. HOLDING J. (1960) The properties and classifications of the predominant Gram negative bacteria occurring in soil. J. uppl. Bact. 23, 51 j-525. JONES P. C. T. and MOLLISON J. E. (1948) A technique folthe quantitative estimation of soil micro-organisms. .J. yen. Microhiol. 2, 54-69. NEWMAN E. I. (1966) A method ofestimating the total length of root in a sample. J. appl. Ecol. 3, 139-145. NEWMAN E. I. and BOWEN H. J. (1974) Patterns of distribution of bacteria on root surfaces. Sail Biol. Biothrw. 6, 205-209.
I’AK~;IMO~
D. and CKOII(.H R. i IYhYi Stud& on fungi in a pinewood solI_ V: Root microHoras of seedlings ofcPiw uiyrtr \ar. laricio. Rev. EA. Biol. Sol. T. VI. 3, 263-275. ROWKA A. D. (19.56) A study of the dcvelopmcnt of the root surface microRol-a during the initial stages of plant growth. J. trp/l/. Bwr. 19, 72 79. Rovri
&r/i. CIAO/.KC,\. C0,W,l. (Sloc~#,o/,r;) 17. 53 50. W,\II) J. S. ( lY57) Distribution offungi &ithin the dccomposmg tlshucb of rkcgrass roots. Tr~rrls. Brit. !zIycol. Sw. 40, 391 306. .ZA(;ALLO A. C. and KAI/NLLSOI H. ( 1957) Metabolic activitl of bacterial isolates from \vheat rhirosphcre and control soil. J. Btrc,r. 73, 760 764.
of Botany.
H. J. BOWEN and R. CAMPBELL
The University.
Bristol BSX IUG. England
Summary- The hacterlal populations on roots of several grassland species were estimated by combining the tcchniqucs of direct counting of stained bacteria itt .siru with standard sampling techniques of plant ecology. The eight plant species examined had bacterial cover of the root surface within the range 4 10 per cent. In a more detailed experiment with two plant species, the method was sufficiently precise to demonstrate that a 7.7 per cent cover of coots of Loliur~l ~CVYVI~~J L. by bacteria was significantly different (P < @05) from a 6.3 per cent cover on Pla~ttrcgo /~IIIL.~U~~IL. The length of fungal mycelium per unit root surface area was estimated hq a modification of the lint intercept method. which was originally developed for mcasurmg root lengths. When hyphal diameters were also measured. fungal cover per cent and volume could hc estimated. Estimates of the numbers of rhiroplnne bacteria and fungi made hq the spread-plate counting method on a non-selective agar showed that the mean number of bacteria from Loliurn was greater (but not sipnificantly ditfcrent) than the counts on Pltr~~tcqc~:houe\er. Lo/k/n did have significantly more Gram-negative bacteria as estimated hq crystal violet agar than did Pltrr~rugo. The numbers of hactcria catimatcd bq direct microscopy were about IO-fold greater than estimates by plate counts. For II given period of time. direct microscopy gave a more prease estimate of total bacterial and fungal abundance than did plate counts hut the technique docs require continuous concentration by the ohscrvcr over long periods
Although direct microscopy has been used to dcmonstrate micro-organisms on the surfaces of roots (Rovira. 1956: Trolldcnicr. 1973). there arc few studies in which the technique has been used to quantitatively assess the microbial populations on roots. Waid (1957) used percent frequency of occurrence of fungal hyphae along roots as observed by direct microscopy to show fungal succession from the root apex while Thornton (1965) used r-tests to compare fungal populations on roots of different ages and species. Parkinson and Crouch (1969) measured hyphal lengths on roots of Pir~rs nigrtr var. laricio and found these to range from 9.2 to 14% mm:mm’. The only quantitative assessment of rhizoplane populations of bacteria by direct microscopy is that of Bowen and Theodorou (1973). who estimated the percentage cover by micro-organisms in rcprcsentative fields of stained roots of Pirnrs ditrtcc Don. The choice of “representative” fields to estimate rhizoplane populations does not enable a valid statistical analysis of population differences. whether due to plant species. soil conditions, root age or any other causes. In this paper we have applied standard sampling techniques of quantitative plant ecology on a micro-
* Permanent address: laide. South ,4ustralia
CSIRO.
Divismn
of Soils. Ade-
scopic scale to estimate the populations of bacteria and fungi on the roots of several plant species. For two of the plant species we have compared these direct counts with the numbers estimated by the conventional dilution plate method. MATE:RIALS AND YIETHODS
The species selected for this study are plants common in permanent grassland on neutral to slightly acid soils in lowland Britain. The species were four grasses. Allfhosur7rhurll odo~turt~ L., C~r10s~ru.s cristatus L.. Holcus lunntu.s L. and LoII’tfntpr~rnnr L., and four dicotqledonous herbs. H~poclwrris ratikuta L.. Plu~tuylo IUK~YI/LI~L~ L., Rurmz IICP~OS~I L., and Trjfi,lium rrprns L. All were grown from seed collected locally, except for Tr$~liur~ which was the commercial variety S. 100. All eight species were included in experiment 1. the results of which were the basis for selecting Loliur,~ and Pl~~~tu(~ofor the more detailed experiment 2. The soil in which the plants were grown came from permanent grassland and had the following properties: mechanical analysis. 7’:” fine gravel (c2.0 mm), 8’>;, sand (0.2- 2 mm), 170o fine sand (0.06-0.2 mm), 46% silt (0.002 -0.06 mm) and 274(, clay ( <0.002 mm); pH 4.5 (in water): 6.2”,, organic matter (wet digestion). For experiment I the plants were grown in &cm free-draining pots standing in saucers. about four
212
A. D. ROVIKA.E. 1. Nr WMAN.H. J. BOWN
plants per pot. with four replicate pots per species. They were grown from August to October in a greenhouse without artificial light. For experiment 2 the plants were grown in 13-cm pots. two plants per pot and nine pots per species. They were grown from December to February with supplementary illumination over an IX-h da!. The soil was kept moist (about 45”,, water) by allowmg the free-draining pots to stand in saucers in which water was maintained at about I cm depth. After I? weeks the plants were graded according to sire and paired pots. one of 0x11 species, were used on each day.
To observe the rhizoplane micro-organisms in situ. it is necessary to wash most of the soil from the root surface. Experiments were conducted with Loliuttt to estimate the loss of root surface bacteria with the gentle washing necessary to clean the roots for direct microscopy. Roots which had been immersed briefly in sterile distilled water (SDW) to remove excess soil were transferred to a conical flask containing 50ml SDW and gently washed to the point at which they could be used for direct microscopy. These roots were transferred to a flask with lOOmI SDW and 5g glass beads (3 4 mm dia) and shaken vigorously for 15 min with a wrist action shaker before preparing dilutions and plate counts. The results showed that the gentle washing removed 3 x IO” bacteria.‘g fresh weight of root lvhile the 15 min shaking removed 90 x 10” bacteria;g fresh weight of root. This result was consistent for several roots and thus WC concluded that the washing required to produce roots for direct examination uould not remove more than S4 per cent of the rhiLoplane population as estimated by the plate count method. Direct examination of roots and plate counts of the suspensions during the shaking period revealed that 40 and 80 prr cent of the bacteria were removed from the roots after 5 and 10 min shaking respectively and that virtually no bacteria or fungi remained on the roots after 15 min. Maceration of the roots after I5 min shaking did not increase plate counts. These experiments indicated that a shaking time of 15 min on a Grifin Flask shaker (Griffin and George Ltd., Birmingham U.K.) with the roots immersed in 100 ml SDW in ;I 250 ml comcal flask containing glass beads is sufficient to remove the rhizoplane microflora. Sektiott o/‘tmrs. The roots which had been gently washed were trimmed to exclude those parts within I cm of the base of the pot. In experiment 1. one piece of root of about 10 cm, subjectively estimated to be of about average diameter, was chosen from each pot. Counts were made on a I-cm portion at each end of the selected root. In cxpcriment 2. the roots wcrc cut into lengths ofabout 2 cm. and spread out in ;I flat-bottomed dish. For the direct counting, twelve Z-cm root segments were then selected randomly. by taking the segments nearest to 12random points on the dish: no distinction was made between the main root ;IY~Sand finer sccondarj roots in this selection of roots.
and
R. CAMIWLI
Staitzitty uttd direct ttric~roscopq of’roots. The 1 or 2 cm root segments were stained with phenolic aniline blue for 3-6 min. The staining time, which varied for fine and coarse roots. was selected to stain bacteria and fungi dark blue against a light blue or clear background (Rovira, 1956). The segments were mounted in water. placed under a no. 1 cover slip glass and examined under high power ( x 675). The eyepiece graticule was divided to give squares of 18 x 18Ilm for counting. In experiment 1. fi\c fields were selected within each l-cm root segment. and counts were made in five squares in each field. In experiment 2 there were again five fields per segment. but onl) three squares per field. The fields were approximately regularly spaced along the root segment. but the obscrvcr was not looking down the microscope while he moved the slide; this avoided any possibility of selecting a particular arca. When the field was obscured by soil or root hairs. the slide was moved again. Only counts of the bacteria on the surface of the root itself were made. with no cstimates of bacteria on root hairs which were sparsely colonized. For further details on the arrangement of the fields and squares. see Newman and Bowen (1974). In experiment 2 the diameter of each root segment was measured. When a square contained 40 or fewer bacteria. clearly separated. the number of individuals was counted to the nearest 5. Where there were tnore than 40 bacteria per square or where tht: individual cells could not be clearly distinguished, the percentage of the square occupied was estimated on the scale I?. 25. 50. 75 or 100 per cent cover. In both experiments all the direct observation was pcrformcd by one person (H.J.B.). to reduce observer error in estimating cover percentage. For calculating the mean cover percent of all squares the numbers of individuals have been converted to cover on the assumption that the mean projccted area of one bacterium is 1/tm’. This figure is based on measurements of the sire of the bacteria on a number of root scpmcnts. By this conversion 40 bactcria per square give 12 per cent cover. In experiment 2. the length of fungal hyphae per unit area of root surface was estimated by a modification of the line intercept technique of Newman (1966). If hyphac are growing on a plane surface, the length of hyphae can be estimated by placing random lines on the surface and counting the number of times hyphae cross these lines. In the experiment. the random lines (each 36,~m long) were provided by two lines on the eyepiece graticule at right angles to each other. For every field, the eyepiece was twisted to give the lines a “random” direction before counting the number of hyphae crossing them. An estimate of L. the mean length of mycelium per unit area, is given by
L = n,l;. 7H M,hcre :L’ is the total number of intersections and H is the total length of the random lines. If II is in mm. L is in mm mm’.
Direct estimation of rhizopfane
To summarize the direct observations on each plant species: experiment l-bacteria, 4 pots x 2 root segments x 5 fields x 5 squares = 200 squares; experiment 2-bacteria, 9 pots x 12 segments x 5 fields x 3 squares = 1620 squares: fungi. 9 pots x 12 segments x 5 fields = 540 fields.
Fifteen clumps of 2cm length of roots were taken from the dish to give a sample of 0&0~6 g fresh weight and transferred to 100 ml SDW with 5 g glass beads in a 250ml conical Bask. Serial dilutions were prepared of the water in which the roots were shaken for 15 min as described earlier. From the appropriate di~utioi~s a drop of 176 ~1 was spread over one half of a Petri dish containing one of three agar media: each drop was duplicated on another plate. Spread plates were used rather than pour plates because of savings in time and materials with no measurable loss in accuracy of counts (Ridge and Rovira, unpublished results). “Total” bacteria were estimated with 0.37; Tryptic Sov Agar (TSA) prepared from Tryptic Soy Broth (Difco Code: 0.370-01) solidified with 1.57; agar; Dr. J. K. Martin (CSIRO Adelaide, personal communication) has shown that for soil and rhizosphere samples. counts with TSA were similar to those obtained with the YPS medium of Bunt and Rovira (1955); TSA has the advantage of being a standard laboratory medium of reproducible composition. Plates were counted after 7 days at 26°C to minimize losses due to spreading bacteria ; Zagallo and Katznelson (I 957) and Rovira and Brisbane (1967) showed that rhizosphere and rhizoplane bacteria have faster growth rates than most soil bacteria and would produce visible colonies in 7 days. Gram-negative bacteria were estimated by incorporating sterile crystal violet solution into TSA at a final
Table
2. Abundance
of bacteria
(numberjmg root dry weight Total bacteria gram-I~egative bacteria Fi~amentous fungi
concentration of 2 ppm immediately before pouring the plates. Comparisons which we had made between this TSA-CV medium and the crystal violet medium of Holding (1960) showed similar counts of Gram-negative bacteria. Plates were incubated at 26-C for 7 days. Filamentous fungi and yeasts were counted on 4’!,, malt agar (Oxoid CM 90) to which streptomycin at 30 ppm was added immediately before pouring plates. The filamentous fungi were counted after 2 days and the yeasts after 7 days at 26’C. In most samples the yeasts were more numerous than filamcntous fungi and were counted at a higher dilution.
Table I. Per cent cover and per cent frequency of b;tctcria on root surfaces ofeight species m experiment 1, determmed by direct observation 0”
species Hypochorris radicata L. Holcus lanucus L. Lolitirn perenne L. Anthomanthum odoratum L. Cynosurus cristatus L. Rumrs acetosa L. Tr$oirtliumrepens L. Piunty ~an~f~olafa L.
cover*
9.3 8.6 8.4 8.1 6.4 6.3 6-2 3.7 10.1 3.2
LSD
12.1 66.7 7.1 1090
‘I,, Frequency? 81 72 64 70 12 61 66
59 < OQ5 19.7
* Per cent cover is the percentage of root area covered by bacterial cells. t Per cent fequency is the percentage of squares containing one or more bacteria. : Results of analysis of variance: P = significance level of differences between species; LSD = least significant difference between species at P = 0.05.
and fungi on root surfaces of Lo/iurtt pr~trtr and Pkzrtta~~o iur~oftrrrr determined by direct observation and plate counts
Direct observation Fungal hyphae. length. mm:mm’ Bacteria. frequency per cent Bacteria, cover per cent Bacteria. number/mm3 root x 10” Plate counts (number/mg root fresh weight “Total” bacteria Gram-negative bacteria Filamentous fungi Yeasts
21.3
microflora
14.3 66.2 6.3 990
in experiment
24 66 I.3
x 10’) 107 48 0.35 26
70 26 032 ‘0.95
< 0.05 to.1
50 21 0.31 24
x 10”)
YedStS
* Statistical significance of ditkrence “r LSD value at P = 0.05.
737 330 2.3 IX.1 between
the species. by unpaired
_ _
t test; P > O-1 is shown as
368 157 1% 14.2
7.
214
A.
D. ROVIRA.E. I.
H. J. BOWEFand R. CAMPIWLI
NEWMAN,
RICSL’LTS
To compare the numbers of bacteria estimated by direct observation and by plate counts. the cover percent values were converted to numbers per unit root volume. To achieve this conversion, certain assumptions needed to be made, firstly that the average cell covered an area of 1 LLrn’and secondly, that the layer of bacteria was only one cell deep. As we found no significant correlation between root diameter and bacterial cover (see below). a mean surface to volume ratio for each species was used to convert bacterial numbers per unit area of root surface to numbers per unit volume. The mean root diameters were 0.24 mm for Lolium and 0.23 for P(ur~cc~~o.By assuming that 1 mm3 fresh root weighs I mg it IS possible to compare the numbers per mm3 root volume directly with the “total” bacteria per mg fresh weight as estimated by plate count. Table 2 shows that when the numbers of bacteria arc estimated in this way, the direct count is about 10 times the plate count. This discrepancy would be greater if the bacteria are sometimes more than one cell deep on the root and less if one assumed a larger average cell six.
Table 1 shows the percentage cover and per cent frequcncy of bacteria on the root surfaces of the plants used in experiment 1. There were significant differences hctwoen species in frequency. The differences in cover did not reach the conventional 5 per cent significance level. but since the species with higher cover tended also to have higher frequency, and since frequency did differ significantly. results suggest that there were real differcnccs in cover between species. Table 2 shows the results of experiment 7. By direct observation. there was no significant difference between Loliurf7 and Pl~tago in frequency of bacteria. The same was true in experiment I. where Loliun~ was lower in frequency than expected from its cover. In experiment 2 bacterial cover differed significantly bctwcen the spccics. Lolir~ having the higher value. as in experiment I. If the data from the two experiments arc combined and treated as if there had been a single cxpcriment with I! replicates, the difference in cover bctwccn f,o/iurr~ and P/LIIITLIMO was significant at P < WE. The length of fungal hyphae did not differ significantly hctween species. The plate counts showed dif-frrences bctwecn species for total bacteria. Gramnegative bacteria and yeasts. but only for the Gramncgativc bacteria was the differcncc statistically significant (at P < 005). Measurements of hyphal diameter were made on nthcr roots of the two species grown in the same soil and gave a mean diameter of 2.7 I’m for Lolim and 2.4 ~mi for P/c~tal/o. Using the length data in Table 2. this indicates th;;t the fungi had about Y,, cover on both species, about half the bacterial values. Rccalculation of the data of Parkinson and Crouch (1969) based on the assumption that the average diameter of fungal h!phac ~vas 2.5 llrn showed from 2.3 to 3.7 per cent of the roots covered by mycelia. The volume of fungal hyphae would bc about 7 x IO5 pm”.‘mm’ root surfi~ce. If the bacteria form a layer 1/lrn thick. their volume would be about 7 x 10”~tm3~mmL.
Table 3. Coefficients
Direct observation Bacteria (cover per cent) Fungi (length per mm’ root) Plate counts (number mg fresh Torul bacteria
weight
of
Rclutioc
&iewy
cfdiwcl
oh.s~wvrtio~~ cmd pht
UMN.\
The efficiency of the methods in detecting differences in microbial populations on roots of different plant species can be assessed from the least significant diffcrcnccs (LSD) in Tables I and 2. A more direct comprison can be made bj, calculating the coefticient of bariation (standard deviation t mean) as shown in Table 3. For plate counts, the calculations arc based on numbers per mg fresh weight: results calculated on a dry weight basis gave similar values. The values are consistently lower for direct observation than for plate counts. indicating a grcuter precision for direct counting in determining mean values and diffcrcnces between means. Against this, one must set the greater working time taken for the direct observations. namclq ;I total of about 45 h as against about 30 h for plate counts for the nine replicates. These times include all
variation (experiment 2)
0. I 60
@‘X8
0,X? 0.26 I
0.123
of root) 0,607
0,372
Gram-negative bxteriu Fllamentous fungi
0.492
0.706
Yeast?
I.044
0.757
0536
0.7
I2
0.653
Direct estimation of rhizoplanc microflora operations, spch as staining the roots for direct observation, and preparation of media. but do not include time for arithmetic calculations. Thus the plate counts took only about two-thirds the time, but on the other hand their mean coefficient of variation is nearly three times as great. Since to increase the precision j-fold (i.e. reduce the standard error to one-third) requires about a 9-fold increase of replication, a very great increase in the time spent on the plate counts would have been necessary to achieve the precision of direct observation.
The cover of bacteria over the surface of the roots of the eight species ranged from 4 to IO per cent; the fungal cover on Loliurn and P/~r~rtr~+~was about 3 per cent. With other species or other growing conditions the values might be different. but it is interesting to note that with Pillars rtrdic~rr Don. Bowen and Theodorou (1973) showed that the cover by bacteria of root segments four days old ranged from I.2 to IS.7 per cent (mean 5.1). while on one segment aged 90 days the cover reached 36.6 per cent. Bowcn cstimatcd by the plate count method the numbers of bactcria’mm’ which had colonized a formerly sterile root in three days and calculated from a mean cell size 3 x I llrn that the bacteria would cover some 15 per cent of the root (Bowen and Rovira. 1973). In view of the man) reports based on plate counts of the rhizosphere of marked stimulation of bacteria by plant roots. it was surprising to find such a relati\cly small proportion of the root surface occupied by bacteria and fungi. Calculations transforming the rhlroplane counts to per cent of root surface occupied by bacteria will help understand the ecology of bacterra and fungi on roots. The estimated IO-fold difference between direct counts and plate counts depends upon the ussumptions that the bacterial colonies are onI4 one cell deep and that each cell covers I Ltrn’. Jones and Mollison (1948) concluded that a major reason for the discrepancy between total counts and plate counts of soil samples was that many bacteria occurred as clumps of cells. each of which would give rise to only one colony on agar while being counted as several bacteria under the microscope. As many bacteria on roots occur in micro-colonies surrounded by mucilage of either plant or microbial origin. the clumping of cells could account for much of the discrcpancq. Numbers of rhiToplane micro-organisms are usually expressed on a root weight basis rather than per unit surface area and it would be of interest to know whether the populations of bacteria arc related to surface area or to volume among roots of varying diameter. The mean cover on each of the 108 root segments and the diameter of the segment for Loliur,~ and PluiltUgo in experiment 2 wcrc used to calculate the correlation between root diameter and bacterial cover. The correlation coeflicicnts were: Loli~nl -0.05. Phtugo -049. For fungal length per mm’ the correlation
115
coefficients were: Lolium -0.07. Pla~tu (I) it I-cq~~ircs wntinuous concentration h> the obscncr wcr long periods: (2) estimation of cover per cent is sub,jcctivc and within each experiment. or at lcast within cnch ropllcate block, all estimating should be done b> the same person: where colonization of the surface is sparse. conversion of counts of bacteria to cover invol\cs an assumption about mean bacterial sire: (3) any convcrsion of cover to numbers. weight or volume of bacteria requires assumptions of cell size ;~nd whether cells occur as a single or multiple layer on the root surliicc. The measurement of length of fungal mycelium on roots by the line intercept method does not suITer the same disadvantages as those outlined for bacteria. Firstly, hyphae are more easily seen than hactcria: secondly. the observer can accurately and easily record the number of times hyphae cross the lines. without having to make any subjective cstimatcs: and thirdly. if mean diameter of hqphac is determined from measurements made during observation, then ;ITC;I covered or volume can be cstimatcd. Although no significant difference in fungal length (mm mm’) on the roots of Lo/i~r~l and Pltr~~rtr,~oWIS detected. the prccision was quite high (L.S.D. = 3.6) and it v.ould have needed the examination of about tn ice the number of fields to cstabtish ahether the t 2.1 and 14.1 mm hyphae/mm’ for the two species were significantly d~fferent. This would not have been unduly time-consuning if the experiment had been designed to study ontb the relative fungal populations and not ken concerned primarily with the bacteria. .~~h,~o~~/ct/~~,~j~~,~~~.\ This rexarch was supported hv a grant from the Nutural Environment Research C’ounc~ilwhich sponsored Dr. A. 11. Rovirn :ls ii VisItins Research F~llob and Mr. H. .I. Bow~n ~1s:l Rcsuarch Assist:int.
216
A. D. ROVIICA.E. I. NI:WMA’V.H. J. Bowr u and R. C~WI%I I I HEFERENCES
BOWEN G. D. and ROVIRA A. D. (1973) Are modelling approaches useful in rhizosphere biology? In Mo&v,~ Methods in the StudY qfMicrohiul Ecoloq~,. (Th. Rosswall. Ed.). Bull. Ecol. Res. Conm. (Stockkh) 17, 443-450. BOWEN G. D. and.THEoix)Kor: C. (I 973) Growth of ectomlcorrhizal fungi around seeds and roots. In Ec~o,n~~corrllix~~--Tkeir Ecolq~~ md Physioloyy. (G. C. Marks and T. T. Kozlowski. Eds.) pp. 107~150. Academic Press. London. BUNT J. S. an’d ROVIKA A. D. (I 955) Microbiological studies of some sub-antarctic soils. J. Soil Sci.. 6. 121~ 12X. HOLDING J. (1960) The properties and classifications of the predominant Gram negative bacteria occurring in soil. J. uppl. Bact. 23, 51 j-525. JONES P. C. T. and MOLLISON J. E. (1948) A technique folthe quantitative estimation of soil micro-organisms. .J. yen. Microhiol. 2, 54-69. NEWMAN E. I. (1966) A method ofestimating the total length of root in a sample. J. appl. Ecol. 3, 139-145. NEWMAN E. I. and BOWEN H. J. (1974) Patterns of distribution of bacteria on root surfaces. Sail Biol. Biothrw. 6, 205-209.
I’AK~;IMO~
D. and CKOII(.H R. i IYhYi Stud& on fungi in a pinewood solI_ V: Root microHoras of seedlings ofcPiw uiyrtr \ar. laricio. Rev. EA. Biol. Sol. T. VI. 3, 263-275. ROWKA A. D. (19.56) A study of the dcvelopmcnt of the root surface microRol-a during the initial stages of plant growth. J. trp/l/. Bwr. 19, 72 79. Rovri
&r/i. CIAO/.KC,\. C0,W,l. (Sloc~#,o/,r;) 17. 53 50. W,\II) J. S. ( lY57) Distribution offungi &ithin the dccomposmg tlshucb of rkcgrass roots. Tr~rrls. Brit. !zIycol. Sw. 40, 391 306. .ZA(;ALLO A. C. and KAI/NLLSOI H. ( 1957) Metabolic activitl of bacterial isolates from \vheat rhirosphcre and control soil. J. Btrc,r. 73, 760 764.